Systematics of the ectomycorrhizal genus Lactariusin the Rocky Mountain alpine zone

Edward G Barge1

Cathy L CrippsPlant Science and Plant Pathology DepartmentMontana State University Bozeman Montana 59717

Todd W OsmundsonBiology Department University of WisconsinndashLa CrosseLa Crosse Wisconsin 54601

Abstract Lactarius (Russulales) is an important com-ponent of ectomycorrhizal fungal communities incold-dominated contiguous arctic and disjunct alpinehabitats where it associates primarily with Betula Dryasand Salix However little is known of this genus in thecentral and southern Rocky Mountain alpine zone(3000ndash3900 m) of North America Molecular phyloge-netic analyses of nuc rDNA ITS1-58S-ITS2 (ITS bar-code) and the second largest subunit of the RNApolymerase II gene (RPB2) partial sequences in con-junction with detailed morphological examinationconfirm at least six species occurring above treelineMost have intercontinental distributions in NorthAmerica and Eurasia according to molecular compari-son with type material and collections from EuropeFennoscandia Svalbard and Alaska Rocky Mountaincollections of L lanceolatus (subgenus Russularia)along with the type from Alaska are paraphyletic withrespect to L aurantiacus and North American taxa Lluculentus and L luculentus v laetus Rocky Mountaincollections of L nanus L glyciosmus L repraesentaneusand L salicis-reticulatae (subgenus Piperites) all formclades with European material from type localitiesand other arctic-alpine habitats The arctic-alpine LpseudouvidusL brunneoviolaceus group appears to bea complex containing additional taxa North Americanmaterial originally described as part of this group iswell-separated phylogenetically and is described hereas L pallidomarginatus sp nov Lactarius lanceolatus Lnanus and L salicis-reticulatae appear largely restrictedto arctic-alpine habitats with Salix Lactarius glyciosmusand L repraesentaneus occur in arctic-alpine subalpineand boreal habitats with Betula and also Picea and pos-sibly Salix for the latter Species distributions arehypothesized to be shaped by host ranges glaciationand long distance dispersal

Key words Arctic-alpine Betula ITS RPB2 Russu-laceae Salix

INTRODUCTION

The arctic-alpine region encompasses cold-dominatedvegetated areas above or beyond the altitudinal or lati-tudinal limit of tree growth (Chapin and Koumlrner1995) Here ectomycorrhizal fungi associate withshrubs including Betula Dryas and Salix and sedgesand herbs such as Kobresia and Bistorta (Cripps andEddington 2005 Dahlberg and Buumlltmann 2013) Theectomycorrhizal basidiomycete genus Lactarius Pers(Russulales Russulaceae) is well-documented in arc-tic-alpine habitats although a comprehensive studyhad not been made of species in this genus occurringin the Rocky Mountain alpine zone As originallydefined Lactarius includes species with sporocarpsthat exude a latex when damaged in addition to theamyloid ornamented basidiospores characteristic ofthe family The genus is now recognized as nonmono-phyletic and has been split into three genera withinRussulaceae Lactarius Lactifluus (Pers) Roussel andMultifurca Buyck amp Hoffstetter (Buyck et al 20082010) Lactarius sensu stricto (ss) contains about80 of the traditional species most of which are tem-perate to boreal in distribution (Buyck et al 20082010 Verbeken and Nuytinck 2013) Most speciesfrom arctic-alpine habitats fall in Lactarius ss subge-nera Piperites (Fr) Kauffman and Russularia (Fr)Kauffman based on morphological characteristicsalthough the monophyly of these subgenera has notbeen thoroughly investigated with molecular data

More than 65 species and varieties of Lactarius havebeen reported from arctic-alpine areas in the northernhemisphere mostly through sporocarp surveys (SUP-

PLEMENTARY TABLE I) However due to synonymynomenclatural misapplications misidentifications andthe possibility that some reports actually came fromsubalpine or subarctic areas it is likely that the actualnumber of species inhabiting arctic-alpine areas ismuch lower

Lactarius species have been reported from arctic-alpine areas in Alaska (Laursen and Ammirati 1982Geml et al 2009) Canada (Ohenoja and Ohenoja1993 2010) Iceland (Heilmann-Clausen et al 1998Eyjolfsdottir pers comm) Greenland (Terkelsen1956 Lange 1957 Kobayasi et al 1971 Knudsen andBorgen 1982 Lamoure et al 1982 Elbourne andKnudsen 1990 Knudsen and Lamoure 1993 Knudsenand Borgen 1994 Watling 1997 Heilmann-Clausenet al 1998 Borgen et al 2006) the British Isles(Watling 1987 Watling et al 2001) the Alps (Favre

Submitted 10 Jul 2015 accepted for publication 2 Dec 20151 Corresponding author E-mail ebarge9gmailcom

Mycologia 108(2) 2016 pp 414ndash440 DOI 10385215-177 2016 by The Mycological Society of America Lawrence KS 66044-8897

414

1955 Kuumlhner 1975 Bon 1985a b 1989 1991 Jamoniand Bon 1991 Senn-Irlet 1993 Jamoni 1995 2008)the Pyrenees (Bon and Ballaraacute 1996 Vila et al 1997Corriol 2008) the Carpathians (Ronikier 2008) Fen-noscandia (Kallio and Kankainen 1964 Gulden et al1985 Heilmann-Clausen et al 1998 Gulden 2005Knudsen and Vesterholt 2008) Svalbard (Gulden andJenssen 1988 Gulden and Torkelsen 1996) northernRussia (Kalamees and Vaasma 1993 Knudsen andMuhkin 1998 Karatygin et al 1999) and the AltaiMountains (Kalamees 2008 Gorbunova 2010 Gorbu-nova and Taylakov 2011)

Many of the Lactarius species reported in these treat-ments appear to have broad intercontinental distribu-tions in North America and Eurasia but thishypothesis remains largely untested In addition somespecies appear largely restricted to arctic-alpine areaswhile reports suggest others have wider distributionsthat extend into subalpine temperate and boreal habi-tats (Knudsen and Borgen 1982 Heilmann-Clausenet al 1998 Knudsen and Vesterholt 2008) A few studieshave confirmed broad intercontinental distributions forspecies in other ectomycorrhizal genera in arctic-alpinehabitats using morphological and molecular data(Peintner 2008 Beker et al 2010 Cripps et al 2010Larsson et al 2014) Environmental sequencing studies(some of which include Lactarius) also suggest intercon-tinental conspecific taxa although they allow less strin-gent species delineation (Geml et al 2012 Timlinget al 2012 2014) In contrast recent molecular phylo-genetic studies of Lactarius ss sampled from subalpinetemperate habitats reveal narrow species ranges and alack of intercontinental conspecificity (Nuytinck et al2007 Stubbe and Verbeken 2012)

Until recently relatively little was known of themacrofungal mycota of the Rocky Mountain alpinezone of North America An extensive sporocarp surveyconducted by Cripps and Horak (2008) in the centraland southern Rocky Mountain alpine zone of USA(Montana Wyoming Colorado) revealed the presenceof more than 165 fungal species in 46 genera and 11families More than 75 appear to be species knownfrom other arctic-alpine regions and the remaindermay be western endemics Detailed studies stemmingfrom this survey include those on the molecular andmorphological systematics of arctic-alpine Cortinarius(Peintner 2008) Hebeloma (Beker et al 2010) Inocybe(Cripps et al 2010 Larsson et al 2014) Laccaria(Osmundson et al 2005) and morphological systema-tics of arctic-alpine Amanita (Cripps and Horak 2010)and Lycoperdaceae (Kasuya 2010)

Lactarius is well-studied in North America (eg Hes-ler and Smith 1979 Methven 1997 Bessette et al2009) however outside Alaska and Canada arctic-alpine taxa are largely unaddressed Preliminary

research tentatively identified six morphologicallydefined Lactarius species from the Rocky Mountainalpine zone (Cripps and Horak 2008 Cripps andBarge 2013) All species originally were describedfrom Europe except for L lanceolatus OK Mill ampLaursen which was described from Alaska Here collec-tions of Lactarius from the Rocky Mountain alpinezone are evaluated through detailed morphologicalstudy and phylogenetic analyses of sequences fromtwo genetic loci nuclear rDNA ITS1ndash58SndashITS2 (ITSbarcode) and the gene region between conserveddomains six and seven of the second largest subunitof the RNA polymerase II (RPB2) Data were com-pared to type specimens specimens from type local-ities collections from arctic-alpine and subalpinehabitats in North America Europe FennoscandiaSvalbard Alaska Greenland and related taxa repre-sented by sequences in databases (GenBank UNITE)Insight into worldwide distribution degree of confine-ment to arctic-alpine habitats and host specificity alsois provided for these and related taxa

MATERIALS AND METHODS

Study sitesmdashThe Rocky Mountain alpine zone occurs asscattered high elevation islands of open landscape abovetreeline on mountaintops in USA and Canada extendingsouthward from arctic Alaska to the Sangre de Cristo Rangein northern New Mexico (Billings 1988) This study wasperformed in alpine areas of the central and southernRocky Mountains as classified by Brouillet and Whetstone(1993) Central Rocky Mountain sites are located abovetreeline on the Beartooth Plateau in southern Montanaand northern Wyoming (FIG 1) and include Birch Site(45u01444N 109u24486W 2990ndash3020 m) Highline Trail(45u00356N 109u24387W 3060ndash3100 m) Frozen Lakes(44u57932N 109u28998W 3190ndash3200 m) Gardner Lake(44u57949N 109u27199W 3030 m) and Solifluction Ter-races (44u58372N 109u26810W 3258 m) Southern RockyMountain sites are all above treeline in Colorado and includeBlue Lake (40u05400N 105u37200W 3560 m) BrainardLake (40u04200N 105u34200W 3300 m) and Loveland Pass(39u39600N 105u52800W 3650m) in theFrontRangeCotton-wood Pass (38u49200N 106u24000W 3700 m) and Indepen-dence Pass (39u06600N 106u33600W 3650 m) in the SawatchRange and Black Bear Pass (37u24000N 107u42000W3900 m) Cinnamon Pass (37u55800N 107u31800W 3840 m)Imogene Pass (37u55800N 107u43200W 3900 m) MaggieGulch (37u51000N 107u34200W 3600 m) and Stony Pass(37u46800N 107u31800W 3700 m) in the San Juan Range(FIG 1) These sites are characterized by a relatively windy drycontinental climate with large diurnal fluctuations in tempera-ture and large differences between winter and summer averagetemperatures although freezing temperatures are often experi-enced even during the growing season The vegetation consistsof low-lying perennial cushion plants grasses rosette plantsand prostrate and erect shrubs Plant community types in theRocky Mountain alpine zone vary often sharply as a result of

BARGE ET AL ROCKY MOUNTAIN ALPINE LACTARIUS 415

changes in the physical environment such as topography aspectandmicrorelief (Johnson and Billings 1962 Cooper et al 1997)Ectomycorrhizal host species in theRockyMountain alpine zoneinclude shrubby willows Salix glauca L and S planifolia Purshdwarf willows S arctica Pall and S reticulata L the bog birchBetula glandulosa Michx the recumbent mat plant Dryas octope-tala L and the perennial sedge Kobresia myosuroides (Vill) Fiori(Schadt 2002 Cripps and Eddington 2005) In general thesame ectomycorrhizal host plants occur across the study siteshowever a greater abundance of Salix glauca was encounteredat sites in Colorado and only one isolatedBetula glandulosa shrubwas encountered on the Beartooth Plateau while B glandulosawas observed at several sites in Colorado

Taxon sampling and processingmdashSporocarps were collectedfrom late Julndashlate Aug 1997ndash2014 All collections weredescribed in detail when fresh and select collections werephotographed Ectomycorrhizal host plants near sporocarpswere noted for each collection Sporocarps were dried on

an electric warm-air dryer and deposited in the MontanaState University herbarium (MONT) Collections initiallywere identified to species primarily using Jamoni (19952008) Bon (1998) Heilmann-Clausen et al (1998) andBasso (1999) Herbarium specimens of type collections puta-tive conspecific and taxonomically similar species wereobtained from the herbarium of the Conservatoire et Jardinbotaniques de la Ville de Genegraveve Geneva Switzerland (G)the herbarium of Universitaumlt Innsbruck Innsbruck Austria(IB) the Herbarium Universitatis Tartuensis Tartu Estonia(TU) the Botanical Museum Oslo Norway (O) HerbariumGB University of Gothenburg Goumlteborg Sweden (GB) theherbarium of Aringbo Akademi University Turku Finland(TURA) the Mycological Herbarium of the Natural HistoryMuseum of Denmark Copenhagen Denmark (C) the her-barium of Universiteacute de Lille France (LIP) the Universityof Michigan fungal herbarium Ann Arbor Michigan(MICH) and the Denver Botanic Gardensrsquo Sam MitchelHerbarium of Fungi Denver Colorado (DBG) The type

FIG 1 Map of the collection locations of specimens that were examined andor included in molecular analyses (if collectionlocations are known) Rocky Mountain alpine study sites are indicated by white dots black dots indicate other arctic-alpine sitesand gray dots indicate subalpine sites Several collections from outside the map area (China Japan Thailand) are not includedThe black dotted line delineates the Rocky Mountains the arctic circle (675uN) is indicated by the dashed gray line and thenorthern tree line which corresponds with the southern extent of arctic tundra is indicated by the solid dark gray line (Brownet al 2001)

416 MYCOLOGIA

collection of L lanceolatus OK Mill amp Laursen was obtainedfrom Massey Herbarium Virginia Tech Blacksburg Virginia(VPI) and is now at the herbarium of the New York BotanicalGarden (NY) Herbarium acronyms follow Thiers (continu-ously updated) The collection locations of specimens thatwere examined andor included in phylogenetic analysesin this study are shown in relation to Rocky Mountain alpinestudy sites (FIG 1)

Morphological descriptionsmdashDescriptions of macromorpho-logical and micromorphological features were made fromfresh and dry material respectively For micromorphologicalexamination dried material was reconstituted in ethanol andthin sections were prepared Basidiospores were examined inMelzerrsquos reagent basidia macrocystidia and pileipellis tissuewere examined in 25 KOH Length and width of micro-morphological features were measured with an ocular micro-meter under an oil-immersion lens at 10006 magnificationfor a random sample of 25 basidiospores per collectionexcluding ornamentation the hilar appendix and giant basi-diospores from two-spored basidia mean basidiospore lengthand width were calculated for each collection The length towidth ratio (Q) was calculated for each basidiospore andmean length to width ratio (Q) was calculated for each col-lection The maximum height of the basidiospore ornamen-tation was measured and recorded and the ornamentationdescribed Length and width (at widest point) were mea-sured at 4006magnification for a random sample of 10 basi-dia (including sterigmata and excluding two-spored basidia)10 pleuromacrocystidia and 10 cheilomacrocystidia per col-lection Drawings of basidiospores pleuromacrocystidia andcheilomacrocystidia were prepared from one representativecollection of each Rocky Mountain alpine Lactarius speciesas well as for their corresponding type specimen (when avail-able) with a Leica drawing tube attached to a Leica DMLSresearch microscope Terminology is according to Heil-mann-Clausen et al (1998)

DNA extraction PCR amplification and sequencingmdashDNAwas extracted from dried ground sporocarp tissue followingOsmundson et al (2013) or using the DNeasy Plant Mini Kit(QIAGEN) following the manufacturerrsquos instructions PCRamplification of the ITS region was performed with primersITS1-F and ITS4 or ITS1-F and ITS2 or ITS3 and ITS4 (Whiteet al 1990 Gardes and Bruns 1993) The RPB2 gene segmentwas amplified with primers bRPB2-6f and fRPB2-7cR orbRPB2-6f and i2r-RPB2 or fRPB2-7cR and ilf-RPB2 (Liu et al1999 Matheny 2005 Stubbe et al 2010) Primer i2r-RPB2 ofStubbe et al (2010) was made slightly less specific by repla-cing primer positions 6 (5 T) 9 (5 C) and 15 (5 T) withY yielding the primer consensus sequence 5acutendashACV GTYTCY TCT TCY TCR GCR TCndash3acute The thermo-cycler programused for ITS amplification was 94 C for 2 min followed by 30cycles of 94 C for 30 s 55 C for 1 min and 72 C for 1 min fol-lowed by a final elongation step of 72 C for 5 min The ther-mos-cycler program used for RPB2 amplification was 94 C for90 s followed by 40 cycles of 94 C for 30 s 55 C for 90 s and 68C for 3 min concluding with a 5 min final extension at 68 CPCR products were cleaned with ExoSAP-IT (USB USA) or

the QIAquick PCR Purification Kit (QIAGEN) following themanufacturerrsquos instructions PCR products were sequencedon both strands with the same primers used for PCR Rawsequences were edited and contigs constructed with eitherGeneious Pro (Drummond et al 2009) or SeqTrace 081(Stucky 2012)

Sequence alignment and phylogenetic analysesmdashIn additionto the sequences generated in this study sequences exhibit-ing 97 or higher BLAST matches to the ITS region of arepresentative specimen of each Rocky Mountain alpinetaxon were downloaded from GenBank (httpwwwncbinlmnihgov) and UNITE (httpsuniteutee) as were anumber of ITS and RPB2 sequences mostly from Buyck et al(2008) Verbeken et al (2014) and the UNITE databaseLocality voucher information and accession numbers ofsequences generated in this study (TABLE I) and of allsequences used in phylogenetic analyses (SUPPLEMENTARY

TABLE II) are provided Due to difficulty unambiguouslyaligning the ITS region across the genus Lactarius four sepa-rate datasets were assembled One broad ITS+RPB2 datasetwas constructed in an attempt to ascertain the phylogeneticposition of each Rocky Mountain alpine taxon within thegenus Lactarius (referred to as the ldquobroad datasetrdquo) Thisresulted in the identification of three clades that served toguide the construction of three subsequent species-levelITS+RPB2 datasets (referred to as ldquodatasets A B Crdquo) Align-ments were performed with MUSCLE (httpwwwebiacukToolsmsamuscle) with default parameters For thebroad dataset ambiguously aligned regions of the ITS align-ment were highlighted and removed with the online versionof GBlocks 091b (Castresana 2000) under the least stringentsettings For datasets A B and C the alignments were editedwith SeAl (Rambaut 1995) For dataset C a 268ndash269 base-pairinsertion present in the ITS1 region of a number of speci-mens (see SUPPLEMENTARY TABLE II) was removed alongwith a small ambiguously aligned region present in taxa lack-ing the insertion

For each dataset topological incongruence between theITS and RPB2 loci was examined with the partition homoge-neity test (Farris et al 1995) as implemented in PAUP(Swofford 2001) The null hypothesis of congruence wasrejected if P 005 Maximum parsimony (MP) analyseswere carried out separately for ITS and RPB2 loci and forthe combined datasets with PAUP using heuristic searchesincluding 100 random addition sequence replicates andtree bisection reconnection branch swapping Clade supportwas assessed with 1000 bootstrap replicates with heuristicsearches tree bisection reconnection branch swapping andone random addition sequence per replicate

Maximum likelihood (ML) analyses were carried out withraxmlGUI 131 (Stamatikis 2006 Silvestro and Michalak2012) An ML search combined with rapid bootstrappingwas run until bootstrap convergence or 1000 bootstrap repli-cates was reached using the autoMRE option TheGTRGAMMA model was estimated separately for ITS1 58SITS2 RPB2 exon first second and third codon positionsand RPB2 introns for the broad dataset For datasets A Band C the GTRCAT model was estimated separately for ITS158S ITS2 RPB2 exon first second and third codon positions

BARGE ET AL ROCKY MOUNTAIN ALPINE LACTARIUS 417

1955 Kuumlhner 1975 Bon 1985a b 1989 1991 Jamoniand Bon 1991 Senn-Irlet 1993 Jamoni 1995 2008)the Pyrenees (Bon and Ballaraacute 1996 Vila et al 1997Corriol 2008) the Carpathians (Ronikier 2008) Fen-noscandia (Kallio and Kankainen 1964 Gulden et al1985 Heilmann-Clausen et al 1998 Gulden 2005Knudsen and Vesterholt 2008) Svalbard (Gulden andJenssen 1988 Gulden and Torkelsen 1996) northernRussia (Kalamees and Vaasma 1993 Knudsen andMuhkin 1998 Karatygin et al 1999) and the AltaiMountains (Kalamees 2008 Gorbunova 2010 Gorbu-nova and Taylakov 2011)

Many of the Lactarius species reported in these treat-ments appear to have broad intercontinental distribu-tions in North America and Eurasia but thishypothesis remains largely untested In addition somespecies appear largely restricted to arctic-alpine areaswhile reports suggest others have wider distributionsthat extend into subalpine temperate and boreal habi-tats (Knudsen and Borgen 1982 Heilmann-Clausenet al 1998 Knudsen and Vesterholt 2008) A few studieshave confirmed broad intercontinental distributions forspecies in other ectomycorrhizal genera in arctic-alpinehabitats using morphological and molecular data(Peintner 2008 Beker et al 2010 Cripps et al 2010Larsson et al 2014) Environmental sequencing studies(some of which include Lactarius) also suggest intercon-tinental conspecific taxa although they allow less strin-gent species delineation (Geml et al 2012 Timlinget al 2012 2014) In contrast recent molecular phylo-genetic studies of Lactarius ss sampled from subalpinetemperate habitats reveal narrow species ranges and alack of intercontinental conspecificity (Nuytinck et al2007 Stubbe and Verbeken 2012)

Until recently relatively little was known of themacrofungal mycota of the Rocky Mountain alpinezone of North America An extensive sporocarp surveyconducted by Cripps and Horak (2008) in the centraland southern Rocky Mountain alpine zone of USA(Montana Wyoming Colorado) revealed the presenceof more than 165 fungal species in 46 genera and 11families More than 75 appear to be species knownfrom other arctic-alpine regions and the remaindermay be western endemics Detailed studies stemmingfrom this survey include those on the molecular andmorphological systematics of arctic-alpine Cortinarius(Peintner 2008) Hebeloma (Beker et al 2010) Inocybe(Cripps et al 2010 Larsson et al 2014) Laccaria(Osmundson et al 2005) and morphological systema-tics of arctic-alpine Amanita (Cripps and Horak 2010)and Lycoperdaceae (Kasuya 2010)

Lactarius is well-studied in North America (eg Hes-ler and Smith 1979 Methven 1997 Bessette et al2009) however outside Alaska and Canada arctic-alpine taxa are largely unaddressed Preliminary

research tentatively identified six morphologicallydefined Lactarius species from the Rocky Mountainalpine zone (Cripps and Horak 2008 Cripps andBarge 2013) All species originally were describedfrom Europe except for L lanceolatus OK Mill ampLaursen which was described from Alaska Here collec-tions of Lactarius from the Rocky Mountain alpinezone are evaluated through detailed morphologicalstudy and phylogenetic analyses of sequences fromtwo genetic loci nuclear rDNA ITS1ndash58SndashITS2 (ITSbarcode) and the gene region between conserveddomains six and seven of the second largest subunitof the RNA polymerase II (RPB2) Data were com-pared to type specimens specimens from type local-ities collections from arctic-alpine and subalpinehabitats in North America Europe FennoscandiaSvalbard Alaska Greenland and related taxa repre-sented by sequences in databases (GenBank UNITE)Insight into worldwide distribution degree of confine-ment to arctic-alpine habitats and host specificity alsois provided for these and related taxa

MATERIALS AND METHODS

Study sitesmdashThe Rocky Mountain alpine zone occurs asscattered high elevation islands of open landscape abovetreeline on mountaintops in USA and Canada extendingsouthward from arctic Alaska to the Sangre de Cristo Rangein northern New Mexico (Billings 1988) This study wasperformed in alpine areas of the central and southernRocky Mountains as classified by Brouillet and Whetstone(1993) Central Rocky Mountain sites are located abovetreeline on the Beartooth Plateau in southern Montanaand northern Wyoming (FIG 1) and include Birch Site(45u01444N 109u24486W 2990ndash3020 m) Highline Trail(45u00356N 109u24387W 3060ndash3100 m) Frozen Lakes(44u57932N 109u28998W 3190ndash3200 m) Gardner Lake(44u57949N 109u27199W 3030 m) and Solifluction Ter-races (44u58372N 109u26810W 3258 m) Southern RockyMountain sites are all above treeline in Colorado and includeBlue Lake (40u05400N 105u37200W 3560 m) BrainardLake (40u04200N 105u34200W 3300 m) and Loveland Pass(39u39600N 105u52800W 3650m) in theFrontRangeCotton-wood Pass (38u49200N 106u24000W 3700 m) and Indepen-dence Pass (39u06600N 106u33600W 3650 m) in the SawatchRange and Black Bear Pass (37u24000N 107u42000W3900 m) Cinnamon Pass (37u55800N 107u31800W 3840 m)Imogene Pass (37u55800N 107u43200W 3900 m) MaggieGulch (37u51000N 107u34200W 3600 m) and Stony Pass(37u46800N 107u31800W 3700 m) in the San Juan Range(FIG 1) These sites are characterized by a relatively windy drycontinental climate with large diurnal fluctuations in tempera-ture and large differences between winter and summer averagetemperatures although freezing temperatures are often experi-enced even during the growing season The vegetation consistsof low-lying perennial cushion plants grasses rosette plantsand prostrate and erect shrubs Plant community types in theRocky Mountain alpine zone vary often sharply as a result of

BARGE ET AL ROCKY MOUNTAIN ALPINE LACTARIUS 415

changes in the physical environment such as topography aspectandmicrorelief (Johnson and Billings 1962 Cooper et al 1997)Ectomycorrhizal host species in theRockyMountain alpine zoneinclude shrubby willows Salix glauca L and S planifolia Purshdwarf willows S arctica Pall and S reticulata L the bog birchBetula glandulosa Michx the recumbent mat plant Dryas octope-tala L and the perennial sedge Kobresia myosuroides (Vill) Fiori(Schadt 2002 Cripps and Eddington 2005) In general thesame ectomycorrhizal host plants occur across the study siteshowever a greater abundance of Salix glauca was encounteredat sites in Colorado and only one isolatedBetula glandulosa shrubwas encountered on the Beartooth Plateau while B glandulosawas observed at several sites in Colorado

Taxon sampling and processingmdashSporocarps were collectedfrom late Julndashlate Aug 1997ndash2014 All collections weredescribed in detail when fresh and select collections werephotographed Ectomycorrhizal host plants near sporocarpswere noted for each collection Sporocarps were dried on

an electric warm-air dryer and deposited in the MontanaState University herbarium (MONT) Collections initiallywere identified to species primarily using Jamoni (19952008) Bon (1998) Heilmann-Clausen et al (1998) andBasso (1999) Herbarium specimens of type collections puta-tive conspecific and taxonomically similar species wereobtained from the herbarium of the Conservatoire et Jardinbotaniques de la Ville de Genegraveve Geneva Switzerland (G)the herbarium of Universitaumlt Innsbruck Innsbruck Austria(IB) the Herbarium Universitatis Tartuensis Tartu Estonia(TU) the Botanical Museum Oslo Norway (O) HerbariumGB University of Gothenburg Goumlteborg Sweden (GB) theherbarium of Aringbo Akademi University Turku Finland(TURA) the Mycological Herbarium of the Natural HistoryMuseum of Denmark Copenhagen Denmark (C) the her-barium of Universiteacute de Lille France (LIP) the Universityof Michigan fungal herbarium Ann Arbor Michigan(MICH) and the Denver Botanic Gardensrsquo Sam MitchelHerbarium of Fungi Denver Colorado (DBG) The type

FIG 1 Map of the collection locations of specimens that were examined andor included in molecular analyses (if collectionlocations are known) Rocky Mountain alpine study sites are indicated by white dots black dots indicate other arctic-alpine sitesand gray dots indicate subalpine sites Several collections from outside the map area (China Japan Thailand) are not includedThe black dotted line delineates the Rocky Mountains the arctic circle (675uN) is indicated by the dashed gray line and thenorthern tree line which corresponds with the southern extent of arctic tundra is indicated by the solid dark gray line (Brownet al 2001)

416 MYCOLOGIA

collection of L lanceolatus OK Mill amp Laursen was obtainedfrom Massey Herbarium Virginia Tech Blacksburg Virginia(VPI) and is now at the herbarium of the New York BotanicalGarden (NY) Herbarium acronyms follow Thiers (continu-ously updated) The collection locations of specimens thatwere examined andor included in phylogenetic analysesin this study are shown in relation to Rocky Mountain alpinestudy sites (FIG 1)

Morphological descriptionsmdashDescriptions of macromorpho-logical and micromorphological features were made fromfresh and dry material respectively For micromorphologicalexamination dried material was reconstituted in ethanol andthin sections were prepared Basidiospores were examined inMelzerrsquos reagent basidia macrocystidia and pileipellis tissuewere examined in 25 KOH Length and width of micro-morphological features were measured with an ocular micro-meter under an oil-immersion lens at 10006 magnificationfor a random sample of 25 basidiospores per collectionexcluding ornamentation the hilar appendix and giant basi-diospores from two-spored basidia mean basidiospore lengthand width were calculated for each collection The length towidth ratio (Q) was calculated for each basidiospore andmean length to width ratio (Q) was calculated for each col-lection The maximum height of the basidiospore ornamen-tation was measured and recorded and the ornamentationdescribed Length and width (at widest point) were mea-sured at 4006magnification for a random sample of 10 basi-dia (including sterigmata and excluding two-spored basidia)10 pleuromacrocystidia and 10 cheilomacrocystidia per col-lection Drawings of basidiospores pleuromacrocystidia andcheilomacrocystidia were prepared from one representativecollection of each Rocky Mountain alpine Lactarius speciesas well as for their corresponding type specimen (when avail-able) with a Leica drawing tube attached to a Leica DMLSresearch microscope Terminology is according to Heil-mann-Clausen et al (1998)

DNA extraction PCR amplification and sequencingmdashDNAwas extracted from dried ground sporocarp tissue followingOsmundson et al (2013) or using the DNeasy Plant Mini Kit(QIAGEN) following the manufacturerrsquos instructions PCRamplification of the ITS region was performed with primersITS1-F and ITS4 or ITS1-F and ITS2 or ITS3 and ITS4 (Whiteet al 1990 Gardes and Bruns 1993) The RPB2 gene segmentwas amplified with primers bRPB2-6f and fRPB2-7cR orbRPB2-6f and i2r-RPB2 or fRPB2-7cR and ilf-RPB2 (Liu et al1999 Matheny 2005 Stubbe et al 2010) Primer i2r-RPB2 ofStubbe et al (2010) was made slightly less specific by repla-cing primer positions 6 (5 T) 9 (5 C) and 15 (5 T) withY yielding the primer consensus sequence 5acutendashACV GTYTCY TCT TCY TCR GCR TCndash3acute The thermo-cycler programused for ITS amplification was 94 C for 2 min followed by 30cycles of 94 C for 30 s 55 C for 1 min and 72 C for 1 min fol-lowed by a final elongation step of 72 C for 5 min The ther-mos-cycler program used for RPB2 amplification was 94 C for90 s followed by 40 cycles of 94 C for 30 s 55 C for 90 s and 68C for 3 min concluding with a 5 min final extension at 68 CPCR products were cleaned with ExoSAP-IT (USB USA) or

the QIAquick PCR Purification Kit (QIAGEN) following themanufacturerrsquos instructions PCR products were sequencedon both strands with the same primers used for PCR Rawsequences were edited and contigs constructed with eitherGeneious Pro (Drummond et al 2009) or SeqTrace 081(Stucky 2012)

Sequence alignment and phylogenetic analysesmdashIn additionto the sequences generated in this study sequences exhibit-ing 97 or higher BLAST matches to the ITS region of arepresentative specimen of each Rocky Mountain alpinetaxon were downloaded from GenBank (httpwwwncbinlmnihgov) and UNITE (httpsuniteutee) as were anumber of ITS and RPB2 sequences mostly from Buyck et al(2008) Verbeken et al (2014) and the UNITE databaseLocality voucher information and accession numbers ofsequences generated in this study (TABLE I) and of allsequences used in phylogenetic analyses (SUPPLEMENTARY

TABLE II) are provided Due to difficulty unambiguouslyaligning the ITS region across the genus Lactarius four sepa-rate datasets were assembled One broad ITS+RPB2 datasetwas constructed in an attempt to ascertain the phylogeneticposition of each Rocky Mountain alpine taxon within thegenus Lactarius (referred to as the ldquobroad datasetrdquo) Thisresulted in the identification of three clades that served toguide the construction of three subsequent species-levelITS+RPB2 datasets (referred to as ldquodatasets A B Crdquo) Align-ments were performed with MUSCLE (httpwwwebiacukToolsmsamuscle) with default parameters For thebroad dataset ambiguously aligned regions of the ITS align-ment were highlighted and removed with the online versionof GBlocks 091b (Castresana 2000) under the least stringentsettings For datasets A B and C the alignments were editedwith SeAl (Rambaut 1995) For dataset C a 268ndash269 base-pairinsertion present in the ITS1 region of a number of speci-mens (see SUPPLEMENTARY TABLE II) was removed alongwith a small ambiguously aligned region present in taxa lack-ing the insertion

For each dataset topological incongruence between theITS and RPB2 loci was examined with the partition homoge-neity test (Farris et al 1995) as implemented in PAUP(Swofford 2001) The null hypothesis of congruence wasrejected if P 005 Maximum parsimony (MP) analyseswere carried out separately for ITS and RPB2 loci and forthe combined datasets with PAUP using heuristic searchesincluding 100 random addition sequence replicates andtree bisection reconnection branch swapping Clade supportwas assessed with 1000 bootstrap replicates with heuristicsearches tree bisection reconnection branch swapping andone random addition sequence per replicate

Maximum likelihood (ML) analyses were carried out withraxmlGUI 131 (Stamatikis 2006 Silvestro and Michalak2012) An ML search combined with rapid bootstrappingwas run until bootstrap convergence or 1000 bootstrap repli-cates was reached using the autoMRE option TheGTRGAMMA model was estimated separately for ITS1 58SITS2 RPB2 exon first second and third codon positionsand RPB2 introns for the broad dataset For datasets A Band C the GTRCAT model was estimated separately for ITS158S ITS2 RPB2 exon first second and third codon positions

BARGE ET AL ROCKY MOUNTAIN ALPINE LACTARIUS 417

changes in the physical environment such as topography aspectandmicrorelief (Johnson and Billings 1962 Cooper et al 1997)Ectomycorrhizal host species in theRockyMountain alpine zoneinclude shrubby willows Salix glauca L and S planifolia Purshdwarf willows S arctica Pall and S reticulata L the bog birchBetula glandulosa Michx the recumbent mat plant Dryas octope-tala L and the perennial sedge Kobresia myosuroides (Vill) Fiori(Schadt 2002 Cripps and Eddington 2005) In general thesame ectomycorrhizal host plants occur across the study siteshowever a greater abundance of Salix glauca was encounteredat sites in Colorado and only one isolatedBetula glandulosa shrubwas encountered on the Beartooth Plateau while B glandulosawas observed at several sites in Colorado

Taxon sampling and processingmdashSporocarps were collectedfrom late Julndashlate Aug 1997ndash2014 All collections weredescribed in detail when fresh and select collections werephotographed Ectomycorrhizal host plants near sporocarpswere noted for each collection Sporocarps were dried on

an electric warm-air dryer and deposited in the MontanaState University herbarium (MONT) Collections initiallywere identified to species primarily using Jamoni (19952008) Bon (1998) Heilmann-Clausen et al (1998) andBasso (1999) Herbarium specimens of type collections puta-tive conspecific and taxonomically similar species wereobtained from the herbarium of the Conservatoire et Jardinbotaniques de la Ville de Genegraveve Geneva Switzerland (G)the herbarium of Universitaumlt Innsbruck Innsbruck Austria(IB) the Herbarium Universitatis Tartuensis Tartu Estonia(TU) the Botanical Museum Oslo Norway (O) HerbariumGB University of Gothenburg Goumlteborg Sweden (GB) theherbarium of Aringbo Akademi University Turku Finland(TURA) the Mycological Herbarium of the Natural HistoryMuseum of Denmark Copenhagen Denmark (C) the her-barium of Universiteacute de Lille France (LIP) the Universityof Michigan fungal herbarium Ann Arbor Michigan(MICH) and the Denver Botanic Gardensrsquo Sam MitchelHerbarium of Fungi Denver Colorado (DBG) The type

FIG 1 Map of the collection locations of specimens that were examined andor included in molecular analyses (if collectionlocations are known) Rocky Mountain alpine study sites are indicated by white dots black dots indicate other arctic-alpine sitesand gray dots indicate subalpine sites Several collections from outside the map area (China Japan Thailand) are not includedThe black dotted line delineates the Rocky Mountains the arctic circle (675uN) is indicated by the dashed gray line and thenorthern tree line which corresponds with the southern extent of arctic tundra is indicated by the solid dark gray line (Brownet al 2001)

416 MYCOLOGIA

collection of L lanceolatus OK Mill amp Laursen was obtainedfrom Massey Herbarium Virginia Tech Blacksburg Virginia(VPI) and is now at the herbarium of the New York BotanicalGarden (NY) Herbarium acronyms follow Thiers (continu-ously updated) The collection locations of specimens thatwere examined andor included in phylogenetic analysesin this study are shown in relation to Rocky Mountain alpinestudy sites (FIG 1)

Morphological descriptionsmdashDescriptions of macromorpho-logical and micromorphological features were made fromfresh and dry material respectively For micromorphologicalexamination dried material was reconstituted in ethanol andthin sections were prepared Basidiospores were examined inMelzerrsquos reagent basidia macrocystidia and pileipellis tissuewere examined in 25 KOH Length and width of micro-morphological features were measured with an ocular micro-meter under an oil-immersion lens at 10006 magnificationfor a random sample of 25 basidiospores per collectionexcluding ornamentation the hilar appendix and giant basi-diospores from two-spored basidia mean basidiospore lengthand width were calculated for each collection The length towidth ratio (Q) was calculated for each basidiospore andmean length to width ratio (Q) was calculated for each col-lection The maximum height of the basidiospore ornamen-tation was measured and recorded and the ornamentationdescribed Length and width (at widest point) were mea-sured at 4006magnification for a random sample of 10 basi-dia (including sterigmata and excluding two-spored basidia)10 pleuromacrocystidia and 10 cheilomacrocystidia per col-lection Drawings of basidiospores pleuromacrocystidia andcheilomacrocystidia were prepared from one representativecollection of each Rocky Mountain alpine Lactarius speciesas well as for their corresponding type specimen (when avail-able) with a Leica drawing tube attached to a Leica DMLSresearch microscope Terminology is according to Heil-mann-Clausen et al (1998)

DNA extraction PCR amplification and sequencingmdashDNAwas extracted from dried ground sporocarp tissue followingOsmundson et al (2013) or using the DNeasy Plant Mini Kit(QIAGEN) following the manufacturerrsquos instructions PCRamplification of the ITS region was performed with primersITS1-F and ITS4 or ITS1-F and ITS2 or ITS3 and ITS4 (Whiteet al 1990 Gardes and Bruns 1993) The RPB2 gene segmentwas amplified with primers bRPB2-6f and fRPB2-7cR orbRPB2-6f and i2r-RPB2 or fRPB2-7cR and ilf-RPB2 (Liu et al1999 Matheny 2005 Stubbe et al 2010) Primer i2r-RPB2 ofStubbe et al (2010) was made slightly less specific by repla-cing primer positions 6 (5 T) 9 (5 C) and 15 (5 T) withY yielding the primer consensus sequence 5acutendashACV GTYTCY TCT TCY TCR GCR TCndash3acute The thermo-cycler programused for ITS amplification was 94 C for 2 min followed by 30cycles of 94 C for 30 s 55 C for 1 min and 72 C for 1 min fol-lowed by a final elongation step of 72 C for 5 min The ther-mos-cycler program used for RPB2 amplification was 94 C for90 s followed by 40 cycles of 94 C for 30 s 55 C for 90 s and 68C for 3 min concluding with a 5 min final extension at 68 CPCR products were cleaned with ExoSAP-IT (USB USA) or

the QIAquick PCR Purification Kit (QIAGEN) following themanufacturerrsquos instructions PCR products were sequencedon both strands with the same primers used for PCR Rawsequences were edited and contigs constructed with eitherGeneious Pro (Drummond et al 2009) or SeqTrace 081(Stucky 2012)

Sequence alignment and phylogenetic analysesmdashIn additionto the sequences generated in this study sequences exhibit-ing 97 or higher BLAST matches to the ITS region of arepresentative specimen of each Rocky Mountain alpinetaxon were downloaded from GenBank (httpwwwncbinlmnihgov) and UNITE (httpsuniteutee) as were anumber of ITS and RPB2 sequences mostly from Buyck et al(2008) Verbeken et al (2014) and the UNITE databaseLocality voucher information and accession numbers ofsequences generated in this study (TABLE I) and of allsequences used in phylogenetic analyses (SUPPLEMENTARY

TABLE II) are provided Due to difficulty unambiguouslyaligning the ITS region across the genus Lactarius four sepa-rate datasets were assembled One broad ITS+RPB2 datasetwas constructed in an attempt to ascertain the phylogeneticposition of each Rocky Mountain alpine taxon within thegenus Lactarius (referred to as the ldquobroad datasetrdquo) Thisresulted in the identification of three clades that served toguide the construction of three subsequent species-levelITS+RPB2 datasets (referred to as ldquodatasets A B Crdquo) Align-ments were performed with MUSCLE (httpwwwebiacukToolsmsamuscle) with default parameters For thebroad dataset ambiguously aligned regions of the ITS align-ment were highlighted and removed with the online versionof GBlocks 091b (Castresana 2000) under the least stringentsettings For datasets A B and C the alignments were editedwith SeAl (Rambaut 1995) For dataset C a 268ndash269 base-pairinsertion present in the ITS1 region of a number of speci-mens (see SUPPLEMENTARY TABLE II) was removed alongwith a small ambiguously aligned region present in taxa lack-ing the insertion

For each dataset topological incongruence between theITS and RPB2 loci was examined with the partition homoge-neity test (Farris et al 1995) as implemented in PAUP(Swofford 2001) The null hypothesis of congruence wasrejected if P 005 Maximum parsimony (MP) analyseswere carried out separately for ITS and RPB2 loci and forthe combined datasets with PAUP using heuristic searchesincluding 100 random addition sequence replicates andtree bisection reconnection branch swapping Clade supportwas assessed with 1000 bootstrap replicates with heuristicsearches tree bisection reconnection branch swapping andone random addition sequence per replicate

Maximum likelihood (ML) analyses were carried out withraxmlGUI 131 (Stamatikis 2006 Silvestro and Michalak2012) An ML search combined with rapid bootstrappingwas run until bootstrap convergence or 1000 bootstrap repli-cates was reached using the autoMRE option TheGTRGAMMA model was estimated separately for ITS1 58SITS2 RPB2 exon first second and third codon positionsand RPB2 introns for the broad dataset For datasets A Band C the GTRCAT model was estimated separately for ITS158S ITS2 RPB2 exon first second and third codon positions

BARGE ET AL ROCKY MOUNTAIN ALPINE LACTARIUS 417

collection of L lanceolatus OK Mill amp Laursen was obtainedfrom Massey Herbarium Virginia Tech Blacksburg Virginia(VPI) and is now at the herbarium of the New York BotanicalGarden (NY) Herbarium acronyms follow Thiers (continu-ously updated) The collection locations of specimens thatwere examined andor included in phylogenetic analysesin this study are shown in relation to Rocky Mountain alpinestudy sites (FIG 1)

Morphological descriptionsmdashDescriptions of macromorpho-logical and micromorphological features were made fromfresh and dry material respectively For micromorphologicalexamination dried material was reconstituted in ethanol andthin sections were prepared Basidiospores were examined inMelzerrsquos reagent basidia macrocystidia and pileipellis tissuewere examined in 25 KOH Length and width of micro-morphological features were measured with an ocular micro-meter under an oil-immersion lens at 10006 magnificationfor a random sample of 25 basidiospores per collectionexcluding ornamentation the hilar appendix and giant basi-diospores from two-spored basidia mean basidiospore lengthand width were calculated for each collection The length towidth ratio (Q) was calculated for each basidiospore andmean length to width ratio (Q) was calculated for each col-lection The maximum height of the basidiospore ornamen-tation was measured and recorded and the ornamentationdescribed Length and width (at widest point) were mea-sured at 4006magnification for a random sample of 10 basi-dia (including sterigmata and excluding two-spored basidia)10 pleuromacrocystidia and 10 cheilomacrocystidia per col-lection Drawings of basidiospores pleuromacrocystidia andcheilomacrocystidia were prepared from one representativecollection of each Rocky Mountain alpine Lactarius speciesas well as for their corresponding type specimen (when avail-able) with a Leica drawing tube attached to a Leica DMLSresearch microscope Terminology is according to Heil-mann-Clausen et al (1998)

DNA extraction PCR amplification and sequencingmdashDNAwas extracted from dried ground sporocarp tissue followingOsmundson et al (2013) or using the DNeasy Plant Mini Kit(QIAGEN) following the manufacturerrsquos instructions PCRamplification of the ITS region was performed with primersITS1-F and ITS4 or ITS1-F and ITS2 or ITS3 and ITS4 (Whiteet al 1990 Gardes and Bruns 1993) The RPB2 gene segmentwas amplified with primers bRPB2-6f and fRPB2-7cR orbRPB2-6f and i2r-RPB2 or fRPB2-7cR and ilf-RPB2 (Liu et al1999 Matheny 2005 Stubbe et al 2010) Primer i2r-RPB2 ofStubbe et al (2010) was made slightly less specific by repla-cing primer positions 6 (5 T) 9 (5 C) and 15 (5 T) withY yielding the primer consensus sequence 5acutendashACV GTYTCY TCT TCY TCR GCR TCndash3acute The thermo-cycler programused for ITS amplification was 94 C for 2 min followed by 30cycles of 94 C for 30 s 55 C for 1 min and 72 C for 1 min fol-lowed by a final elongation step of 72 C for 5 min The ther-mos-cycler program used for RPB2 amplification was 94 C for90 s followed by 40 cycles of 94 C for 30 s 55 C for 90 s and 68C for 3 min concluding with a 5 min final extension at 68 CPCR products were cleaned with ExoSAP-IT (USB USA) or

the QIAquick PCR Purification Kit (QIAGEN) following themanufacturerrsquos instructions PCR products were sequencedon both strands with the same primers used for PCR Rawsequences were edited and contigs constructed with eitherGeneious Pro (Drummond et al 2009) or SeqTrace 081(Stucky 2012)

Sequence alignment and phylogenetic analysesmdashIn additionto the sequences generated in this study sequences exhibit-ing 97 or higher BLAST matches to the ITS region of arepresentative specimen of each Rocky Mountain alpinetaxon were downloaded from GenBank (httpwwwncbinlmnihgov) and UNITE (httpsuniteutee) as were anumber of ITS and RPB2 sequences mostly from Buyck et al(2008) Verbeken et al (2014) and the UNITE databaseLocality voucher information and accession numbers ofsequences generated in this study (TABLE I) and of allsequences used in phylogenetic analyses (SUPPLEMENTARY

TABLE II) are provided Due to difficulty unambiguouslyaligning the ITS region across the genus Lactarius four sepa-rate datasets were assembled One broad ITS+RPB2 datasetwas constructed in an attempt to ascertain the phylogeneticposition of each Rocky Mountain alpine taxon within thegenus Lactarius (referred to as the ldquobroad datasetrdquo) Thisresulted in the identification of three clades that served toguide the construction of three subsequent species-levelITS+RPB2 datasets (referred to as ldquodatasets A B Crdquo) Align-ments were performed with MUSCLE (httpwwwebiacukToolsmsamuscle) with default parameters For thebroad dataset ambiguously aligned regions of the ITS align-ment were highlighted and removed with the online versionof GBlocks 091b (Castresana 2000) under the least stringentsettings For datasets A B and C the alignments were editedwith SeAl (Rambaut 1995) For dataset C a 268ndash269 base-pairinsertion present in the ITS1 region of a number of speci-mens (see SUPPLEMENTARY TABLE II) was removed alongwith a small ambiguously aligned region present in taxa lack-ing the insertion

For each dataset topological incongruence between theITS and RPB2 loci was examined with the partition homoge-neity test (Farris et al 1995) as implemented in PAUP(Swofford 2001) The null hypothesis of congruence wasrejected if P 005 Maximum parsimony (MP) analyseswere carried out separately for ITS and RPB2 loci and forthe combined datasets with PAUP using heuristic searchesincluding 100 random addition sequence replicates andtree bisection reconnection branch swapping Clade supportwas assessed with 1000 bootstrap replicates with heuristicsearches tree bisection reconnection branch swapping andone random addition sequence per replicate

Maximum likelihood (ML) analyses were carried out withraxmlGUI 131 (Stamatikis 2006 Silvestro and Michalak2012) An ML search combined with rapid bootstrappingwas run until bootstrap convergence or 1000 bootstrap repli-cates was reached using the autoMRE option TheGTRGAMMA model was estimated separately for ITS1 58SITS2 RPB2 exon first second and third codon positionsand RPB2 introns for the broad dataset For datasets A Band C the GTRCAT model was estimated separately for ITS158S ITS2 RPB2 exon first second and third codon positions

BARGE ET AL ROCKY MOUNTAIN ALPINE LACTARIUS 417

TABLE I Voucher locality information and GenBank accession numbers for DNA sequences generated in this study Herbar-ium acronyms follow Thiers httpsweetgumnybgorgih (continuously updated) Rocky Mountain alpine specimens areshown in boldface

Taxon Voucher Location ITS RPB2

L aspideoides RL Shaffer 6957 (MICH) Emmet County MI USA KR090893 NAL aspideus JV24534 (TURA) Varsinais-Suomi Finland KR090894 KR090972L aurantiacus JV94-422 (C) Uppland Sweden (type country) KR090896 KR090974L cf aurantiacus1 JV15112F (TURA) Nordland Norway KR090895 KR090973L cf aurantiacus1 CLC1885 (MONT) Longyearbyen Svalbard KR090897 KR090975L cf aurantiacus1 CLC2743 (MONT) Palmer Creek Road AK USA KR090898 KR090976L aff brunneoviolaceus CLC2133 (MONT) Finse Norway KR090899 KR090977L aff brunneoviolaceus2 P Larsen 361395 (O) Stordal Norway KR090900 KR090978L dryadophilus CLC2729 (MONT) AK USA KR090901 KR090979L dryadophilus EL57-10 (GB) Latnjavagge Sweden KR090902 KR090980L dryadophilus CLC2744 (MONT) Palmer Creek Road AK USA KR090903 KR090981L flavopalustris JV23334 (TURA) Koillismaa Finland (type country) KR090904 KR090982L glyciosmus TWO269 (MONT) Beartooth Plateau MT USA KR090905 KR090983L glyciosmus ZT12723 (MONT) Beartooth Plateau MT USA KR090906 NAL glyciosmus CLC1624 (MONT) Front Range CO USA KR090907 KR090984L glyciosmus 20923 (DBG) Brainard Lake Front Range CO USA KR090908 KR090985L glyciosmus EB133 (MONT) San Juan Range (subalpine) CO USA KR090909 KR090986L glyciosmus M Moser 19810148 (IB) Femsjouml Sweden (type locality) KR090910 NAL glyciosmus M Moser 19780234 (IB) Femsjouml Sweden (type locality) KR090911 NAL glyciosmus M Moser 19780191 (IB) Femsjouml Sweden (type locality) KR090912 NAL glyciosmus CLC2874 (MONT) Fjell Ailigas Finland KR090913 KR090987L hysginoides JV28432 (TURA) Koillismaa Finland (type country) KR090914 KR090988L lanceolatus (HOLOTYPE) F4239 (VPI) Beaufort Lagoon AK USA KR090915 KR090989L lanceolatus CLC1389 (MONT) Beartooth Plateau MT USA KR090916 KR090990L lanceolatus CLC2319 (MONT) Beartooth Plateau WY USA KR090917 KR090991L lanceolatus CLC2358 (MONT) Beartooth Plateau MT USA KR090918 KR090992L lanceolatus EB105-13 (MONT) Beartooth Plateau WY USA KR090919 KR090993L luculentus AH Smith 79943 (MICH) OR USA (near type locality) KR090920 NAL luculentus AH Smith 90905 (MICH) Tucannon River WA USA KR090921 NAL luculentus v laetus DBG-F-024643 (DBG) CO USA (near type locality) KR090922 KR090994L luculentus v laetus DBG-F-022653 (DBG) El Paso County CO (near type locality) KR090923 KR090995L montanus (PARATYPE) AH Smith 81954 (MICH) Bonner County ID USA KR090924 NAL montanus EB120-13 (MONT) Madison Range MT USA KR090925 KR090996L montanus CLC3001 (MONT) Tobacco Root Mountains MT USA KR090926 KR090997L nanus CLC1403 (MONT) Beartooth Plateau WY USA KR090927 KR090998L nanus EB125 (MONT) Beartooth Plateau WY USA NA KR090999L nanus EB106-13 (MONT) Beartooth Plateau MT USA KR090928 KR091000L nanus CLC1716 (MONT) San Juan Range CO USA KR090929 KR091001L nanus CLC1801 (MONT) San Juan Range CO USA KR090930 KR091002L nanus EB138 (MONT) San Juan Range CO USA KR090931 KR091003L nanus CLC1829 (MONT) San Juan Range CO USA KR090932 KR091004L nanus CLC1471 (MONT) Sawatch Range CO USA KR090933 KR091005L nanus CLC2134 (MONT) Finse Norway KR090934 KR091006L nanus Bon 89093 (LIP) Savoie France (near type locality) KR090935 NAL nanus JV15148 (TURA) Torne Lappmark Sweden KR090936 KR091007

418 MYCOLOGIA

and RPB2 introns Single-gene and combined ML analyseswere performed on each dataset

For Bayesian inference (BI) analyses the best-fitting substi-tution model for the evolution of ITS1 58S ITS2 RPB2exon first second and third codon positions and RPB2 intronwas estimated from 24 models by the Akaike information

criterion (AIC) with MrModeltest 23 (Nylander 2004) Mod-els selected for each partition within each dataset are pro-vided (SUPPLEMENTARY TABLE III) BI analyses were executedin MrBayes 32 (Ronquist and Huelsenbeck 2003) Para-meters were unlinked between partitions and the substitu-tion rate was allowed to vary across partitions Preliminary

TABLE I Continued

Taxon Voucher Location ITS RPB2

L nanus CLC1896 (MONT) Longyearbyen Svalbard KR090937 KR091008L pallescens (HOLOTYPE) AH Smith 81936 (MICH) Boundary County ID USA KR090938 NAL pallidomarginatus sp nov CLC1470 (MONT) Sawatch Range CO USA KR090939 KR091009L pallidomarginatus sp nov EB0041 (MONT) San Juan Range CO USA KR090940 KR091010L aff pseudouvidus U Peintner 20070035 (IB) Tyrol Austria (near type locality) NA KR091011L aff pseudouvidus3 JV28448F (TURA) Koillismaa Finland NA KR091012L aff pseudouvidus JV10468 (TURA) Inarin Lappi Finland KR090941 KR091013L aff pseudouvidus4 U Peintner 20040156 (IB) Trentino Italy (near type locality) KR090942 KR091014L aff pseudouvidus E Soslashyland 73867 (O) Ulvik Norway KR090943 KR091015L aff pseudouvidus EL101-11 (GB) Latnjajaure Sweden KR090944 KR091016L aff pseudouvidus EL63-10 (GB) Latnjavagge Sweden KR090945 KR091017L aff pseudouvidus TWO809 (MONT) Svalbard KR090946 KR091018L repraesentaneus CLC2318 (MONT) Beartooth Plateau MT USA KR090948 KR091020L repraesentaneus EB107-13 (MONT) Beartooth Plateau MT USA KR090949 KR091021L repraesentaneus EB0048 (MONT) Sawatch Range CO USA KR090950 KR091022L repraesentaneus CLC1747 (MONT) Sawatch Range CO USA KR090951 KR091023L repraesentaneus JV21671 (TURA) Enontakiouml Finland KR090952 KR091024L repraesentaneus EL92-07 (GB) Latnjavagge Sweden KR090953 KR091025L repraesentaneus JV13837F (TURA) Varsinais-Suomi Finland KR090954 KR091026L salicis-herbaceae CLC1536 (MONT) Sismiut Greenland KR090955 KR091027L salicis-reticulatae CLC1211 (MONT) Beartooth Plateau MT USA KR090956 NAL salicis-reticulatae CLC2776 (MONT) Beartooth Plateau MT USA KR090957 KR091028L salicis-reticulatae EB0057-14 (MONT) Beartooth Plateau WY USA KR090958 KR091029L salicis-reticulatae CLC2885 (MONT) Near Utsjoki Finland KR090959 KR091030L salicis-reticulatae CLC2745 (MONT) Palmer Creek Road Alaska USA KR090960 KR091031L salicis-reticulatae JV15133 (TURA) Sweden (near type locality) KR090961 KR091032L aff salicis-reticulatae CLC1710 (MONT) San Juan Range CO USA KR090962 KR091033L aff salicis-reticulatae CLC1741 (MONT) Sawatch Range CO USA KR090963 KR091034L aff salicis-reticulatae EB0036 (MONT) San Juan Range CO USA KR090964 KR091035L aff salicis-reticulatae CLC1689 (MONT) San Juan Range CO USA KR090965 KR091036L aff salicis-reticulatae EB0039 (MONT) San Juan Range CO USA KR090966 KR091037L subflammeus (HOLOTYPE) AH Smith 83602 (MICH) Tillamook County OR USA KR090967 NAL substriatus AH Smith 83693 (MICH) Tillamook County OR USA KR090968 NAL substriatus AH Smith 83694 (MICH) Tillamook County OR USA KR090969 NAL subviscidus (PARATYPE) AH Smith 83066 (MICH) Lewis County WA USA KR090970 NAL subviscidus (PARATYPE) AH Smith 83331 (MICH) Lewis County WA USA KR090971 NALactarius sp CLC1910 (MONT) Svalbard KR090947 KR091019

1 Originally identified as L lanceolatus2 Originally identified as L pseudouvidus3 Originally identified as L brunneoviolaceus4 Originally identified as L robertianus

BARGE ET AL ROCKY MOUNTAIN ALPINE LACTARIUS 419

Bayesian trees were an order of magnitude longer but hadnearly identical topologies to maximum-likelihood treesThis issue was addressed by setting a branch length prior of001 for all analyses (see Marshall 2010 for a discussion ofthis problem) BI was executed separately for ITS and RPB2and also on the combined datasets All BI analyses consistedof two independent runs with four chains (three heated onecold) each run for 10 million generations sampling every500th tree The plot of generation number versus the logprobability of observing the data output by MrBayes wasused to determine how many samples to discard as burn-inFor all runs the default burn-in value of 25 was deemed suf-ficient Topological convergence of independent programruns was assessed with AWTY (Nylander et al 2008) The pro-gram TreeGraph 2 (Stoumlver and Muumlller 2010) was used todraw edit and finalize the resulting trees

RESULTS

In total 79 ITS sequences and 66 RPB2 sequences from82 collections were generated in this study (TABLE I)Sequences were obtained from six type collectionsincluding that of Lactarius lanceolatus OK Mill amp Laur-sen Sequences were not successfully obtained fromtypes of L nanus J Favre L pseudouvidus Kuumlhner andL salicis-reticulatae Kuumlhner L glyciosmus (Fr) Fr andL repraesentaneus Britzelm lack physical type materialhowever sequences were obtained from type localitymaterial for all five taxa An overview of the datasetsused for phylogenetic analyses is provided (SUPPLEMEN-

TARY TABLE IV) Alignments and multigene (ITS+RPB2)trees were deposited in TreeBASE (httppurlorgphylotreebasephylowsstudyTB2S17922)

Convergence diagnostics for the Bayesian analyses ofthe combined (ITS+RPB2) datasets indicated that eachanalysis achieved topological convergence althoughsliding window analyses indicate a moderate degreeof within-run instability the cumulative posterior prob-ability plots and between-run comparison plot indicateconvergence (SUPPLEMENTARY FIGS 1ndash4)

For the broad dataset (Lactarius and related genera)the partition homogeneity test indicated that the ITSand RPB2 loci were resolving significantly differentphylogenetic relationships (P 5 003) However theseconflicts generally received low support (bootstrapsupport 75 posterior probability 095) and per-tained only to the placement of a few terminalbranches Therefore the broad combined (ITS+RPB2) dataset was analyzed and the Bayesian 50majority-rule consensus tree is displayed (FIG 2) Twoclades corresponding with Lactarius subgenera Plintho-galus (Burl) Hesler amp AH Sm (well-supported) andRussularia (well-supported) as recognized in Verbekenet al (2014) and a monophyletic clade correspondingto section Uvidi (Konr) Bon (poorly supported) wereobtained as were clades that were used to guide theconstruction of datasets A B and C Lactarius subgenus

Piperites was not resolved as monophyletic in the Baye-sian (FIG 2) or MP (not shown) analyses however itwas resolved as monophyletic with low support in theML analysis (not shown)

For datasets A (P 5 044) B (P 5 014) and C(P 5 041) the partition homogeneity test indicatedthat the ITS and RPB2 loci did not produce conflictingsignals thus combined datasets were analyzed Com-bined (ITS+RPB2) Bayesian 50 majority-rule consen-sus trees for datasets A (FIG 3) B (FIG 4) and C(FIG 5) are shown

For dataset A (subgenus Russularia pp) collectionsoriginally identified as L lanceolatus from the RockyMountain alpine zone as well as the holotype collec-tion from Alaska form a polytomy within a well-sup-ported clade that also includes Lactarius sp 2 Lluculentus Burl L luculentus v laetus Hesler amp AHSm and L aurantiacus (Pers) Gray (FIG 3) Severalarctic-alpine collections originally identified as L lan-ceolatus from Norway (JV15112F) Svalbard (CLC1885IA-F20) and Alaska (CLC2743) group with L aurantia-cus (syn L mitissimus [Fr] Fr) from the type countryin a well-supported clade Two collections identified asL luculentus v laetus (DBG-F-024643 DBG-F-022653)from the type locality form a well-supported clade sis-ter to a well-supported clade containing two collectionsof L luculentus (AH Smith 79943 from type locality AHSmith 90905) and a collection identified as L luculen-tus v laetus (SMI221) from British Columbia suggest-ing there may be two species in this group Otherspecies closely related to L lanceolatus include L sub-striatus AH SmL subflammeus Hesler amp AH Sm(taxa that may be conspecific) and Lactarius sp 1represented by unidentified environmental GenBanksequences from subalpine Alaska and arctic-alpineNorway with Dryas octopetala GenBank sequenceDQ974746 (voucher src438 Lactarius sp 2) is likelymisidentified as L substriatus because specimens col-lected by the author of L substriatus form a separateclade (FIG 3) Results of the ITS (SUPPLEMENTARY

FIG 5) analyses are similar to the combined analysishowever in the RPB2 analysis (SUPPLEMENTARY FIG 6)L aurantiacus does not form a clade separate fromthe Rocky Mountain alpine and holotype L lanceolatuscollections

For dataset B (subgenus Piperites pp) Rocky Moun-tain alpine collections initially identified as L nanusoccur in a well-supported clade with an alpine speci-men collected by Marcel Bon from the French Alps(type locality) along with a number of collectionsfrom arctic-alpine areas elsewhere in Europe (FIG 4)sequencing of the holotype was unsuccessful In multi-gene (FIG 4) ITS (SUPPLEMENTARY FIG 7) and RPB2(SUPPLEMENTARY FIG 8) analyses L nanus is paraphy-letic with respect to L hysginoides and the two

420 MYCOLOGIA

Bayesian trees were an order of magnitude longer but hadnearly identical topologies to maximum-likelihood treesThis issue was addressed by setting a branch length prior of001 for all analyses (see Marshall 2010 for a discussion ofthis problem) BI was executed separately for ITS and RPB2and also on the combined datasets All BI analyses consistedof two independent runs with four chains (three heated onecold) each run for 10 million generations sampling every500th tree The plot of generation number versus the logprobability of observing the data output by MrBayes wasused to determine how many samples to discard as burn-inFor all runs the default burn-in value of 25 was deemed suf-ficient Topological convergence of independent programruns was assessed with AWTY (Nylander et al 2008) The pro-gram TreeGraph 2 (Stoumlver and Muumlller 2010) was used todraw edit and finalize the resulting trees

RESULTS

In total 79 ITS sequences and 66 RPB2 sequences from82 collections were generated in this study (TABLE I)Sequences were obtained from six type collectionsincluding that of Lactarius lanceolatus OK Mill amp Laur-sen Sequences were not successfully obtained fromtypes of L nanus J Favre L pseudouvidus Kuumlhner andL salicis-reticulatae Kuumlhner L glyciosmus (Fr) Fr andL repraesentaneus Britzelm lack physical type materialhowever sequences were obtained from type localitymaterial for all five taxa An overview of the datasetsused for phylogenetic analyses is provided (SUPPLEMEN-

TARY TABLE IV) Alignments and multigene (ITS+RPB2)trees were deposited in TreeBASE (httppurlorgphylotreebasephylowsstudyTB2S17922)

Convergence diagnostics for the Bayesian analyses ofthe combined (ITS+RPB2) datasets indicated that eachanalysis achieved topological convergence althoughsliding window analyses indicate a moderate degreeof within-run instability the cumulative posterior prob-ability plots and between-run comparison plot indicateconvergence (SUPPLEMENTARY FIGS 1ndash4)

For the broad dataset (Lactarius and related genera)the partition homogeneity test indicated that the ITSand RPB2 loci were resolving significantly differentphylogenetic relationships (P 5 003) However theseconflicts generally received low support (bootstrapsupport 75 posterior probability 095) and per-tained only to the placement of a few terminalbranches Therefore the broad combined (ITS+RPB2) dataset was analyzed and the Bayesian 50majority-rule consensus tree is displayed (FIG 2) Twoclades corresponding with Lactarius subgenera Plintho-galus (Burl) Hesler amp AH Sm (well-supported) andRussularia (well-supported) as recognized in Verbekenet al (2014) and a monophyletic clade correspondingto section Uvidi (Konr) Bon (poorly supported) wereobtained as were clades that were used to guide theconstruction of datasets A B and C Lactarius subgenus

Piperites was not resolved as monophyletic in the Baye-sian (FIG 2) or MP (not shown) analyses however itwas resolved as monophyletic with low support in theML analysis (not shown)

For datasets A (P 5 044) B (P 5 014) and C(P 5 041) the partition homogeneity test indicatedthat the ITS and RPB2 loci did not produce conflictingsignals thus combined datasets were analyzed Com-bined (ITS+RPB2) Bayesian 50 majority-rule consen-sus trees for datasets A (FIG 3) B (FIG 4) and C(FIG 5) are shown

For dataset A (subgenus Russularia pp) collectionsoriginally identified as L lanceolatus from the RockyMountain alpine zone as well as the holotype collec-tion from Alaska form a polytomy within a well-sup-ported clade that also includes Lactarius sp 2 Lluculentus Burl L luculentus v laetus Hesler amp AHSm and L aurantiacus (Pers) Gray (FIG 3) Severalarctic-alpine collections originally identified as L lan-ceolatus from Norway (JV15112F) Svalbard (CLC1885IA-F20) and Alaska (CLC2743) group with L aurantia-cus (syn L mitissimus [Fr] Fr) from the type countryin a well-supported clade Two collections identified asL luculentus v laetus (DBG-F-024643 DBG-F-022653)from the type locality form a well-supported clade sis-ter to a well-supported clade containing two collectionsof L luculentus (AH Smith 79943 from type locality AHSmith 90905) and a collection identified as L luculen-tus v laetus (SMI221) from British Columbia suggest-ing there may be two species in this group Otherspecies closely related to L lanceolatus include L sub-striatus AH SmL subflammeus Hesler amp AH Sm(taxa that may be conspecific) and Lactarius sp 1represented by unidentified environmental GenBanksequences from subalpine Alaska and arctic-alpineNorway with Dryas octopetala GenBank sequenceDQ974746 (voucher src438 Lactarius sp 2) is likelymisidentified as L substriatus because specimens col-lected by the author of L substriatus form a separateclade (FIG 3) Results of the ITS (SUPPLEMENTARY

FIG 5) analyses are similar to the combined analysishowever in the RPB2 analysis (SUPPLEMENTARY FIG 6)L aurantiacus does not form a clade separate fromthe Rocky Mountain alpine and holotype L lanceolatuscollections

For dataset B (subgenus Piperites pp) Rocky Moun-tain alpine collections initially identified as L nanusoccur in a well-supported clade with an alpine speci-men collected by Marcel Bon from the French Alps(type locality) along with a number of collectionsfrom arctic-alpine areas elsewhere in Europe (FIG 4)sequencing of the holotype was unsuccessful In multi-gene (FIG 4) ITS (SUPPLEMENTARY FIG 7) and RPB2(SUPPLEMENTARY FIG 8) analyses L nanus is paraphy-letic with respect to L hysginoides and the two

420 MYCOLOGIA

consistently differ at only one nucleotide position inthe ITS region and at four positions and one indel inthe RPB2 locus Despite high sequence similarity thetwo taxa are maintained as distinct in this studybecause basidiospore size and habitat appear to beconsistently different

Also in dataset B Rocky Mountain alpine collec-tions originally identified as L glyciosmus occur in awell-supported clade with several specimens col-lected by Meinhard Moser from Femsjouml Sweden

(type locality) as well as with collections from arc-tic-alpine and subalpine areas elsewhere in NorthAmerica and Europe (FIG 4) Several GenBank andUNITE sequences from specimens identified as Lglyciosmus (labeled as L aff glyciosmus in FIG 4)from subalpine Alaska Europe and Japan occur ina separate poorly supported clade and appear torepresent a distinct cryptic species No definitivemorphological or geographical differences weredetected between L glyciosmus and L aff glyciosmus

FIG 2 Combined (ITS+RPB2) Bayesian 50 majority-rule consensus tree based on analysis of the broad dataset(Lactarius and related genera) Thickened branches lead to clades receiving $ 75 bootstrap support from either PAUP orRAxML and Bayesian posterior probabilities $ 095 Boldface tip labels represent Rocky Mountain alpine specimens AA 5arctic-alpine

BARGE ET AL ROCKY MOUNTAIN ALPINE LACTARIUS 421

however the L aff glyciosmus clade does not containany arctic-alpine specimens (FIG 4)

In dataset C (subgenus Piperites section Uvidi) col-lections originally identified as L aff pseudouvidusfrom the Rocky Mountain alpine zone are well-sepa-rated from the majority of collections (mostly fromEurope) labeled the L pseudouvidus group (FIG 5)the latter appear in the BI consensus tree as paraphy-letic to L brunneoviolaceus MP Christ and L luridus(Pers) Gray Instead Rocky Mountain alpine materialforms a well-supported clade close to the morphologi-cally very different species L aspideus (Fr) Fr and anarctic-alpine environmental ITS sequence isolatedfrom the roots of Salix polaris Wahlenb in Svalbard(AB588960 Fujiyoshi et al 2012) (FIG 5) Thus based

on morphological and molecular differences theRocky Mountain alpine material is recognized here asthe new species L pallidomarginatus Barge amp Crippssp nov (see TAXONOMY) An additional collectionfrom Svalbard with Salix polaris (labeled Lactarius sp3 in FIG 5) is sister to the clade containing L aspi-deusL pallidomarginatus

The ITS region of the European L pseudouvidusgroup has little variation and is paraphyletic withrespect to L montanus (Hesler amp AH Sm) Montoyaamp Bandala (SUPPLEMENTARY FIG 9) However theRPB2 locus is highly variable both between and withinmost collections examined (SUPPLEMENTARY FIG 10) Itis not possible to definitively provide names to taxawithin this group in that sequencing of the L

FIG 3 Combined (ITS+RPB2) Bayesian 50 majority-rule consensus tree of dataset A (Lactarius subgenus Russularia pp)Support values (PAUP bootstrapRAxML bootstrapBayesian posterior probability) $ 60 (bootstrap) and $ 080 (posteriorprobability) are indicated above or below branches leading to clades Thickened branches lead to clades receiving $ 75bootstrap support and Bayesian posterior probabilities $ 095 Boldface tip labels represent Rocky Mountain alpine specimensAA 5 arctic-alpine Originally identified as L lanceolatus

422 MYCOLOGIA

pseudouvidus holotype was unsuccessful and cryptictaxa may be present Collections matching the mor-phological concept of the similar arctic-alpine taxonL brunneoviolaceus (syn L robertianus Bon) occur in a

moderately supported clade (labeled L aff brunneovio-laceus)in the combined (FIG 5) and ITS analyses (SUPPLEMEN-

TARY FIG 9) but these are paraphyletic with the L

FIG 4 Combined (ITS+RPB2) Bayesian 50 majority-rule consensus tree of dataset B (Lactarius subgenus Piperites pp)Support values (PAUP bootstrapRAxML bootstrapBayesian posterior probability) $ 60 (bootstrap) and $ 080 (posteriorprobability) are indicated above or below branches leading up to clades Thickened branches lead to clades receiving $ 75bootstrap support and Bayesian posterior probabilities $ 095 Boldface tip labels represent Rocky Mountain alpine specimensAA 5 arctic-alpine

BARGE ET AL ROCKY MOUNTAIN ALPINE LACTARIUS 423

FIG 5 Combined (ITS+RPB2) Bayesian 50 majority-rule consensus tree of dataset C (Lactarius subgenus Piperites sectionUvidi) Support values (PAUP bootstrapRAxML bootstrapBayesian posterior probability) $ 60 (bootstrap) and $ 080(posterior probability) are indicated above or below branches leading up to clades Thickened branches lead to clades receiving

424 MYCOLOGIA

pseudouvidus group in RPB2 analyses (SUPPLEMENTARY

FIG 10) Collections of Lactarius pallidomarginatus Lac-tarius sp 3 L aspideus L montanus L luridus L affpseudouvidus and L aff brunneoviolaceus for which ITSdata are available all have a similar 268ndash269 base-pairinsertion in the ITS1 region There is variation withinthe base-pair content of the insertion between collec-tions however inclusion of this insertion did notincrease phylogenetic resolution in preliminary ana-lyses and it therefore was omitted The insertion itselfis highly similar to an insertion in Russula olivacea (eg GenBank accessions AF418634 AY061699) as tolength sequence (approximately 90 similar) andplacement within the ITS1 region

Also in dataset C Rocky Mountain alpine collectionsinitially identified as L repraesentaneus form a well-supported clade with a collection of L repraesentaneusfrom Germany (the lectotype country) as well as withnumerous collections from subalpine and arctic-alpineareas elsewhere in Eurasia and Alaska (FIG 5) Thisclade is sister to a poorly supported L dryadophilusKuumlhner clade The two GenBank sequences JX630967from arctic Canada with Dryas and JF394416 from arcticSvalbard which occur just outside the L dryadophilusclade are likely conspecific with L dryadophilusalthough this cannot be confirmed based on presentresults Lactarius repraesentaneus and L dryadophilus con-sistently differ at two nucleotide positions in the ITSlocus and at four positions in the RPB2 locus and haveconsistent morphological and host differences

In dataset C collections originally identified as L sal-icis-reticulatae from alpine areas in the Rocky Mountainsare paraphyletic with regard to L salicis-herbaceaeKuumlhner L aspideoides BurlL flavopalustris Kytoumlvand L aff flavopalustris (FIG 5) Collections of Lsalicis-reticulatae from alpine areas on the BeartoothPlateau (central Rocky Mountains) form a poorlysupported clade with an alpine collection of L salicis-reticulatae from Mt Laringktatjaringkka Sweden the type local-ity (sequencing of the holotype of L salicis-reticulataewas not successful) as well as with other arctic-alpinecollections of L salicis-reticulatae from Finland andAlaska and an unidentified collection from Svalbard(FIG 5) this clade is hypothesized to represent Lsalicis-reticulatae Collections from southern RockyMountain alpine areas in Colorado are larger thanreported for L salicis-reticulatae and form an unre-solved group that consistently differs at one nucleotideposition in the ITS region but no positions in the RPB2

locus they are tentatively referred to as L aff salicis-reticulatae pending data from additional loci

Overall phylogenetic analyses support the recogni-tion of six to seven Lactarius species from the RockyMountain alpine zone Lactarius lanceolatus (FIG 3)L nanus L glyciosmus (FIG 4) L pallidomarginatus spnov L repraesentaneus L salicis-reticulatae and possiblyL aff salicis-reticulatae (FIG 5) Violet-staining taxa Lpallidomarginatus L repraesentaneus L salicis-reticulataeand L aff salicis-reticulatae are resolved as a poorly sup-ported clade exclusively containing other members ofthe violet-staining section Uvidi supporting the mono-phyly of this section (FIG 2)

TAXONOMY

All descriptions are based on Rocky Mountain alpinematerial

Lactarius lanceolatus OK Mill amp Laursen FIGS 6 7Short description Lactarius lanceolatus is recognized by

its small to medium size and bright light to deeporange pileus pale cream to pale orange lamellae dis-coloring brownish in age or where damaged paleorange stipe watery unchanging latex and mild flavorgrowing in arctic-alpine (possibly also subalpine) areaswith Salix species

Pileus 10ndash45 mm diam convex at first becomingplane to infundibuliform with a depressed centerwith or without a small papilla smooth sometimesfaintly scaly toward center viscid deep orange-brownto deep orange when immature becoming light todeep orange often blotchy often darker toward cen-ter margin incurved to straight when immaturebecoming straight to upturned and often slightlywavy when mature Lamellae adnate to subdecur-rent slightly crowded pale cream to pale yellow topale orange discoloring brownish orange in age orwhere damaged Stipe 10ndash20 6 25ndash75 mm equalto clavate central smooth dry at first covered byfaint whitish pubescence pale orange discoloringdingy orange to dingy light brown where damagedhollow Latex scarce watery unchanging Contextof pileus and stipe pale orange Odor mild Flavormild Basidiospore deposit not observed Basidios-pores 8ndash10 6 6ndash8 mm average 5 86 6 7 mmQ 5 114ndash138 Q5 124 hyaline in 25 KOHbroadly ellipsoid ornamentation amyloid of isolatedwarts connected by fine lines up to 05 mm highBasidia 381ndash660 6 89ndash102 mm four-spored Pleur-omacrocystidia 737ndash127 6 64ndash102 mm narrowly

r$75 bootstrap support and Bayesian posterior probabilities $ 095 Boldface tip labels represent Rocky Mountain alpinespecimens AA 5 arctic-alpine See TABLE I for original identifications

BARGE ET AL ROCKY MOUNTAIN ALPINE LACTARIUS 425

fusiform to lanceolate with acute to moniliformapex thin-walled scattered to abundant Cheiloma-crocystidia 356ndash89 6 51ndash102 mm conical to fusi-form with acute to moniliform apex thin-walledsparse to abundant Pileipellis an ixotrichodermwith well-developed gelatinous layer

Distribution and habitat Lactarius lanceolatus origin-ally was described from arctic-alpine Alaska (Millerand Laursen 1973) and phylogenetic analyses(including the holotype) further confirm its pre-sence in the central Rocky Mountain alpine zoneand Svalbard In alpine areas on the Beartooth Pla-teau it occurs with Salix reticulata and possibly otherSalix species (S arctica S planifolia) it is notreported from the southern Rocky Mountains Over-all L lanceolatus appears largely confined to arctic-alpine habitats however an ITS sequence from amaritime forest in Alaska (GAL18572) clusters withthe arctic-alpine collections in phylogenetic analyses(FIG 3)

Previous arctic-alpine reports include Canada (Ohe-noja and Ohenoja 1993 2010) Alaska (Miller andLaursen 1973 Laursen and Ammirati 1982 Heil-mann-Clausen et al 1998) Beartooth Plateau in theRocky Mountain alpine zone (Cripps and Barge2013) Greenland (Heilmann-Clausen et al 1998 Bor-gen et al 2006) Fennoscandia (Bon 1998 Heilmann-Clausen et al 1998 Knudsen and Vesterholt 2008)Svalbard (Gulden and Jenssen 1988 Geml et al2012) the British Isles (Watling et al 2001) andSiberia (Nezdoiminogo 1997 Heilmann-Clausen et al1998 Karatygin et al 1999 Knudsen and Mukhin1998) however phylogenetic analyses (FIG 3) suggestthat some of these reports may represent L aurantiacus(Pers Fr) Gray

Specimens examined USA ALASKA North Slope CountyBeaufort Lagoon 30 Jul 1971 F4239 (Holotype VPI) MON-TANA Carbon County Beartooth Plateau Highline Trail 1Aug 1997 ZT6214 (MONT) 7 Aug 1998 ZT6412 (MONT)20 Aug 1999 CLC 1389 (MONT) 5 Aug 2008 CLC 2358(MONT) 27 Jul 1997 CLC 1139 (MONT) WYOMINGPark County Beartooth Plateau Solifluction Terraces 20July 2001 CLC 1578 (MONT) 13 Aug 2007 CLC 2319(MONT) 17 Aug 2013 EB 105-13 (MONT)

Other specimens examined DENMARK Broby Vesterskov 16Oct 1997 JHC97-251 (L aurantiacus C) NORWAY Leikan-ger 11 Sept 1999 JHC00-057 (L aurantiacus C) Nordland14 Aug 1999 JV15112F (L cf aurantiacus originally identi-fied as L lanceolatus TURA) Svalbard Longyearbyen 18Aug 2003 CLC 1885 (L cf aurantiacus originally identifiedas L lanceolatus MONT) SWEDEN Uppland 8 Sept 1994JV94-422 (L aurantiacus C) USA ALASKA Kenai PeninsulaCounty Palmer Creek Road 7 Aug 2011 CLC 2743 (L cfaurantiacus originally identified as L lanceolatus MONT)COLORADO El Paso County 27 Aug 2011 DBG-F-022653(L luculentus v laetus DBG) Gilpin County Perigo 13 Aug1974 AH Smith 84923 (Paratype of L luculentus v laetus

MICH) Larimer County 23 Aug 2008 DBG-F-024643 (Lluculentus v laetus DBG) OREGON Polk County 14 Nov1970 AH Smith 79943 (L luculentus MICH) WASHING-TON Lewis County 30 Oct 1972 AH Smith 83066 (Paratypeof L subviscidus MICH) 1 Nov 1972 AH Smith 83331 (Para-type of L subviscidus MICH) Tucannon River 11 Oct1980 AH Smith 90905 (L luculentus MICH)

Comments Lactarius lanceolatus belongs to a taxono-mically problematic group of species with orangeorange-brown or orange-red pilei including L auran-tiacus L luculentus L luculentus v laetus L subflammeusand L substriatus

Traditionally L lanceolatus and L aurantiacus havebeen separated mainly by ecology with L aurantiacusbeing restricted to a variety of subalpine habitats(although it has been reported from arctic-alpine habi-tats) and L lanceolatus to arctic-alpine habitats withSalix spp however it is clear from phylogenetic ana-lyses (FIG 3) that L aurantiacus also may occur in arc-tic-alpine habitats likely associating with Salix as well asKobresia and that the ranges of the two taxa overlap atleast in arctic-alpine areas of Alaska and Svalbard Ithas been suggested that L lanceolatus has longermacrocystidia and larger basidiospores than L auran-tiacus (eg Heilmann-Clausen et al 1998) howevercollections designated as L aurantiacus measured inthis study have pleuromacrocystidia that overlap andactually exceed the upper limit measured for L lanceo-latus including the holotype (see Barge 2015) Basi-diospore sizes for L aurantiacus also overlap andactually exceed the upper limit measured for L lanceo-latus in the collections examined (Barge 2015) Overallsolid morphological differences between the two taxawere not detected and further study may reveal thatthey are conspecific however provisionally L lanceola-tus may tend to have a thicker gelatinous layer over thepileipellis as well as a smooth pileus margin in contrastto the crenulate pileus margin reported for L aurantia-cus Lactarius aurantiacus is not reported from theRocky Mountains

For North American taxa Lactarius luculentus has abrowner pileus a slowly acrid flavor shorter cheilo-macrocystidia (23ndash45 6 4ndash6 mm) and exsiccati arepaler and more yellow-tan (Hesler and Smith 1979)Lactarius luculentus v laetus is similar in appearanceto L lanceolatus but also has shorter cheilomacrocysti-dia (15ndash30 6 4ndash8 mm) and possibly more crowdedlamellae (Hesler and Smith 1979) Lactarius substria-tus has a redder pileus that is glutinous when wetwith a translucent-striate margin latex that slowlybecomes straw yellow shorter cheilomacrocystidiaand an acrid flavor (Hesler and Smith 1979) Lactariussubflammeus also has a redder pileus (at least whenyoung) short translucent striations along its pileusmargin shorter cheilomacrocystidia and a slowly

426 MYCOLOGIA

FIG 6 A L lanceolatus (EB105-13) B L nanus (EB106-13) C L nanus (EB138) D L glyciosmus (ZT12723) E Lpallidomarginatus Barge amp Cripps sp nov (EB0041 HOLOTYPE) F L repraesentaneus (EB107-13) G L salicis-reticulatae(EB0057-14) H L aff salicis-reticulatae (EB0036) Photographs by E Barge (A-C E-H) E Horak (D) Bars 5 2 cm

BARGE ET AL ROCKY MOUNTAIN ALPINE LACTARIUS 427

acrid taste (pers obs of holotype AH Smith 83602Hesler and Smith 1979) Molecular phylogenetic ana-lyses (FIG 3) suggest that L subflammeus is conspecificwith L substriatus however sequencing of the holo-type of L substriatus is needed to confirm this observa-tion Lactarius luculentus L luculentus v laetus Lsubflammeus and L substriatus have been reportedonly from subalpine areas with conifers in westernNorth America (Hesler and Smith 1979)

Lactarius duplicatus AH Sm (syn L lapponicus Har-maja) a North American and European Betula associ-ate is also morphologically close to L lanceolatusHowever it can be distinguished by the productionof strongly yellowing latex We did not procure anymolecular data from material representing this taxonand no molecular data is publicly available on Gen-Bank or UNITE However L duplicatus should beincluded in future phylogenetic analyses investigatingthe L lanceolatusL aurantiacus group

Lactarius nanus J Favre FIGS 6 7Short description Lactarius nanus is recognized by its

typically small size gray-brown to liver brown pileustypically with a glaucous coating at least when youngcream to pinkish tan lamellae pale cream-tan to paleapricot stipe watery unchanging latex mild odormild to slightly acrid flavor and occurrence with Salixspecies in arctic-alpine areas

Pileus 10ndash50 mm diam shallowly convex to plano-convex becoming plane to infundibuliform with orwithout a depressed center and small papilla smoothslightly viscid to dry with whitish glaucous coatingwhen immature easily rubbing away or fading in ageoften in patches deep brown to gray-brown to liverbrown with lighter and darker areas present oftenlighter toward margin becoming lighter overall upondessication margin straight when immature becomingupturned and wavy to convoluted when matureLamellae adnate to subdecurrent subdistant to slightlycrowded cream when immature becoming dingycream to light tan in age sometimes with a faint pink-ish tinge discoloring to dingy tan where damagedStipe 5ndash30 6 3ndash15 mm equal to clavate centralsmooth dry at first covered with whitish glaucous coat-ing as in pileus pale apricot to dingy cream-tan hol-low Latex scarce watery unchanging Context ofpileus and stipe dingy cream to brownish Odor mildFlavor mild to slightly acrid Basidiospore deposit notobserved Basidiospores 7ndash105(ndash12) 6 5ndash8 mm av 584 6 66 mm Q 5 107ndash160 Q 5 127 hyaline in25 KOH subglobose to ellipsoid ornamentationof amyloid scattered ridges and warts forming anincomplete reticulum with some completely reticulateareas to 07 mm high Basidia 406ndash762 6 89ndash152 mm mostly four-spored some two-spored

Pleuromacrocystidia 533ndash965(ndash1143) 6 64ndash114mm lanceolate with acute to moniliform apexscattered to abundant Cheilomacrocystidia 260ndash9146 76ndash102 mm subulate to linear with undulatingmargin with acute to rounded apex scattered toabundant Pileipellis an (ixo-) cutis with some ascend-ing hyphae embedded in a very faint gelatinouslayer

Distribution and habitat Lactarius nanus originally wasdescribed by Jules Favre from the Swiss Alps with Salixherbacea L (Favre 1955) Phylogenetic analyses includ-ing a specimen from near the type locality confirmthis species in the Rocky Mountain alpine zone whereit is one of the most commonly encountered Lactariusspecies it associates with Salix arctica S reticulata andS planifolia Overall L nanus appears to be largelyrestricted to arctic-alpine areas with Salix

Previous arctic-alpine reports include Canada (Ohe-noja and Ohenoja 1993 2010) the Rocky Mountainalpine zone (Moser and McKnight 1987 Cripps andHorak 2008 Cripps and Barge 2013) Greenland(Knudsen and Borgen 1982 Lamoure et al 1982 Heil-mann-Clausen et al 1998 Borgen et al 2006) Iceland(Eyjolfsdottir pers comm Knudsen and Vesterholt2008) the European Alps (Favre 1955 Kuumlhner 1975Kuumlhner and Lamoure 1986 Senn-Irlet 1993 Bon1985a 1998 Heilmann-Clausen et al 1998 Jamoni1995) the Pyrenees (Bon and Ballaraacute 1996) the Car-pathians (Ronikier 2008) Fennoscandia (Kuumlhner1975 Gulden et al 1985 Heilmann-Clausen et al1998 Gulden 2005 Knudsen and Vesterholt 2008)Svalbard (Gulden et al 1985) Siberia (Nezdoiminogo1997) and alpine areas in the Altai Mountains (Karaty-gin et al 1999)

Specimens examined FRANCE Savoie 24 Aug 1989 Bon89093 (LIP) NORWAY Finse 5 Aug 2010 CLC 2134(MONT) Svalbard Longyearbyen 20 Aug 2002 CLC 1896(MONT) SWEDEN Torne Lappmark 15 Aug 1999JV15148 (TURA) SWITZERLAND Grisons 20 Aug 1943ZA192c (Holotype G) USA COLORADO Pitkin CountySawatch Range Independence Pass 6 Aug 2000 CLC 1471(MONT) San Juan County San Juan Range Black BearPass 11 Aug 2001 CLC 1716 (MONT) 11 Aug 2001ZT9529 (MONT) San Juan County San Juan Range Cinna-mon Pass 27 July 2002 CLC 1801 (MONT) 15 Aug 2012EB138 (MONT) San Miguel County San Juan Range Imo-gene Pass 29 July 2002 CLC 1829 (MONT) MONTANACarbon County Beartooth Plateau Highline Trail 7 Aug1998 CLC 1221 (MONT) 8 Aug 1998 ZT6422 (MONT) 18Aug 2013 EB106-13 (MONT) WYOMING Park CountyBeartooth Plateau Frozen Lakes 21 Aug 1999 CLC 1403(MONT) Park County Beartooth Plateau Solifluction Ter-races 11 Aug 2012 EB125 (MONT)

Other specimens examined FINLAND Koillismaa 28 Aug2011 JV28432 (L hysginoides TURA)

428 MYCOLOGIA

Comments Lactarius nanus is morphologically andphylogenetically close to L hysginoides however thelatter has a more cinnamon-toned pileus smaller basi-diospores (6ndash8 6 5ndash7 mm av 68 6 57 mm) and isgenerally reported from boreal areas with Betula Piceaand Salix in Europe (Heilmann-Clausen et al 1998)although phylogenetic placement of environmentalsequences extend its range into boreal Alaska (FIG4) Its range likely overlaps that of L nanus in alpine-subalpine transition areas It is not reported from theRocky Mountains Lactarius mammosus is a more robustspecies with a coconut odor (Heilmann-Clausen et al1998) that associates with members of the Pinaceaeit has not been reported from the Rocky MountainsDifferences between L glyciosmus and L nanus are dis-cussed in the taxonomy section for L glyciosmus

Lactarius glyciosmus (Fr) Fr FIGS 6 7Short description Lactarius glyciosmus is recognized by

its small to medium size pale gray-brown to mauvepileus typically with a glaucous coating at least whenyoung cream pale pinkish or pale yellow-orangelamellae buff or pale salmon stipe watery unchan-ging latex odor of coconuts mild flavor and associa-tion with Betula

Pileus 15ndash50 mm diam broadly convex later becom-ing plane with or without a depressed center and smallpapilla smooth dry pale gray-brown to mauve (violet-brown) with whitish glaucous coating when immaturefading in age azonate to lightly zoned especially nearmargin margin incurved when young and remainingso or becoming straight when mature Lamellaeadnate to subdecurrent slightly crowded to crowdedcream to pale pinkish to pale yellow-orange Stipe10ndash40 6 3ndash12 mm equal to slightly clavate oftencurved central to eccentric smooth dry buff or palesalmon with faint glaucous coating at first as in pileusstuffed or hollow often white-mycelioid toward baseLatex scarce watery unchanging Context of pileusand stipe pale cream Odor of coconut Flavor mildBasidiospore deposit not observed Basidiospores 7ndash96 5ndash7 mm av 5 77 6 60 mm Q 5 117ndash142 Q 5

128 hyaline in 25 KOH broadly ellipsoid to ellip-soid ornamentation of amyloid scattered ridges andwarts forming an incomplete to nearly complete reticu-lum to 05 mm high Basidia 381ndash635 6 76ndash102 mmmostly four-spored some two-spored Pleuromacrocys-tidia 457ndash635 6 64ndash76 mm subclavate to lanceolatewith rounded to occasionally mucronate apex scarceto scattered Cheilomacrocystidia 33ndash66 6 51ndash89mm cylindrical to subclavate with rounded apex scat-tered to abundant Pileipellis a cutis with tufts ofascending hyphae

Distribution and habitat Lactarius glyciosmus was firstdescribed by Fries from a hemiboreal to boreal habitat

in the Femsjouml region of Sweden and the species lacks atype specimen Phylogenetic analyses including speci-mens from the type locality confirm this species inthe central and southern Rocky Mountain alpinezone and suggest it has a broad ecological and geogra-phical distribution confirmed from subalpine and arc-tic-alpine areas in Alaska western Canada IdahoWisconsin New Hampshire Svalbard and Fennoscan-dia In subalpine and alpine areas of the Rocky Moun-tains L glyciosmus associates with Betula glandulosaPhylogenetic placement (FIG 4) of ectomycorrhizalroot tip sequences confirm Betula nana L as a host inAlaska (GU998223 GU998695) and likely B occidenta-lis Hook in Idaho (JX198526 Bogar amp Kennedy2013) Elsewhere it is reported with B pubescens Ehrh(eg Elborne amp Knudsen 1990) and B papyrifera Mar-shall (eg Durall et al 2006)

Previous reports from arctic-alpine areas includeCanada (Ohenoja and Ohenoja 1993 2010) theRocky Mountain alpine zone (Cripps and Horak2008 Cripps and Barge 2013) Greenland (Lange1957 Knudsen and Borgen 1982 Lamoure et al1982 Elbourne and Knudsen 1990 Borgen 2006 Bor-gen et al 2006) Iceland (Eyjolfsdottir pers comm) theAlps (Bon 1998) Fennoscandia (Kallio and Kankainen1964 Kuumlhner 1975 Bon 1998) Svalbard (Ohenoja1971 Geml et al 2012) and Siberia (Karatygin et al1999) At least some reports might be L aff glyciosmusas defined here

Specimens examined FINLAND Fjell Ailigas 27 Aug 2012CLC 2874 (MONT) SWEDEN Femsjouml 13 Aug 1978 MMoser 19780191 (Type locality IB) 16 Aug 1978 M Moser19780234 (Type locality IB) 9 Aug 1981 M Moser19810148 (Type locality IB) USA COLORADO BoulderCounty Front Range Blue Lake 2 Aug 2001 CLC 1624(MONT) Boulder County Front Range Brainard Lake 13Aug 2000 20923 (DBG) Ouray County San Juan RangeIronton Park 13 Aug 2012 EB133 (MONT) MONTANACarbon County Beartooth Plateau Birch Site 29 July 1997CLC 1134 5 ZT6096 (MONT) 8 Aug 1998 CLC 1217(MONT) 10 Aug 1999 TWO 269 (MONT) 19 Aug 1999CLC 1380 (MONT) 13 Aug 2007 ZT12723 (MONT)

Other specimens examined ESTONIA Saare CountyLuumlmanda Commune 17 Sep 2012 TU118535 (L aff glycios-mus TU) Tartu County Votildennu Commune 12 Sept 2010TU106737 (L aff glyciosmus TU) 13 Sept 2010 TU106724(L aff glyciosmus TU)

Comments Lactarius glyciosmus is morphologically andphylogenetically similar to L mammosus however thelatter has a browner pileus stouter stature and moreelongate more reticulate basidiospores (Heilmann-Clausen et al 1998) Phylogenetic placement ofsequences isolated from ectomycorrhizal root tips ofPinus sylvestris L (AJ633116 Heinonsalo et al 2007)and Picea abies (L) Karst (FM992937 Kjoslashller andClemmensen 2009) (FIG 4) confirm that L mammosus

BARGE ET AL ROCKY MOUNTAIN ALPINE LACTARIUS 429

FIG 7 Comparison of micromorphological features of Rocky Mountain alpine specimens and Holotypes (where available)Basidiospores pleuromacrocystidia (left) and cheilomacrocystidia (right) of A Lactarius lanceolatus (CLC2319) B L lanceolatus(F 4239 HOLOTYPE) C L nanus (CLC1471) D L nanus (ZA 192c HOLOTYPE) E L pallidomarginatus Barge amp Cripps spnov (EB0041 HOLOTYPE) F L pseudouvidus (Va 71971 HOLOTYPE) G L salicis-reticulatae (CLC2776) H L salicis-reticulatae

430 MYCOLOGIA

associates with members of the Pinaceae however italso has been reported with Betula mainly in subalpineareas (Heilmann-Clausen et al 1998) Lactarius vietus(Fr) Fr is also similar but has a distinct pale zonenear the stipe apex latex that dries greenish graylonger pleuromacrocystidia and a mild to slightly fruityodor (Heilmann-Clausen et al 1998) It has not beenreported from alpine areas in the Rocky Mountainsalthough it has been reported from elsewhere (mostlynorthern areas) in North America with Betula (Heslerand Smith 1979) The similar-looking L nanus alsooccurs in alpine areas of the Rocky Mountains andhas larger basidiospores larger and differently shapedmacrocystidia lacks an odor of coconut and associateswith Salix Lactarius spinosulus is phylogenetically closeto L glyciosmus (as well as L nanus) (FIG 4) howevermorphologically they are very different Lactarius spino-sulus has a distinctly zoned coarsely squamulose pink-ish salmon- or brick-colored pileus with a finely hairymargin and fruity odor (Heilmann-Clausen et al1998) it has not been reported from North AmericaNo definitive morphological differences were detectedbetween L glyciosmus and L aff glyciosmus and the lat-ter is not reported from the Rocky Mountainalpine zone

Lactarius pallidomarginatus Barge amp CL Cripps spnov FIGS 6 7Mycobank MB812246

Typification USA COLORADO San Juan CountySan Juan Range Stony Pass scattered solitary onsoil under Salix planifolia 13 Aug 2011 EB0041 (holo-type MONT) GenBank KR090940 (ITS) andKR091010 (RPB2)

Etymology Referring to the pale pileus marginDiagnosis Lactarius pallidomarginatus is recognized by

its small to medium size blotchy light tan to lightbrown to gray-brown pileus which is typically lightertoward the margin white to pale yellow-cream lamel-lae pale cream stipe violet staining tissue mild odorand flavor and association with Salix Morphologicallyit is close to L pseudouvidus Kuumlhner but differs by hav-ing smaller basidiospores with an ornamentation ofthinner ridges as well as macrocystidia that becomemore lanceolate (as opposed to subfusiform) and atte-nuated Phylogenetically it is close to L aspideus (Fr)Fr but differs consistently in two nucleotide positionsin the rDNA ITS locus and at eight nucleotide posi-tions in the RPB2 locus morphologically L aspideushas larger yellow basidiomata

Pileus 20ndash50 mm diam convex to broadly convexbecoming broadly convex to plane with or without adepressed center smooth dry to slightly viscid azo-nate blotchy light tan to light brown to gray-browndeveloping violet stains lighter (to cream) toward mar-gin margin incurved to downturned when youngremaining downturned or becoming nearly straightwhen mature Lamellae adnate to subdecurrent subdi-stant to slightly crowded white to pale yellow-creamstaining violet where damaged Stipe 10ndash40 6 5ndash10mm equal to slightly clavate central smooth drywhite to cream staining violet where damaged hollowLatex scarce watery staining tissue violet Context ofpileus and stipe white to cream staining violet wheredamaged Odor mild Flavor mild Basidiosporedeposit not observed Basidiospores 8ndash10 6 65ndash8mm av 5 87 6 71 mm Q 5 114ndash136 Q5 123 hya-line in 25 KOH broadly ellipsoid to ellipsoid orna-mentation amyloid of thin ridges and a few isolatedwarts forming an incomplete to dense reticulum to05 mm high Basidia 508ndash711 6 102ndash127 mm four-spored Pleuromacrocystidia 813ndash1118 6 89ndash102mm cylindrical to lanceolate with acute to moniliformapex scarce to scattered Cheilomacrocystidia 483ndash1016 6 76ndash127 mm cylindrical to lanceolate withacute to moniliform apex scattered Pileipellis an(ixo-)cutis with some ascending hyphae embedded ina faint gelatinous layer

Distribution and habitat Rare in low alpine areas withSalix planifolia reported from Independence Pass inthe Sawatch Range and Stony Pass in the San JuanRange in Colorado An additional collection(ZT5229) from Union Peak in the Wind River Rangeof Wyoming with Salix glauca is likely conspecific how-ever this hypothesis has not been confirmed withmolecular data Due to confusion with L pseudouvidusit is possible that L pallidomarginatus is more wide-spread than confirmed here

Specimens examined USA COLORADO Pitkin CountySawatch Range Independence Pass 6 Aug 2000 CLC 1470(MONT) 9 Aug 2000 ZT9093 (MONT) San Juan CountySan Juan Range Stony Pass 13 Aug 2011 EB0041 (HolotypeMONT) WYOMING Sublette County Wind River RangeUnion Peak 22 Aug 1994 ZT5229 (MONT)

Other specimens examined AUSTRIA Tyrol 3 July 2007 UPeintner 20070035 (L aff pseudouvidus from near type local-ity IB) FINLAND Inarin Lappi 16 Aug 1995 JV10468 (Laff pseudouvidus TURA) Koillismaa JV28448F 30 Aug 2011(L aff pseudouvidus originally identified as L brunneoviola-ceus TURA) FRANCE Isegravere 13 Aug 1971 Va 7 1971 (Holo-type of L pseudouvidus G) ITALY Trentino 20 Aug 2004 U

r(K72-104 HOLOTYPE no cheilocystidia drawn) I L glyciosmus (TWO269) J L repraesentaneus (CLC1747) Bars 5 10 mm forbasidiospores and 20 mm for cystidia Drawings by E Barge

BARGE ET AL ROCKY MOUNTAIN ALPINE LACTARIUS 431

Peintner 20040156 (L aff pseudouvidus from near type local-ity originally identified as L robertianus IB) NORWAYFinse 10 Aug 2005 CLC 2133 (Lactarius aff brunneoviolaceusMONT) Stordal 26 Aug 2006 P Larsen 361395 (L aff brun-neoviolaceus originally identified as L pseudouvidus O) Sval-bard 18 Aug 2002 TWO809 (L aff pseudouvidus MONT)20 Aug 2002 CLC 1910 (Lactarius sp 5 MONT) Ulvik 11Aug 2011 E Soslashyland 73867 (L aff pseudouvidus O) SWE-DEN Latnjajaure 4 Aug 2011 EL101-11 (L aff pseudouvidusGB) Latnjavagge 17 Aug 2010 EL63-10 (L aff pseudouvi-dus GB)

Comments Morphological features of L pallidomar-ginatus fit the holotype description of L pseudouvidusand both are described as lighter near the pileus mar-gin (Kuumlhner 1975 Basso 1999) however basidios-pores of L pallidomarginatus are slightly smaller (8ndash10 6 65ndash8 mm as opposed to 75ndash12 6 65ndash8(85)mm) and have narrower ridges that form a less densereticulum and macrocystidia take on a more lanceo-late form with greater attenuation of the apex (FIG7) In phylogenetic analyses exsiccati identified as Lpseudouvidus from Europe (including specimensfrom near the type locality) appear as distinct fromRocky Mountain alpine material and may representa species complex Lactarius sp 3 (CLC1910) col-lected in Svalbard has darker more vinaceous pileithan L pallidomarginatus and may represent anotherundescribed species although only one collectionwas morphologically examined Members of the Laff brunneoviolaceus clade (FIG 5) that were exam-ined differ from L pallidomarginatus in having dar-ker more vinaceous pilei larger more elongatebasidiospores (to 13 mm) and green staining of exsic-cati in 25 KOH a feature not mentioned previouslyfor L brunneoviolaceus but which could be of taxo-nomic interest Based on taxa included in the phylo-genetic analyses (FIG 5) Lactarius aspideus appearsclosely related to L pallidomarginatus however theformer has more yellow coloration and typically lar-ger sporocarps (pileus 10ndash70 mm diam) and smallerbasidiospores (67ndash95 6 56ndash78 mm) traditionallyL aspideus has been placed with other yellow violet-staining taxa such as L flavidus Boud L salicis-reticu-latae and L salicis-herbaceae (Heilmann-Clausen et al1998) but it is separated here

Lactarius montanus another violet-staining speciesoriginally described from the Rocky Mountains dif-fers from L pallidomarginatus by its larger staturedarker grayish vinaceous to vinaceous-brown pileusand green staining flesh in 25 KOH it is typicallyfound in subalpine habitats with conifers but alsooccurs in the krummholz zone The violet-stainingLactarius pallescens Hesler amp AH Sm occurs in theRocky Mountains but its sporocarps are larger andlighter in color and it is reported from subalpineconifer forests (Hesler and Smith 1979) Other

gray-brown to brownish violet-staining species suchas L luridus L uvidus (Fr) Fr L violascens (JOtto) Fr L pallescens v palmerensis Hesler amp AHSm and L cordovaensis Hesler amp AH Sm are alsolarger and have a subalpine ecology none havebeen reported from the central or southern RockyMountains (Hesler and Smith 1979 Heilmann-Clau-sen et al 1998) Lactarius cistophilus Bon amp Trimbachtypically has larger basidiomata larger basidiospores(10ndash14 6 72ndash9 mm) and occurs under Cistus inthe Mediterranean region (Basso 1999) The smallpale gray-brown European alpine taxon L uvidus falpigines Jamoni amp Bon has microscopic featuresthat match those of L uvidus (Jamoni and Bon1991) but not L pallidomarginatus The small non-violet staining L nanus has basidiospores withthicker more jagged ridges and differently shapedmacrocystidia (often with more rounded apices)(FIG 7)

Lactarius repraesentaneus Britzelm FIGS 6 7Short description Lactarius repraesentaneus is recog-

nized by its medium to large size orange-brown to yel-low-brown hairy pileus with a heavily bearded margincream to pale-yellow lamellae stout cream to pale-yel-low often pitted stipe violet staining latex and tissuespicy-floral odor slightly acrid flavor and associationwith Betula Picea and possibly shrubby Salix speciessuch as S glauca

Pileus 60ndash100 mm diam convex to plano-convexusually with a depressed center and sometimes with apapilla smooth at center becoming increasingly hairyto bearded toward margin dry to viscid azonate sur-face orange-brown at center becoming pale yellow-brown to cream toward margin hairs pale yellow-brown to red-brown margin strongly incurvedremaining so or becoming nearly straight whenmature Lamellae adnate to decurrent crowdedcream to pale-yellow staining violet where damagedStipe 30ndash70 6 15ndash35 mm stout equal to clavate cen-tral dry to viscid cream to pale yellow surface oftenwith numerous dingy yellow golden yellow yellow-brown or light orange-brown pits hollow Latex scarceto abundant white becoming violet Context of pileusand stipe white staining violet where damaged Odorspicy-floral Flavor slightly acrid Basidiospore depositnot observed Basidiospores 8ndash105 6 6ndash85 mm av5 93 6 73 mm Q 5 113ndash143 Q5 127 hyaline in25 KOH broadly ellipsoid to ellipsoid ornamenta-tion amyloid of narrow to broad ridges and isolatedwarts forming an incomplete to dense reticulum to1 mm high Basidia 610ndash762 6 102ndash114 mm four-spored Pleuromacrocystidia 787ndash1448 6 89ndash127 mm subfusiform to lanceolate thin-walled withacute to moniliform apex scattered to abundant

432 MYCOLOGIA

Cheilomacrocystidia 559ndash1397 6 76ndash127 mm subfu-siform to lanceolate thin-walled with acute to monili-form apex scattered to abundant Pileipellis anixotrichoderm with well-developed gelatinous layer

Distribution and habitat Lactarius repraesentaneus firstwas described from Germany (Britzelmayr 1885) Phy-logenetic analyses including material from Germanyconfirm L repraesentaneus from alpine areas of theRocky Mountains and overall reinforce the traditionalconcept that this species has a broad ecological andgeographic range in arctic-alpine and subalpine habi-tats in North America northern central and southernEurope In alpine areas of the Rocky Mountains itassociates with Salix glauca however krummholz Piceaalso is typically (maybe always) present it is reportedwith Picea and Betula elsewhere

Previous reports from arctic-alpine areas mainlywith Betula include Alaska (Laursen and Ammirati1982 Geml et al 2009) the Rocky Mountain alpinezone (Cripps and Horak 2008 Cripps and Barge2013) Greenland (Knudsen and Borgen 1982 Watling1983 Elbourne and Knudsen 1990 Borgen 2006 Bor-gen et al 2006) Iceland (Eyjolfsdottir pers comm)Fennoscandia (Kallio and Kankainen 1964 Kuumlhner1975 Bon 1998) Siberia (Karatygin et al 1999) andthe Altai Mountains (Gorbunova 2010)

Specimens examined FINLAND Enontakiouml 16 Aug 2004JV21671 (TURA) Varsinais-Suomi 9 Aug 1998 JV13837F(TURA) SWEDEN Latnjavagge 12 Aug 2007 EL92-07(GB) USA COLORADO Chaffee County Sawatch RangeCottonwood Pass 4 Aug 2001 CLC 1643 (MONT) 16 Aug2011 EB0048 (MONT) Pitkin County Sawatch Range Inde-pendence Pass 14 Aug 2001 CLC 1747 (MONT) ZT9837(MONT) 15 Aug 2001 ZT9537 (MONT) MONTANA Car-bon County Beartooth Plateau Birch Site 12 Aug 2002CLC 1971 (MONT) 13 Aug 2007 CLC 2318 (MONT) 18Aug 2013 EB107-13 (MONT) WYOMING Park CountyBeartooth Plateau Frozen Lakes 21 Aug 1999 CLC1394 (MONT)

Other specimens examined SWEDEN Latnjavagge 14 Aug2010 EL57-10 (L dryadophilus GB) USA ALASKA 5 Aug2011 CLC 2729 (L dryadophilus MONT) Kenai PeninsulaCounty Palmer Creek Road 7 Aug 2011 CLC 2744 (L drya-dophilus MONT)

Comments The combination of a large pale yellow-brown to orange-brown pileus with a bearded marginand violet staining latex clearly separates L repraesenta-neus from other Lactarius species of the central andsouthern Rocky Mountain alpine zone Phylogeneti-cally close the arctic-alpine taxon L dryadophilus ismorphologically similar but has a cream to yellowsmoother pileus with hairs restricted to near the mar-gin basidiospore ornamentation consisting of sparsethinner ridges and ecological associations with Dryasand Salix It has not been reported from the RockyMountains

Lactarius salicis-reticulatae Kuumlhner FIGS 6 7Short description Lactarius salicis-reticulatae is recog-

nized by its small to medium cream pale yellow orpale ocher sporocarps with pale yellowish well-spacedlamellae often with a pinkish buff tint cream to pale yel-low stipe violet staining latex and tissue mild to slightlysweet odor mild flavor and association with dwarf (Sreticulata) and possibly also shrubby Salix species

Pileus 20ndash40 mm diam convex to broadly convexusually with a depressed center smooth dry to viscidazonate cream pale yellow or pale ocher sometimesdarker toward center staining violet where damagedmargin invcurved remaining so or becoming down-turned when mature Lamellae adnate to subdecur-rent fairly well-spaced cream pale yellow or paleorange-ocher often with a pinkish-buff tint stainingviolet where damaged Stipe 15ndash20 6 10ndash15 mmequal to clavate central dry to viscid cream to pale-yellow staining violet where damaged hollow Latexscarce watery-white becoming violet Context ofpileus and stipe white staining violet where damagedOdor mild to slightly sweet Flavor mild Basidiosporedeposit not observed Basidiospores (7ndash)85ndash115 6(7ndash)8ndash10 mm av 5 100 6 88 mm Q 5 (100ndash)113ndash143 Q 5 125 hyaline in 25 KOH sublgobose toellipsoid ornamentation amyloid of narrow ridgesand a few isolated warts forming an incomplete reticu-lum to 03 mm high Basidia 737ndash889 6 102ndash127 mm four-spored Pleuromacrocystidia 762ndash10166 76ndash114 mm subfusiform to fusiform thin-walledwith rounded to acute to moniliform apex scarceCheilomacrocystidia 686ndash9146 76ndash102 mm subfusi-form to fusiform thin-walled with acute to rounded tomoniliform apex scattered to abundant Pileipellis anixocutis

Distribution and habitat Lactarius salicis-reticulatae firstwas described from alpine tundra with Salix on MtLaringktatjaringkka Sweden (Kuumlhner 1975) Phylogenetic ana-lyses including material from the type locality confirmthis species from alpine areas on the Beartooth Plateauwith Salix reticulata and shrubby Salix spp and in arctic-alpine areas of Alaska Svalbard and Finland OverallL salicis-reticulatae appears largely confined to arctic-alpine habitats Colorado collections with S reticulataand S planifolia may represent a distinct taxonalthough support is low

Previous arctic-alpine reports include Greenland(Lamoure et al 1982 Heilmann-Clausen et al1998 Borgen et al 2006) the Rocky Mountainalpine zone (Cripps and Horak 2008 Cripps andBarge 2013) the Alps (Kuumlhner 1975 Kuumlhner andLamoure 1986 Jamoni 1995 2008 Bon 1998 Heil-mann-Clausen et al 1998) the Pyrenees (Ballaraacute1997) Fennoscandia (Kuumlhner 1975 Heilmann-Clau-sen et al 1998 Gulden 2005 Knudsen and

BARGE ET AL ROCKY MOUNTAIN ALPINE LACTARIUS 433

Vesterholt 2008) and Siberia (Nezdoiminogo 1997Karatygin et al 1999) previous reports of L aspi-deoides Burl from arctic-alpine areas in Alaska (Laur-sen and Ammirati 1982) Greenland (Knudsen andBorgen 1982) and the British Isles (Watling 1987)may refer to this species

Specimens examined FINLAND Near Utsjoki 28 Aug 2012CLC 2885 (MONT) SWEDEN Mt Laringktatjaringkka 12 Aug1972 K72-104 (Holotype G) 15 Aug 1999 JV15133 (Typelocality TURA) USA ALASKA Kenai Peninsula County Pal-mer Creek Road 7 Aug 2011 CLC 2745 (MONT) MON-TANA Carbon County Beartooth Plateau Birch Site 17Aug 2011 CLC 2776 (MONT) Carbon County BeartoothPlateau Highline Trail 8 Aug 1998 CLC 1211 (MONT)WYOMING Park County Beartooth Plateau GardnerLake 16 Aug 2014 EB0057-14 (MONT)

Other specimens examined FINLAND Koillismaa 31 Aug2005 JV23334 (L flavopalustris TURA) GREENLAND Sis-miut 19 Aug 2000 CLC 1536 (L salicis-herbaceae MONT)USA COLORADO Pitkin County Sawatch Range Indepen-dence Pass 13 Aug 2001 CLC 1741 (L aff salicis-reticulataeMONT) San Juan County San Juan Range CinnamonPass 10 Aug 2001 CLC 1710 (L aff salicis-reticulataeMONT) San Juan County San Juan Range Maggie Gulch11 Aug 2011 EB0036 (L aff salicis-reticulatae MONT)San Juan County San Juan Range Stony Pass 9 Aug 2001CLC 1689 (L aff salicis-reticulatae MONT) 13 Aug 2011EB0039 (L aff salicis-reticulatae MONT) MICHIGANEmmet County 10 Aug 1973 RL Shaffer 6957 (L aspi-deoides MICH)

Comments Some taxonomic confusion has beencaused by the fact that Kuumlhner (1975) originallynamed this species L aspideoides Kuumlhner but subse-quently renamed it because the name had been pre-viously applied L aspideoides Burl (1907) is generallylarger with a somewhat zonate pileus more crowdedlamellae that lack salmon-colored hues slightly smallerbasidiospores with broader ridges a bitter to slightlyacrid flavor and a subalpine ecology apparently ineastern and western North America (pers obs of RLShaffer 6957 Hesler and Smith 1979) Lactarius fla-vopalustris is also similar however it has a largermore brightly yellow pileus with faint zonationmore crowded lamellae a weakly scrobiculate stipe(sometimes) smaller basidiospores with thickerridges and a subalpine ecology (Kytoumlvuori 2009)Phylogenetic analyses (FIG 5) reveal three collec-tions of L flavopalustris (two from near the typelocality in Finland) nested in a well-supported cladewith L aspideoides from a subalpine forest in north-ern Michigan suggesting the possible conspecificityof these taxa Neither is reported from the RockyMountains Two other collections identified as L fla-vopalustris (from Estonia) were resolved as distinctfrom the L aspideoidesflavopalustris clade (FIG 5)their identity is unclear

Lactarius aspideus is differentiated from L salicis-reti-culate by its smaller basidiospores with denser reticula-tion more crowded creamier-colored lamellae andhabitat mainly in subalpine areas (Heilmann-Clausenet al 1998) although an environmental sequence iso-lated from an S polaris root tip from arctic Svalbardclusters with L aspideus in phylogenetic analyses (FIG5) Lactarius dryadophilus is often larger with a cream-colored pileus that has a tomentose to bearded margin(Heilmann-Clausen et al 1998) Lactarius flavidus is lar-ger with a sparsely zonate pileus more crowdedcream-colored lamellae darker violet-staining contextbasidiospores with more prominent ornamentationand a distally amyloid plage and subalpine ecology(Heilmann-Clausen et al 1998) Lactarius flavoaspideusKytoumlv has slightly smaller basidiospores on averagewith a denser reticulum and subalpine ecology withSalix (Kytoumlvuori 2009) it has been reported onlyfrom northern Europe Lactarius salicis-herbaceae asimilar arctic-alpine Salix associate has smaller (typi-cally) darker yellow to yellow-brown sporocarpspale cream to grayish buff lamellae and more denselyreticulate basidiospores (pers obs Heilmann-Clausenet al 1998) It is fairly widespread in arctic-alpineareas but is not reported from alpine areas in the cen-tral and southern Rocky Mountains Lactarius aff sali-cis-reticulatae from Colorado may be a distinct taxonand sporocarps can obtain a larger size (pileus to 90mm diam) but otherwise morphological featuresstrongly overlap

KEY TO ROCKY MOUNTAIN ALPINE LACTARIUS SPECIES

1 Latex white turning violet to dull lilac or flesh stain-ing violet to dull lilac 2

1 Latex watery to white unchanging and flesh notstaining violet to dull lilac 42 Fruiting body robust pileus 60ndash100 mm diam

yellow-brown to orange-brown hairy especiallynear margin lamellae cream to pale-yellow stipestout cream to pale-yellow often with numerousyellow-brown pits odor spicy-floral flavor mildlyacrid basidiospores 8ndash105 6 6ndash85 mm puta-tively associated with Betula and Picea (possiblyalso Salix) in Rocky Mountain alpine zone andsubalpine and arctic-alpine areas in North Amer-ica and Eurasia L repraesentaneus

2 Fruiting body smaller 20ndash50 mm diam pileus andmargin smooth 3

3 Pileus pale yellow to pale ocher lamellae cream paleyellow or pale orange-ocher stipe cream to pale yel-low odor mild to slightly sweet flavor mild basidios-pores 85ndash115 6 8ndash10 mm in Rocky Mountain alpinezone and other arctic-alpine areas in North Americaand Eurasia with Salix L salicis-reticulatae

3 Pileus blotchy light tan to light brown to gray-brownmargin lighter lamellae white to pale yellow-cream

434 MYCOLOGIA

stipe white to cream odor mild flavor mild basi-

diospores 8ndash10 6 65ndash8 mm in low alpine areas inColorado with Salix planifolia and possibly Wyom-

ing with Salix glauca L pallidomarginatus4 Pileus bright orange to orange-brown 10ndash45

mm diam margin smooth lamellae pale creamto pale orange stipe pale orange odor mildflavor mild in Rocky Mountain alpine zonearctic-alpine Alaska (possibly also subalpine)to Svalbard with Salix L lanceolatus

4 Pileus pinkish buff gray-brown brown ormauve 5

5 With odor of coconuts pileus 15ndash50 mm diam palepinkish buff to pale gray-brown with glaucous coat-ing lamellae cream to pale yellow-orange stipe buffor pale salmon flavor mild basidiospores 7ndash9 6 5ndash7mm in Rocky Mountain alpine zone and subalpinearctic-alpine areas in North America and Eurasiawith Betula L glyciosmus

5 Without odor of coconuts pileus 10ndash50 mm diamgray-brown to liver-brown with whitish glaucous coat-ing lamellae cream to light tan sometimes with pink-ish tinge discoloring to dingy tan where damagedStipe pale apricot to dingy cream-tan flavor mild toslightly acrid basidiospores 7ndash105 6 5ndash8 mm inRocky Mountain alpine zone and arctic-alpine areasin Eurasia and likely other arctic-alpine areas in NorthAmerica with Salix L nanus

DISCUSSION

The number of Lactarius species reported from theRocky Mountain alpine zone represents only a fractionof the total number of Lactarius species reported else-where from arctic-alpine areas however nearly halfof the core arctic-alpine species are now confirmedfor continental North America Core species oftencited as being restricted to arctic-alpine areas withdwarf and shrubby Salix andor Dryas species includeL brunneoviolaceus L dryadophilus L lanceolatus Lnanus L pseudouvidus L salicis-herbaceae and L salicis-reticulatae (Knudsen and Borgen 1982 Heilmann-Clau-sen et al 1998 Basso 1999 Knudsen and Vesterholt2008) Phylogenetic analyses and morphological exam-ination confirm three out of seven of these species forthe Rocky Mountain alpine zone (L lanceolatus Lnanus L salicis-reticulatae) and support the hypothesisthat these species are largely restricted to arctic-alpineareas with Salix One definitive new species L pallido-marginatus (with Salix) was revealed along with a possi-ble cryptic species L aff salicis-reticulatae (also withSalix) Many Lactarius species previously reportedfrom arctic-alpine areas are also commonly reportedfrom subalpine areas with Betulaceae andor Pinaceae

as well as with other plant families (Heilmann-Clausenet al 1998) From this set of species we report L gly-ciosmus which associates with Betula glandulosa in theRocky Mountain alpine zone and Betula spp acrossthe rest of its distribution and L repraesentaneus whichis commonly reported with Betula and Picea (Heslerand Smith 1979 Heilmann-Clausen et al 1998) andappears to associate with Salix glauca andor perhapskrummholz Picea in the Rocky Mountain alpine zone

Several taxa of interest were not molecularly distinctIn dataset A L lanceolatus did not form a monophyleticgroup but was paraphyletic with respect to Lactarius sp2 the L luculentus group and L aurantiacus In datasetB L nanus and L hysginoides were not clearly distinctIn dataset C L salicis-reticulatae did not form a clearmonophyletic group and L pseudouvidus L brunneovio-laceus and L luridus were not clearly distinguishedfrom each other especially at the RPB2 locus poormolecular resolution also has been encountered forthe latter three taxa by Das et al (2015)

Overall there was little genetic variation distinguish-ing closely related species in this study These findingsare similar to reports from other researchers who sug-gest that there is in general relatively little molecularvariation yet high morphological variation in genusLactarius (Verbeken and Nuytinck 2013) While manyof the species could be morphologically distinguishedall of the Rocky Mountain alpine taxa delineated inthis study are 97 or more similar at the ITS regionto one or more species For example L repraesentaneusand L dryadophilus are as much as 994 similar L sal-icis-reticulatae is approximately 98 similar to L salicis-herbaceae and L flavopalustris L pallidomarginatus isapproximately 97ndash98 similar to L aff brunneoviola-ceus L luridus L montanus and members of the Laff pseudouvidus group and approximately 99 similarto L aspideus L glyciosmus is approximately 98 simi-lar to L mammosus L nanus is approximately 98similar to L hysginoides and L lanceolatus is approxi-mately 97 similar to L substriatussubflammeus L aur-antiacus L luculentus v laetus and Lactarius sp 1 and 2and approximately 98 similar to L luculentus Thishigh degree of similarity could be problematic forenvironmental sequencing studies that use a strict97 ITS region similarity cutoff for recognizing spe-cies (eg Geml et al 2012 Timling et al 2012 2014)In addition Tedersoo et al (2014) suggested that theITS region in the sebacina lineage evolves moreslowly in high latitude areas which could be true forother genera The RPB2 locus proved better for differ-entiating some species and overall phylogenetic ana-lyses revealed several taxonomic issues that we werenot able to resolve A search for additional phylogen-etically informative loci for Lactarius should beinitiated along with more species-level phylogenetic

BARGE ET AL ROCKY MOUNTAIN ALPINE LACTARIUS 435

studies to provide a high quality molecular databasefor ecological studies Furthermore improvement ofmethods for sequencing older material such as typespecimens is critical

Most of the Rocky Mountain alpine Lactarius speciesdelineated in this study (except L pallidomarginatusand L aff salicis-reticulatae for which further studycould reveal broader ranges) appear to have broadintercontinental distributions in North America andEurasia The current distributions of these specieslikely were shaped by a number of biotic and abioticfactors that include host dispersal and distribution cli-mate glaciation and geography Because the genusLactarius is obligately ectomycorrhizal it is likely thatthe distribution of each fungal species was shaped lar-gely by the distribution of suitable host plants manyof which also have broad intercontinental distribu-tions Long-distance dispersal is hypothesized to haveplayed an important role in structuring present distri-bution patterns of arctic-alpine plants although manyappear clonal (Abbott and Brochmann 2003 Alsoset al 2007) and ectomycorrhizal fungi (Geml et al2012 Timling et al 2014) It also has been suggestedthat long-distance dispersal mechanisms have beenselected for in arctic flora due to repeated glaciationsand the need to track potential niches (Alsos et al2007) Pollen data from Blacktail Pond YellowstoneNational Park suggest that montane mid-elevationsin the northern greater Yellowstone ecosystem (Mon-tana Wyoming) were occupied by alpine tundra(Betula Salix many herbaceous taxa) similar to thatof the present Rocky Mountain alpine vegetation dur-ing the last glacial period (14 000ndash11 500 calibratedyears before present) (Krause and Whitlock 2013)Alpine vegetation also appears to have dominatedareas in the southern Rocky Mountains (WyomingColorado) 17 500ndash12 600 calibrated years before pre-sent (Minckley 2014) Both studies suggest the pre-sence of broad low-elevation glacial refugia for arctic-alpine life in the Rocky Mountains along the glacialfront and along with this study lend support to theidea that arctic-alpine Lactarius species (and manyother ectomycorrhizal fungi) may have occupied tun-dra areas south of the icesheets during cooler weatherduring the last glacial maximum and then dispersedupward in elevation and northward as the glaciersand ectomycorrhizal host plants retreated Broaderecological tolerances of taxa such as L glyciosmus andL repraesentaneus which are not restricted to arctic-alpine habitats likely let these species persist in tundraand non-tundra areas during the last glacial maximumas well as to track arctic-alpine vegetation as it movedupward in elevation and northward following theretreat of the icesheets For these species to haveachieved and maintained such broad distributions

gene flow must have been possible over long distancesduring the Pleistocene perhaps mediated by long-dis-tance dispersal (Geml et al 2012) range shifts due toclimate change and glacial expansion and retreat(Birks 2008) glacial and interglacial refugia (Billings1974) and the Bering land bridge (Shapiro et al2004 Geml et al 2006)

ACKNOWLEDGMENTS

We thank Dr Ellen Larsson Dr Pierre-Arthur Moreau JukkaVauras Dr Ursula Peintner Dr Egon Horak and herbaria GIB TU O GB TURA C LIP MICH DBG VPI and NY forloan of specimens Carol Johnson and Dr Matt Lavin areacknowledged for reviewing early stages of the manuscriptWe thank Don Bachman Marcy Barge and Charissa Bujakfor assistance with field work We also gratefully acknowledgesupport from the Montana Institute on Ecosystems graduateenhancement fund

LITERATURE CITED

Abbott RJ Brochmann C 2003 History and evolution of thearctic flora in the footsteps of Eric Hulteacuten Mol Ecol12299ndash313 doi101046j1365-294X200301731x

Alsos IG Eidesen PB Ehrich D Skrede I Westergaard KJacobsen GH Landvik JY Taberlet P Brochmann C2007 Frequent long-distance plant colonization in thechanging arctic Science 3161606ndash1609 doi101126science1139178

Ballaraacute J 1997 Nou estudi drsquoespegravecies fuacutengiques interessantsdels estatges alpiacute i subalpiacute dels Pirineus catalans RevCatalana Micol 201ndash24

Barge E 2015 Systematics of Lactarius in the Rocky Moun-tain alpine zone (masterrsquos thesis) Bozeman MontanaMontana State University 265 p

Basso MT 1999 Lactarius Pers Fungi Europaei 7 AlassioMykoflora (Italy) 845 p

Beker HJ Eberhardt U Vesterholt J 2010 Hebeloma hiemaleBres In arcticalpine habitats N Am Fungi 551ndash65

Bessette AE Harris DB Bessette AR 2009 Milk mushroomsof North America a field identification guide to thegenus Lactarius Syracuse New York Syracuse UniversityPress 297 p

Billings WD 1974 Adaptations and origins of alpine plantsArctic Alpine Res 6129ndash142 doi1023071550081

mdashmdashmdash 1988 Alpine vegetation In Barbour MG BillingsWD eds North American terrestrial vegetation NewYork Cambridge University Press p 392ndash420

Birks HH 2008 The late-quaternary history of arctic andalpine plants Plant Ecol Divers 1135ndash146 doi10108017550870802328652

Bogar LM Kennedy PG 2013 New wrinkles in an old para-digm neighborhood effects can modify the structureand specificity of Alnus-associated ectomycorrhizal fun-gal communities FEMS Microbiol Ecol 83767ndash777doi1011111574-694112032

Bon M 1985a Stage mycologie alpine Lanslebourg (Savoie)du 1 aug 3 sep 1984 Bull Feacuted Mycol Daupineacute-Savoie9619ndash25

436 MYCOLOGIA

however the L aff glyciosmus clade does not containany arctic-alpine specimens (FIG 4)

In dataset C (subgenus Piperites section Uvidi) col-lections originally identified as L aff pseudouvidusfrom the Rocky Mountain alpine zone are well-sepa-rated from the majority of collections (mostly fromEurope) labeled the L pseudouvidus group (FIG 5)the latter appear in the BI consensus tree as paraphy-letic to L brunneoviolaceus MP Christ and L luridus(Pers) Gray Instead Rocky Mountain alpine materialforms a well-supported clade close to the morphologi-cally very different species L aspideus (Fr) Fr and anarctic-alpine environmental ITS sequence isolatedfrom the roots of Salix polaris Wahlenb in Svalbard(AB588960 Fujiyoshi et al 2012) (FIG 5) Thus based

on morphological and molecular differences theRocky Mountain alpine material is recognized here asthe new species L pallidomarginatus Barge amp Crippssp nov (see TAXONOMY) An additional collectionfrom Svalbard with Salix polaris (labeled Lactarius sp3 in FIG 5) is sister to the clade containing L aspi-deusL pallidomarginatus

The ITS region of the European L pseudouvidusgroup has little variation and is paraphyletic withrespect to L montanus (Hesler amp AH Sm) Montoyaamp Bandala (SUPPLEMENTARY FIG 9) However theRPB2 locus is highly variable both between and withinmost collections examined (SUPPLEMENTARY FIG 10) Itis not possible to definitively provide names to taxawithin this group in that sequencing of the L

FIG 3 Combined (ITS+RPB2) Bayesian 50 majority-rule consensus tree of dataset A (Lactarius subgenus Russularia pp)Support values (PAUP bootstrapRAxML bootstrapBayesian posterior probability) $ 60 (bootstrap) and $ 080 (posteriorprobability) are indicated above or below branches leading to clades Thickened branches lead to clades receiving $ 75bootstrap support and Bayesian posterior probabilities $ 095 Boldface tip labels represent Rocky Mountain alpine specimensAA 5 arctic-alpine Originally identified as L lanceolatus

422 MYCOLOGIA

pseudouvidus holotype was unsuccessful and cryptictaxa may be present Collections matching the mor-phological concept of the similar arctic-alpine taxonL brunneoviolaceus (syn L robertianus Bon) occur in a

moderately supported clade (labeled L aff brunneovio-laceus)in the combined (FIG 5) and ITS analyses (SUPPLEMEN-

TARY FIG 9) but these are paraphyletic with the L

FIG 4 Combined (ITS+RPB2) Bayesian 50 majority-rule consensus tree of dataset B (Lactarius subgenus Piperites pp)Support values (PAUP bootstrapRAxML bootstrapBayesian posterior probability) $ 60 (bootstrap) and $ 080 (posteriorprobability) are indicated above or below branches leading up to clades Thickened branches lead to clades receiving $ 75bootstrap support and Bayesian posterior probabilities $ 095 Boldface tip labels represent Rocky Mountain alpine specimensAA 5 arctic-alpine

BARGE ET AL ROCKY MOUNTAIN ALPINE LACTARIUS 423

FIG 5 Combined (ITS+RPB2) Bayesian 50 majority-rule consensus tree of dataset C (Lactarius subgenus Piperites sectionUvidi) Support values (PAUP bootstrapRAxML bootstrapBayesian posterior probability) $ 60 (bootstrap) and $ 080(posterior probability) are indicated above or below branches leading up to clades Thickened branches lead to clades receiving

424 MYCOLOGIA

pseudouvidus group in RPB2 analyses (SUPPLEMENTARY

FIG 10) Collections of Lactarius pallidomarginatus Lac-tarius sp 3 L aspideus L montanus L luridus L affpseudouvidus and L aff brunneoviolaceus for which ITSdata are available all have a similar 268ndash269 base-pairinsertion in the ITS1 region There is variation withinthe base-pair content of the insertion between collec-tions however inclusion of this insertion did notincrease phylogenetic resolution in preliminary ana-lyses and it therefore was omitted The insertion itselfis highly similar to an insertion in Russula olivacea (eg GenBank accessions AF418634 AY061699) as tolength sequence (approximately 90 similar) andplacement within the ITS1 region

Also in dataset C Rocky Mountain alpine collectionsinitially identified as L repraesentaneus form a well-supported clade with a collection of L repraesentaneusfrom Germany (the lectotype country) as well as withnumerous collections from subalpine and arctic-alpineareas elsewhere in Eurasia and Alaska (FIG 5) Thisclade is sister to a poorly supported L dryadophilusKuumlhner clade The two GenBank sequences JX630967from arctic Canada with Dryas and JF394416 from arcticSvalbard which occur just outside the L dryadophilusclade are likely conspecific with L dryadophilusalthough this cannot be confirmed based on presentresults Lactarius repraesentaneus and L dryadophilus con-sistently differ at two nucleotide positions in the ITSlocus and at four positions in the RPB2 locus and haveconsistent morphological and host differences

In dataset C collections originally identified as L sal-icis-reticulatae from alpine areas in the Rocky Mountainsare paraphyletic with regard to L salicis-herbaceaeKuumlhner L aspideoides BurlL flavopalustris Kytoumlvand L aff flavopalustris (FIG 5) Collections of Lsalicis-reticulatae from alpine areas on the BeartoothPlateau (central Rocky Mountains) form a poorlysupported clade with an alpine collection of L salicis-reticulatae from Mt Laringktatjaringkka Sweden the type local-ity (sequencing of the holotype of L salicis-reticulataewas not successful) as well as with other arctic-alpinecollections of L salicis-reticulatae from Finland andAlaska and an unidentified collection from Svalbard(FIG 5) this clade is hypothesized to represent Lsalicis-reticulatae Collections from southern RockyMountain alpine areas in Colorado are larger thanreported for L salicis-reticulatae and form an unre-solved group that consistently differs at one nucleotideposition in the ITS region but no positions in the RPB2

locus they are tentatively referred to as L aff salicis-reticulatae pending data from additional loci

Overall phylogenetic analyses support the recogni-tion of six to seven Lactarius species from the RockyMountain alpine zone Lactarius lanceolatus (FIG 3)L nanus L glyciosmus (FIG 4) L pallidomarginatus spnov L repraesentaneus L salicis-reticulatae and possiblyL aff salicis-reticulatae (FIG 5) Violet-staining taxa Lpallidomarginatus L repraesentaneus L salicis-reticulataeand L aff salicis-reticulatae are resolved as a poorly sup-ported clade exclusively containing other members ofthe violet-staining section Uvidi supporting the mono-phyly of this section (FIG 2)

TAXONOMY

All descriptions are based on Rocky Mountain alpinematerial

Lactarius lanceolatus OK Mill amp Laursen FIGS 6 7Short description Lactarius lanceolatus is recognized by

its small to medium size and bright light to deeporange pileus pale cream to pale orange lamellae dis-coloring brownish in age or where damaged paleorange stipe watery unchanging latex and mild flavorgrowing in arctic-alpine (possibly also subalpine) areaswith Salix species

Pileus 10ndash45 mm diam convex at first becomingplane to infundibuliform with a depressed centerwith or without a small papilla smooth sometimesfaintly scaly toward center viscid deep orange-brownto deep orange when immature becoming light todeep orange often blotchy often darker toward cen-ter margin incurved to straight when immaturebecoming straight to upturned and often slightlywavy when mature Lamellae adnate to subdecur-rent slightly crowded pale cream to pale yellow topale orange discoloring brownish orange in age orwhere damaged Stipe 10ndash20 6 25ndash75 mm equalto clavate central smooth dry at first covered byfaint whitish pubescence pale orange discoloringdingy orange to dingy light brown where damagedhollow Latex scarce watery unchanging Contextof pileus and stipe pale orange Odor mild Flavormild Basidiospore deposit not observed Basidios-pores 8ndash10 6 6ndash8 mm average 5 86 6 7 mmQ 5 114ndash138 Q5 124 hyaline in 25 KOHbroadly ellipsoid ornamentation amyloid of isolatedwarts connected by fine lines up to 05 mm highBasidia 381ndash660 6 89ndash102 mm four-spored Pleur-omacrocystidia 737ndash127 6 64ndash102 mm narrowly

r$75 bootstrap support and Bayesian posterior probabilities $ 095 Boldface tip labels represent Rocky Mountain alpinespecimens AA 5 arctic-alpine See TABLE I for original identifications

BARGE ET AL ROCKY MOUNTAIN ALPINE LACTARIUS 425

fusiform to lanceolate with acute to moniliformapex thin-walled scattered to abundant Cheiloma-crocystidia 356ndash89 6 51ndash102 mm conical to fusi-form with acute to moniliform apex thin-walledsparse to abundant Pileipellis an ixotrichodermwith well-developed gelatinous layer

Distribution and habitat Lactarius lanceolatus origin-ally was described from arctic-alpine Alaska (Millerand Laursen 1973) and phylogenetic analyses(including the holotype) further confirm its pre-sence in the central Rocky Mountain alpine zoneand Svalbard In alpine areas on the Beartooth Pla-teau it occurs with Salix reticulata and possibly otherSalix species (S arctica S planifolia) it is notreported from the southern Rocky Mountains Over-all L lanceolatus appears largely confined to arctic-alpine habitats however an ITS sequence from amaritime forest in Alaska (GAL18572) clusters withthe arctic-alpine collections in phylogenetic analyses(FIG 3)

Previous arctic-alpine reports include Canada (Ohe-noja and Ohenoja 1993 2010) Alaska (Miller andLaursen 1973 Laursen and Ammirati 1982 Heil-mann-Clausen et al 1998) Beartooth Plateau in theRocky Mountain alpine zone (Cripps and Barge2013) Greenland (Heilmann-Clausen et al 1998 Bor-gen et al 2006) Fennoscandia (Bon 1998 Heilmann-Clausen et al 1998 Knudsen and Vesterholt 2008)Svalbard (Gulden and Jenssen 1988 Geml et al2012) the British Isles (Watling et al 2001) andSiberia (Nezdoiminogo 1997 Heilmann-Clausen et al1998 Karatygin et al 1999 Knudsen and Mukhin1998) however phylogenetic analyses (FIG 3) suggestthat some of these reports may represent L aurantiacus(Pers Fr) Gray

Specimens examined USA ALASKA North Slope CountyBeaufort Lagoon 30 Jul 1971 F4239 (Holotype VPI) MON-TANA Carbon County Beartooth Plateau Highline Trail 1Aug 1997 ZT6214 (MONT) 7 Aug 1998 ZT6412 (MONT)20 Aug 1999 CLC 1389 (MONT) 5 Aug 2008 CLC 2358(MONT) 27 Jul 1997 CLC 1139 (MONT) WYOMINGPark County Beartooth Plateau Solifluction Terraces 20July 2001 CLC 1578 (MONT) 13 Aug 2007 CLC 2319(MONT) 17 Aug 2013 EB 105-13 (MONT)

Other specimens examined DENMARK Broby Vesterskov 16Oct 1997 JHC97-251 (L aurantiacus C) NORWAY Leikan-ger 11 Sept 1999 JHC00-057 (L aurantiacus C) Nordland14 Aug 1999 JV15112F (L cf aurantiacus originally identi-fied as L lanceolatus TURA) Svalbard Longyearbyen 18Aug 2003 CLC 1885 (L cf aurantiacus originally identifiedas L lanceolatus MONT) SWEDEN Uppland 8 Sept 1994JV94-422 (L aurantiacus C) USA ALASKA Kenai PeninsulaCounty Palmer Creek Road 7 Aug 2011 CLC 2743 (L cfaurantiacus originally identified as L lanceolatus MONT)COLORADO El Paso County 27 Aug 2011 DBG-F-022653(L luculentus v laetus DBG) Gilpin County Perigo 13 Aug1974 AH Smith 84923 (Paratype of L luculentus v laetus

MICH) Larimer County 23 Aug 2008 DBG-F-024643 (Lluculentus v laetus DBG) OREGON Polk County 14 Nov1970 AH Smith 79943 (L luculentus MICH) WASHING-TON Lewis County 30 Oct 1972 AH Smith 83066 (Paratypeof L subviscidus MICH) 1 Nov 1972 AH Smith 83331 (Para-type of L subviscidus MICH) Tucannon River 11 Oct1980 AH Smith 90905 (L luculentus MICH)

Comments Lactarius lanceolatus belongs to a taxono-mically problematic group of species with orangeorange-brown or orange-red pilei including L auran-tiacus L luculentus L luculentus v laetus L subflammeusand L substriatus

Traditionally L lanceolatus and L aurantiacus havebeen separated mainly by ecology with L aurantiacusbeing restricted to a variety of subalpine habitats(although it has been reported from arctic-alpine habi-tats) and L lanceolatus to arctic-alpine habitats withSalix spp however it is clear from phylogenetic ana-lyses (FIG 3) that L aurantiacus also may occur in arc-tic-alpine habitats likely associating with Salix as well asKobresia and that the ranges of the two taxa overlap atleast in arctic-alpine areas of Alaska and Svalbard Ithas been suggested that L lanceolatus has longermacrocystidia and larger basidiospores than L auran-tiacus (eg Heilmann-Clausen et al 1998) howevercollections designated as L aurantiacus measured inthis study have pleuromacrocystidia that overlap andactually exceed the upper limit measured for L lanceo-latus including the holotype (see Barge 2015) Basi-diospore sizes for L aurantiacus also overlap andactually exceed the upper limit measured for L lanceo-latus in the collections examined (Barge 2015) Overallsolid morphological differences between the two taxawere not detected and further study may reveal thatthey are conspecific however provisionally L lanceola-tus may tend to have a thicker gelatinous layer over thepileipellis as well as a smooth pileus margin in contrastto the crenulate pileus margin reported for L aurantia-cus Lactarius aurantiacus is not reported from theRocky Mountains

For North American taxa Lactarius luculentus has abrowner pileus a slowly acrid flavor shorter cheilo-macrocystidia (23ndash45 6 4ndash6 mm) and exsiccati arepaler and more yellow-tan (Hesler and Smith 1979)Lactarius luculentus v laetus is similar in appearanceto L lanceolatus but also has shorter cheilomacrocysti-dia (15ndash30 6 4ndash8 mm) and possibly more crowdedlamellae (Hesler and Smith 1979) Lactarius substria-tus has a redder pileus that is glutinous when wetwith a translucent-striate margin latex that slowlybecomes straw yellow shorter cheilomacrocystidiaand an acrid flavor (Hesler and Smith 1979) Lactariussubflammeus also has a redder pileus (at least whenyoung) short translucent striations along its pileusmargin shorter cheilomacrocystidia and a slowly

426 MYCOLOGIA

FIG 6 A L lanceolatus (EB105-13) B L nanus (EB106-13) C L nanus (EB138) D L glyciosmus (ZT12723) E Lpallidomarginatus Barge amp Cripps sp nov (EB0041 HOLOTYPE) F L repraesentaneus (EB107-13) G L salicis-reticulatae(EB0057-14) H L aff salicis-reticulatae (EB0036) Photographs by E Barge (A-C E-H) E Horak (D) Bars 5 2 cm

BARGE ET AL ROCKY MOUNTAIN ALPINE LACTARIUS 427

acrid taste (pers obs of holotype AH Smith 83602Hesler and Smith 1979) Molecular phylogenetic ana-lyses (FIG 3) suggest that L subflammeus is conspecificwith L substriatus however sequencing of the holo-type of L substriatus is needed to confirm this observa-tion Lactarius luculentus L luculentus v laetus Lsubflammeus and L substriatus have been reportedonly from subalpine areas with conifers in westernNorth America (Hesler and Smith 1979)

Lactarius duplicatus AH Sm (syn L lapponicus Har-maja) a North American and European Betula associ-ate is also morphologically close to L lanceolatusHowever it can be distinguished by the productionof strongly yellowing latex We did not procure anymolecular data from material representing this taxonand no molecular data is publicly available on Gen-Bank or UNITE However L duplicatus should beincluded in future phylogenetic analyses investigatingthe L lanceolatusL aurantiacus group

Lactarius nanus J Favre FIGS 6 7Short description Lactarius nanus is recognized by its

typically small size gray-brown to liver brown pileustypically with a glaucous coating at least when youngcream to pinkish tan lamellae pale cream-tan to paleapricot stipe watery unchanging latex mild odormild to slightly acrid flavor and occurrence with Salixspecies in arctic-alpine areas

Pileus 10ndash50 mm diam shallowly convex to plano-convex becoming plane to infundibuliform with orwithout a depressed center and small papilla smoothslightly viscid to dry with whitish glaucous coatingwhen immature easily rubbing away or fading in ageoften in patches deep brown to gray-brown to liverbrown with lighter and darker areas present oftenlighter toward margin becoming lighter overall upondessication margin straight when immature becomingupturned and wavy to convoluted when matureLamellae adnate to subdecurrent subdistant to slightlycrowded cream when immature becoming dingycream to light tan in age sometimes with a faint pink-ish tinge discoloring to dingy tan where damagedStipe 5ndash30 6 3ndash15 mm equal to clavate centralsmooth dry at first covered with whitish glaucous coat-ing as in pileus pale apricot to dingy cream-tan hol-low Latex scarce watery unchanging Context ofpileus and stipe dingy cream to brownish Odor mildFlavor mild to slightly acrid Basidiospore deposit notobserved Basidiospores 7ndash105(ndash12) 6 5ndash8 mm av 584 6 66 mm Q 5 107ndash160 Q 5 127 hyaline in25 KOH subglobose to ellipsoid ornamentationof amyloid scattered ridges and warts forming anincomplete reticulum with some completely reticulateareas to 07 mm high Basidia 406ndash762 6 89ndash152 mm mostly four-spored some two-spored

Pleuromacrocystidia 533ndash965(ndash1143) 6 64ndash114mm lanceolate with acute to moniliform apexscattered to abundant Cheilomacrocystidia 260ndash9146 76ndash102 mm subulate to linear with undulatingmargin with acute to rounded apex scattered toabundant Pileipellis an (ixo-) cutis with some ascend-ing hyphae embedded in a very faint gelatinouslayer

Distribution and habitat Lactarius nanus originally wasdescribed by Jules Favre from the Swiss Alps with Salixherbacea L (Favre 1955) Phylogenetic analyses includ-ing a specimen from near the type locality confirmthis species in the Rocky Mountain alpine zone whereit is one of the most commonly encountered Lactariusspecies it associates with Salix arctica S reticulata andS planifolia Overall L nanus appears to be largelyrestricted to arctic-alpine areas with Salix

Previous arctic-alpine reports include Canada (Ohe-noja and Ohenoja 1993 2010) the Rocky Mountainalpine zone (Moser and McKnight 1987 Cripps andHorak 2008 Cripps and Barge 2013) Greenland(Knudsen and Borgen 1982 Lamoure et al 1982 Heil-mann-Clausen et al 1998 Borgen et al 2006) Iceland(Eyjolfsdottir pers comm Knudsen and Vesterholt2008) the European Alps (Favre 1955 Kuumlhner 1975Kuumlhner and Lamoure 1986 Senn-Irlet 1993 Bon1985a 1998 Heilmann-Clausen et al 1998 Jamoni1995) the Pyrenees (Bon and Ballaraacute 1996) the Car-pathians (Ronikier 2008) Fennoscandia (Kuumlhner1975 Gulden et al 1985 Heilmann-Clausen et al1998 Gulden 2005 Knudsen and Vesterholt 2008)Svalbard (Gulden et al 1985) Siberia (Nezdoiminogo1997) and alpine areas in the Altai Mountains (Karaty-gin et al 1999)

Specimens examined FRANCE Savoie 24 Aug 1989 Bon89093 (LIP) NORWAY Finse 5 Aug 2010 CLC 2134(MONT) Svalbard Longyearbyen 20 Aug 2002 CLC 1896(MONT) SWEDEN Torne Lappmark 15 Aug 1999JV15148 (TURA) SWITZERLAND Grisons 20 Aug 1943ZA192c (Holotype G) USA COLORADO Pitkin CountySawatch Range Independence Pass 6 Aug 2000 CLC 1471(MONT) San Juan County San Juan Range Black BearPass 11 Aug 2001 CLC 1716 (MONT) 11 Aug 2001ZT9529 (MONT) San Juan County San Juan Range Cinna-mon Pass 27 July 2002 CLC 1801 (MONT) 15 Aug 2012EB138 (MONT) San Miguel County San Juan Range Imo-gene Pass 29 July 2002 CLC 1829 (MONT) MONTANACarbon County Beartooth Plateau Highline Trail 7 Aug1998 CLC 1221 (MONT) 8 Aug 1998 ZT6422 (MONT) 18Aug 2013 EB106-13 (MONT) WYOMING Park CountyBeartooth Plateau Frozen Lakes 21 Aug 1999 CLC 1403(MONT) Park County Beartooth Plateau Solifluction Ter-races 11 Aug 2012 EB125 (MONT)

Other specimens examined FINLAND Koillismaa 28 Aug2011 JV28432 (L hysginoides TURA)

428 MYCOLOGIA

Comments Lactarius nanus is morphologically andphylogenetically close to L hysginoides however thelatter has a more cinnamon-toned pileus smaller basi-diospores (6ndash8 6 5ndash7 mm av 68 6 57 mm) and isgenerally reported from boreal areas with Betula Piceaand Salix in Europe (Heilmann-Clausen et al 1998)although phylogenetic placement of environmentalsequences extend its range into boreal Alaska (FIG4) Its range likely overlaps that of L nanus in alpine-subalpine transition areas It is not reported from theRocky Mountains Lactarius mammosus is a more robustspecies with a coconut odor (Heilmann-Clausen et al1998) that associates with members of the Pinaceaeit has not been reported from the Rocky MountainsDifferences between L glyciosmus and L nanus are dis-cussed in the taxonomy section for L glyciosmus

Lactarius glyciosmus (Fr) Fr FIGS 6 7Short description Lactarius glyciosmus is recognized by

its small to medium size pale gray-brown to mauvepileus typically with a glaucous coating at least whenyoung cream pale pinkish or pale yellow-orangelamellae buff or pale salmon stipe watery unchan-ging latex odor of coconuts mild flavor and associa-tion with Betula

Pileus 15ndash50 mm diam broadly convex later becom-ing plane with or without a depressed center and smallpapilla smooth dry pale gray-brown to mauve (violet-brown) with whitish glaucous coating when immaturefading in age azonate to lightly zoned especially nearmargin margin incurved when young and remainingso or becoming straight when mature Lamellaeadnate to subdecurrent slightly crowded to crowdedcream to pale pinkish to pale yellow-orange Stipe10ndash40 6 3ndash12 mm equal to slightly clavate oftencurved central to eccentric smooth dry buff or palesalmon with faint glaucous coating at first as in pileusstuffed or hollow often white-mycelioid toward baseLatex scarce watery unchanging Context of pileusand stipe pale cream Odor of coconut Flavor mildBasidiospore deposit not observed Basidiospores 7ndash96 5ndash7 mm av 5 77 6 60 mm Q 5 117ndash142 Q 5

128 hyaline in 25 KOH broadly ellipsoid to ellip-soid ornamentation of amyloid scattered ridges andwarts forming an incomplete to nearly complete reticu-lum to 05 mm high Basidia 381ndash635 6 76ndash102 mmmostly four-spored some two-spored Pleuromacrocys-tidia 457ndash635 6 64ndash76 mm subclavate to lanceolatewith rounded to occasionally mucronate apex scarceto scattered Cheilomacrocystidia 33ndash66 6 51ndash89mm cylindrical to subclavate with rounded apex scat-tered to abundant Pileipellis a cutis with tufts ofascending hyphae

Distribution and habitat Lactarius glyciosmus was firstdescribed by Fries from a hemiboreal to boreal habitat

in the Femsjouml region of Sweden and the species lacks atype specimen Phylogenetic analyses including speci-mens from the type locality confirm this species inthe central and southern Rocky Mountain alpinezone and suggest it has a broad ecological and geogra-phical distribution confirmed from subalpine and arc-tic-alpine areas in Alaska western Canada IdahoWisconsin New Hampshire Svalbard and Fennoscan-dia In subalpine and alpine areas of the Rocky Moun-tains L glyciosmus associates with Betula glandulosaPhylogenetic placement (FIG 4) of ectomycorrhizalroot tip sequences confirm Betula nana L as a host inAlaska (GU998223 GU998695) and likely B occidenta-lis Hook in Idaho (JX198526 Bogar amp Kennedy2013) Elsewhere it is reported with B pubescens Ehrh(eg Elborne amp Knudsen 1990) and B papyrifera Mar-shall (eg Durall et al 2006)

Previous reports from arctic-alpine areas includeCanada (Ohenoja and Ohenoja 1993 2010) theRocky Mountain alpine zone (Cripps and Horak2008 Cripps and Barge 2013) Greenland (Lange1957 Knudsen and Borgen 1982 Lamoure et al1982 Elbourne and Knudsen 1990 Borgen 2006 Bor-gen et al 2006) Iceland (Eyjolfsdottir pers comm) theAlps (Bon 1998) Fennoscandia (Kallio and Kankainen1964 Kuumlhner 1975 Bon 1998) Svalbard (Ohenoja1971 Geml et al 2012) and Siberia (Karatygin et al1999) At least some reports might be L aff glyciosmusas defined here

Specimens examined FINLAND Fjell Ailigas 27 Aug 2012CLC 2874 (MONT) SWEDEN Femsjouml 13 Aug 1978 MMoser 19780191 (Type locality IB) 16 Aug 1978 M Moser19780234 (Type locality IB) 9 Aug 1981 M Moser19810148 (Type locality IB) USA COLORADO BoulderCounty Front Range Blue Lake 2 Aug 2001 CLC 1624(MONT) Boulder County Front Range Brainard Lake 13Aug 2000 20923 (DBG) Ouray County San Juan RangeIronton Park 13 Aug 2012 EB133 (MONT) MONTANACarbon County Beartooth Plateau Birch Site 29 July 1997CLC 1134 5 ZT6096 (MONT) 8 Aug 1998 CLC 1217(MONT) 10 Aug 1999 TWO 269 (MONT) 19 Aug 1999CLC 1380 (MONT) 13 Aug 2007 ZT12723 (MONT)

Other specimens examined ESTONIA Saare CountyLuumlmanda Commune 17 Sep 2012 TU118535 (L aff glycios-mus TU) Tartu County Votildennu Commune 12 Sept 2010TU106737 (L aff glyciosmus TU) 13 Sept 2010 TU106724(L aff glyciosmus TU)

Comments Lactarius glyciosmus is morphologically andphylogenetically similar to L mammosus however thelatter has a browner pileus stouter stature and moreelongate more reticulate basidiospores (Heilmann-Clausen et al 1998) Phylogenetic placement ofsequences isolated from ectomycorrhizal root tips ofPinus sylvestris L (AJ633116 Heinonsalo et al 2007)and Picea abies (L) Karst (FM992937 Kjoslashller andClemmensen 2009) (FIG 4) confirm that L mammosus

BARGE ET AL ROCKY MOUNTAIN ALPINE LACTARIUS 429

FIG 7 Comparison of micromorphological features of Rocky Mountain alpine specimens and Holotypes (where available)Basidiospores pleuromacrocystidia (left) and cheilomacrocystidia (right) of A Lactarius lanceolatus (CLC2319) B L lanceolatus(F 4239 HOLOTYPE) C L nanus (CLC1471) D L nanus (ZA 192c HOLOTYPE) E L pallidomarginatus Barge amp Cripps spnov (EB0041 HOLOTYPE) F L pseudouvidus (Va 71971 HOLOTYPE) G L salicis-reticulatae (CLC2776) H L salicis-reticulatae

430 MYCOLOGIA

associates with members of the Pinaceae however italso has been reported with Betula mainly in subalpineareas (Heilmann-Clausen et al 1998) Lactarius vietus(Fr) Fr is also similar but has a distinct pale zonenear the stipe apex latex that dries greenish graylonger pleuromacrocystidia and a mild to slightly fruityodor (Heilmann-Clausen et al 1998) It has not beenreported from alpine areas in the Rocky Mountainsalthough it has been reported from elsewhere (mostlynorthern areas) in North America with Betula (Heslerand Smith 1979) The similar-looking L nanus alsooccurs in alpine areas of the Rocky Mountains andhas larger basidiospores larger and differently shapedmacrocystidia lacks an odor of coconut and associateswith Salix Lactarius spinosulus is phylogenetically closeto L glyciosmus (as well as L nanus) (FIG 4) howevermorphologically they are very different Lactarius spino-sulus has a distinctly zoned coarsely squamulose pink-ish salmon- or brick-colored pileus with a finely hairymargin and fruity odor (Heilmann-Clausen et al1998) it has not been reported from North AmericaNo definitive morphological differences were detectedbetween L glyciosmus and L aff glyciosmus and the lat-ter is not reported from the Rocky Mountainalpine zone

Lactarius pallidomarginatus Barge amp CL Cripps spnov FIGS 6 7Mycobank MB812246

Typification USA COLORADO San Juan CountySan Juan Range Stony Pass scattered solitary onsoil under Salix planifolia 13 Aug 2011 EB0041 (holo-type MONT) GenBank KR090940 (ITS) andKR091010 (RPB2)

Etymology Referring to the pale pileus marginDiagnosis Lactarius pallidomarginatus is recognized by

its small to medium size blotchy light tan to lightbrown to gray-brown pileus which is typically lightertoward the margin white to pale yellow-cream lamel-lae pale cream stipe violet staining tissue mild odorand flavor and association with Salix Morphologicallyit is close to L pseudouvidus Kuumlhner but differs by hav-ing smaller basidiospores with an ornamentation ofthinner ridges as well as macrocystidia that becomemore lanceolate (as opposed to subfusiform) and atte-nuated Phylogenetically it is close to L aspideus (Fr)Fr but differs consistently in two nucleotide positionsin the rDNA ITS locus and at eight nucleotide posi-tions in the RPB2 locus morphologically L aspideushas larger yellow basidiomata

Pileus 20ndash50 mm diam convex to broadly convexbecoming broadly convex to plane with or without adepressed center smooth dry to slightly viscid azo-nate blotchy light tan to light brown to gray-browndeveloping violet stains lighter (to cream) toward mar-gin margin incurved to downturned when youngremaining downturned or becoming nearly straightwhen mature Lamellae adnate to subdecurrent subdi-stant to slightly crowded white to pale yellow-creamstaining violet where damaged Stipe 10ndash40 6 5ndash10mm equal to slightly clavate central smooth drywhite to cream staining violet where damaged hollowLatex scarce watery staining tissue violet Context ofpileus and stipe white to cream staining violet wheredamaged Odor mild Flavor mild Basidiosporedeposit not observed Basidiospores 8ndash10 6 65ndash8mm av 5 87 6 71 mm Q 5 114ndash136 Q5 123 hya-line in 25 KOH broadly ellipsoid to ellipsoid orna-mentation amyloid of thin ridges and a few isolatedwarts forming an incomplete to dense reticulum to05 mm high Basidia 508ndash711 6 102ndash127 mm four-spored Pleuromacrocystidia 813ndash1118 6 89ndash102mm cylindrical to lanceolate with acute to moniliformapex scarce to scattered Cheilomacrocystidia 483ndash1016 6 76ndash127 mm cylindrical to lanceolate withacute to moniliform apex scattered Pileipellis an(ixo-)cutis with some ascending hyphae embedded ina faint gelatinous layer

Distribution and habitat Rare in low alpine areas withSalix planifolia reported from Independence Pass inthe Sawatch Range and Stony Pass in the San JuanRange in Colorado An additional collection(ZT5229) from Union Peak in the Wind River Rangeof Wyoming with Salix glauca is likely conspecific how-ever this hypothesis has not been confirmed withmolecular data Due to confusion with L pseudouvidusit is possible that L pallidomarginatus is more wide-spread than confirmed here

Specimens examined USA COLORADO Pitkin CountySawatch Range Independence Pass 6 Aug 2000 CLC 1470(MONT) 9 Aug 2000 ZT9093 (MONT) San Juan CountySan Juan Range Stony Pass 13 Aug 2011 EB0041 (HolotypeMONT) WYOMING Sublette County Wind River RangeUnion Peak 22 Aug 1994 ZT5229 (MONT)

Other specimens examined AUSTRIA Tyrol 3 July 2007 UPeintner 20070035 (L aff pseudouvidus from near type local-ity IB) FINLAND Inarin Lappi 16 Aug 1995 JV10468 (Laff pseudouvidus TURA) Koillismaa JV28448F 30 Aug 2011(L aff pseudouvidus originally identified as L brunneoviola-ceus TURA) FRANCE Isegravere 13 Aug 1971 Va 7 1971 (Holo-type of L pseudouvidus G) ITALY Trentino 20 Aug 2004 U

r(K72-104 HOLOTYPE no cheilocystidia drawn) I L glyciosmus (TWO269) J L repraesentaneus (CLC1747) Bars 5 10 mm forbasidiospores and 20 mm for cystidia Drawings by E Barge

BARGE ET AL ROCKY MOUNTAIN ALPINE LACTARIUS 431

Peintner 20040156 (L aff pseudouvidus from near type local-ity originally identified as L robertianus IB) NORWAYFinse 10 Aug 2005 CLC 2133 (Lactarius aff brunneoviolaceusMONT) Stordal 26 Aug 2006 P Larsen 361395 (L aff brun-neoviolaceus originally identified as L pseudouvidus O) Sval-bard 18 Aug 2002 TWO809 (L aff pseudouvidus MONT)20 Aug 2002 CLC 1910 (Lactarius sp 5 MONT) Ulvik 11Aug 2011 E Soslashyland 73867 (L aff pseudouvidus O) SWE-DEN Latnjajaure 4 Aug 2011 EL101-11 (L aff pseudouvidusGB) Latnjavagge 17 Aug 2010 EL63-10 (L aff pseudouvi-dus GB)

Comments Morphological features of L pallidomar-ginatus fit the holotype description of L pseudouvidusand both are described as lighter near the pileus mar-gin (Kuumlhner 1975 Basso 1999) however basidios-pores of L pallidomarginatus are slightly smaller (8ndash10 6 65ndash8 mm as opposed to 75ndash12 6 65ndash8(85)mm) and have narrower ridges that form a less densereticulum and macrocystidia take on a more lanceo-late form with greater attenuation of the apex (FIG7) In phylogenetic analyses exsiccati identified as Lpseudouvidus from Europe (including specimensfrom near the type locality) appear as distinct fromRocky Mountain alpine material and may representa species complex Lactarius sp 3 (CLC1910) col-lected in Svalbard has darker more vinaceous pileithan L pallidomarginatus and may represent anotherundescribed species although only one collectionwas morphologically examined Members of the Laff brunneoviolaceus clade (FIG 5) that were exam-ined differ from L pallidomarginatus in having dar-ker more vinaceous pilei larger more elongatebasidiospores (to 13 mm) and green staining of exsic-cati in 25 KOH a feature not mentioned previouslyfor L brunneoviolaceus but which could be of taxo-nomic interest Based on taxa included in the phylo-genetic analyses (FIG 5) Lactarius aspideus appearsclosely related to L pallidomarginatus however theformer has more yellow coloration and typically lar-ger sporocarps (pileus 10ndash70 mm diam) and smallerbasidiospores (67ndash95 6 56ndash78 mm) traditionallyL aspideus has been placed with other yellow violet-staining taxa such as L flavidus Boud L salicis-reticu-latae and L salicis-herbaceae (Heilmann-Clausen et al1998) but it is separated here

Lactarius montanus another violet-staining speciesoriginally described from the Rocky Mountains dif-fers from L pallidomarginatus by its larger staturedarker grayish vinaceous to vinaceous-brown pileusand green staining flesh in 25 KOH it is typicallyfound in subalpine habitats with conifers but alsooccurs in the krummholz zone The violet-stainingLactarius pallescens Hesler amp AH Sm occurs in theRocky Mountains but its sporocarps are larger andlighter in color and it is reported from subalpineconifer forests (Hesler and Smith 1979) Other

gray-brown to brownish violet-staining species suchas L luridus L uvidus (Fr) Fr L violascens (JOtto) Fr L pallescens v palmerensis Hesler amp AHSm and L cordovaensis Hesler amp AH Sm are alsolarger and have a subalpine ecology none havebeen reported from the central or southern RockyMountains (Hesler and Smith 1979 Heilmann-Clau-sen et al 1998) Lactarius cistophilus Bon amp Trimbachtypically has larger basidiomata larger basidiospores(10ndash14 6 72ndash9 mm) and occurs under Cistus inthe Mediterranean region (Basso 1999) The smallpale gray-brown European alpine taxon L uvidus falpigines Jamoni amp Bon has microscopic featuresthat match those of L uvidus (Jamoni and Bon1991) but not L pallidomarginatus The small non-violet staining L nanus has basidiospores withthicker more jagged ridges and differently shapedmacrocystidia (often with more rounded apices)(FIG 7)

Lactarius repraesentaneus Britzelm FIGS 6 7Short description Lactarius repraesentaneus is recog-

nized by its medium to large size orange-brown to yel-low-brown hairy pileus with a heavily bearded margincream to pale-yellow lamellae stout cream to pale-yel-low often pitted stipe violet staining latex and tissuespicy-floral odor slightly acrid flavor and associationwith Betula Picea and possibly shrubby Salix speciessuch as S glauca

Pileus 60ndash100 mm diam convex to plano-convexusually with a depressed center and sometimes with apapilla smooth at center becoming increasingly hairyto bearded toward margin dry to viscid azonate sur-face orange-brown at center becoming pale yellow-brown to cream toward margin hairs pale yellow-brown to red-brown margin strongly incurvedremaining so or becoming nearly straight whenmature Lamellae adnate to decurrent crowdedcream to pale-yellow staining violet where damagedStipe 30ndash70 6 15ndash35 mm stout equal to clavate cen-tral dry to viscid cream to pale yellow surface oftenwith numerous dingy yellow golden yellow yellow-brown or light orange-brown pits hollow Latex scarceto abundant white becoming violet Context of pileusand stipe white staining violet where damaged Odorspicy-floral Flavor slightly acrid Basidiospore depositnot observed Basidiospores 8ndash105 6 6ndash85 mm av5 93 6 73 mm Q 5 113ndash143 Q5 127 hyaline in25 KOH broadly ellipsoid to ellipsoid ornamenta-tion amyloid of narrow to broad ridges and isolatedwarts forming an incomplete to dense reticulum to1 mm high Basidia 610ndash762 6 102ndash114 mm four-spored Pleuromacrocystidia 787ndash1448 6 89ndash127 mm subfusiform to lanceolate thin-walled withacute to moniliform apex scattered to abundant

432 MYCOLOGIA

Cheilomacrocystidia 559ndash1397 6 76ndash127 mm subfu-siform to lanceolate thin-walled with acute to monili-form apex scattered to abundant Pileipellis anixotrichoderm with well-developed gelatinous layer

Distribution and habitat Lactarius repraesentaneus firstwas described from Germany (Britzelmayr 1885) Phy-logenetic analyses including material from Germanyconfirm L repraesentaneus from alpine areas of theRocky Mountains and overall reinforce the traditionalconcept that this species has a broad ecological andgeographic range in arctic-alpine and subalpine habi-tats in North America northern central and southernEurope In alpine areas of the Rocky Mountains itassociates with Salix glauca however krummholz Piceaalso is typically (maybe always) present it is reportedwith Picea and Betula elsewhere

Previous reports from arctic-alpine areas mainlywith Betula include Alaska (Laursen and Ammirati1982 Geml et al 2009) the Rocky Mountain alpinezone (Cripps and Horak 2008 Cripps and Barge2013) Greenland (Knudsen and Borgen 1982 Watling1983 Elbourne and Knudsen 1990 Borgen 2006 Bor-gen et al 2006) Iceland (Eyjolfsdottir pers comm)Fennoscandia (Kallio and Kankainen 1964 Kuumlhner1975 Bon 1998) Siberia (Karatygin et al 1999) andthe Altai Mountains (Gorbunova 2010)

Specimens examined FINLAND Enontakiouml 16 Aug 2004JV21671 (TURA) Varsinais-Suomi 9 Aug 1998 JV13837F(TURA) SWEDEN Latnjavagge 12 Aug 2007 EL92-07(GB) USA COLORADO Chaffee County Sawatch RangeCottonwood Pass 4 Aug 2001 CLC 1643 (MONT) 16 Aug2011 EB0048 (MONT) Pitkin County Sawatch Range Inde-pendence Pass 14 Aug 2001 CLC 1747 (MONT) ZT9837(MONT) 15 Aug 2001 ZT9537 (MONT) MONTANA Car-bon County Beartooth Plateau Birch Site 12 Aug 2002CLC 1971 (MONT) 13 Aug 2007 CLC 2318 (MONT) 18Aug 2013 EB107-13 (MONT) WYOMING Park CountyBeartooth Plateau Frozen Lakes 21 Aug 1999 CLC1394 (MONT)

Other specimens examined SWEDEN Latnjavagge 14 Aug2010 EL57-10 (L dryadophilus GB) USA ALASKA 5 Aug2011 CLC 2729 (L dryadophilus MONT) Kenai PeninsulaCounty Palmer Creek Road 7 Aug 2011 CLC 2744 (L drya-dophilus MONT)

Comments The combination of a large pale yellow-brown to orange-brown pileus with a bearded marginand violet staining latex clearly separates L repraesenta-neus from other Lactarius species of the central andsouthern Rocky Mountain alpine zone Phylogeneti-cally close the arctic-alpine taxon L dryadophilus ismorphologically similar but has a cream to yellowsmoother pileus with hairs restricted to near the mar-gin basidiospore ornamentation consisting of sparsethinner ridges and ecological associations with Dryasand Salix It has not been reported from the RockyMountains

Lactarius salicis-reticulatae Kuumlhner FIGS 6 7Short description Lactarius salicis-reticulatae is recog-

nized by its small to medium cream pale yellow orpale ocher sporocarps with pale yellowish well-spacedlamellae often with a pinkish buff tint cream to pale yel-low stipe violet staining latex and tissue mild to slightlysweet odor mild flavor and association with dwarf (Sreticulata) and possibly also shrubby Salix species

Pileus 20ndash40 mm diam convex to broadly convexusually with a depressed center smooth dry to viscidazonate cream pale yellow or pale ocher sometimesdarker toward center staining violet where damagedmargin invcurved remaining so or becoming down-turned when mature Lamellae adnate to subdecur-rent fairly well-spaced cream pale yellow or paleorange-ocher often with a pinkish-buff tint stainingviolet where damaged Stipe 15ndash20 6 10ndash15 mmequal to clavate central dry to viscid cream to pale-yellow staining violet where damaged hollow Latexscarce watery-white becoming violet Context ofpileus and stipe white staining violet where damagedOdor mild to slightly sweet Flavor mild Basidiosporedeposit not observed Basidiospores (7ndash)85ndash115 6(7ndash)8ndash10 mm av 5 100 6 88 mm Q 5 (100ndash)113ndash143 Q 5 125 hyaline in 25 KOH sublgobose toellipsoid ornamentation amyloid of narrow ridgesand a few isolated warts forming an incomplete reticu-lum to 03 mm high Basidia 737ndash889 6 102ndash127 mm four-spored Pleuromacrocystidia 762ndash10166 76ndash114 mm subfusiform to fusiform thin-walledwith rounded to acute to moniliform apex scarceCheilomacrocystidia 686ndash9146 76ndash102 mm subfusi-form to fusiform thin-walled with acute to rounded tomoniliform apex scattered to abundant Pileipellis anixocutis

Distribution and habitat Lactarius salicis-reticulatae firstwas described from alpine tundra with Salix on MtLaringktatjaringkka Sweden (Kuumlhner 1975) Phylogenetic ana-lyses including material from the type locality confirmthis species from alpine areas on the Beartooth Plateauwith Salix reticulata and shrubby Salix spp and in arctic-alpine areas of Alaska Svalbard and Finland OverallL salicis-reticulatae appears largely confined to arctic-alpine habitats Colorado collections with S reticulataand S planifolia may represent a distinct taxonalthough support is low

Previous arctic-alpine reports include Greenland(Lamoure et al 1982 Heilmann-Clausen et al1998 Borgen et al 2006) the Rocky Mountainalpine zone (Cripps and Horak 2008 Cripps andBarge 2013) the Alps (Kuumlhner 1975 Kuumlhner andLamoure 1986 Jamoni 1995 2008 Bon 1998 Heil-mann-Clausen et al 1998) the Pyrenees (Ballaraacute1997) Fennoscandia (Kuumlhner 1975 Heilmann-Clau-sen et al 1998 Gulden 2005 Knudsen and

BARGE ET AL ROCKY MOUNTAIN ALPINE LACTARIUS 433

Vesterholt 2008) and Siberia (Nezdoiminogo 1997Karatygin et al 1999) previous reports of L aspi-deoides Burl from arctic-alpine areas in Alaska (Laur-sen and Ammirati 1982) Greenland (Knudsen andBorgen 1982) and the British Isles (Watling 1987)may refer to this species

Specimens examined FINLAND Near Utsjoki 28 Aug 2012CLC 2885 (MONT) SWEDEN Mt Laringktatjaringkka 12 Aug1972 K72-104 (Holotype G) 15 Aug 1999 JV15133 (Typelocality TURA) USA ALASKA Kenai Peninsula County Pal-mer Creek Road 7 Aug 2011 CLC 2745 (MONT) MON-TANA Carbon County Beartooth Plateau Birch Site 17Aug 2011 CLC 2776 (MONT) Carbon County BeartoothPlateau Highline Trail 8 Aug 1998 CLC 1211 (MONT)WYOMING Park County Beartooth Plateau GardnerLake 16 Aug 2014 EB0057-14 (MONT)

Other specimens examined FINLAND Koillismaa 31 Aug2005 JV23334 (L flavopalustris TURA) GREENLAND Sis-miut 19 Aug 2000 CLC 1536 (L salicis-herbaceae MONT)USA COLORADO Pitkin County Sawatch Range Indepen-dence Pass 13 Aug 2001 CLC 1741 (L aff salicis-reticulataeMONT) San Juan County San Juan Range CinnamonPass 10 Aug 2001 CLC 1710 (L aff salicis-reticulataeMONT) San Juan County San Juan Range Maggie Gulch11 Aug 2011 EB0036 (L aff salicis-reticulatae MONT)San Juan County San Juan Range Stony Pass 9 Aug 2001CLC 1689 (L aff salicis-reticulatae MONT) 13 Aug 2011EB0039 (L aff salicis-reticulatae MONT) MICHIGANEmmet County 10 Aug 1973 RL Shaffer 6957 (L aspi-deoides MICH)

Comments Some taxonomic confusion has beencaused by the fact that Kuumlhner (1975) originallynamed this species L aspideoides Kuumlhner but subse-quently renamed it because the name had been pre-viously applied L aspideoides Burl (1907) is generallylarger with a somewhat zonate pileus more crowdedlamellae that lack salmon-colored hues slightly smallerbasidiospores with broader ridges a bitter to slightlyacrid flavor and a subalpine ecology apparently ineastern and western North America (pers obs of RLShaffer 6957 Hesler and Smith 1979) Lactarius fla-vopalustris is also similar however it has a largermore brightly yellow pileus with faint zonationmore crowded lamellae a weakly scrobiculate stipe(sometimes) smaller basidiospores with thickerridges and a subalpine ecology (Kytoumlvuori 2009)Phylogenetic analyses (FIG 5) reveal three collec-tions of L flavopalustris (two from near the typelocality in Finland) nested in a well-supported cladewith L aspideoides from a subalpine forest in north-ern Michigan suggesting the possible conspecificityof these taxa Neither is reported from the RockyMountains Two other collections identified as L fla-vopalustris (from Estonia) were resolved as distinctfrom the L aspideoidesflavopalustris clade (FIG 5)their identity is unclear

Lactarius aspideus is differentiated from L salicis-reti-culate by its smaller basidiospores with denser reticula-tion more crowded creamier-colored lamellae andhabitat mainly in subalpine areas (Heilmann-Clausenet al 1998) although an environmental sequence iso-lated from an S polaris root tip from arctic Svalbardclusters with L aspideus in phylogenetic analyses (FIG5) Lactarius dryadophilus is often larger with a cream-colored pileus that has a tomentose to bearded margin(Heilmann-Clausen et al 1998) Lactarius flavidus is lar-ger with a sparsely zonate pileus more crowdedcream-colored lamellae darker violet-staining contextbasidiospores with more prominent ornamentationand a distally amyloid plage and subalpine ecology(Heilmann-Clausen et al 1998) Lactarius flavoaspideusKytoumlv has slightly smaller basidiospores on averagewith a denser reticulum and subalpine ecology withSalix (Kytoumlvuori 2009) it has been reported onlyfrom northern Europe Lactarius salicis-herbaceae asimilar arctic-alpine Salix associate has smaller (typi-cally) darker yellow to yellow-brown sporocarpspale cream to grayish buff lamellae and more denselyreticulate basidiospores (pers obs Heilmann-Clausenet al 1998) It is fairly widespread in arctic-alpineareas but is not reported from alpine areas in the cen-tral and southern Rocky Mountains Lactarius aff sali-cis-reticulatae from Colorado may be a distinct taxonand sporocarps can obtain a larger size (pileus to 90mm diam) but otherwise morphological featuresstrongly overlap

KEY TO ROCKY MOUNTAIN ALPINE LACTARIUS SPECIES

1 Latex white turning violet to dull lilac or flesh stain-ing violet to dull lilac 2

1 Latex watery to white unchanging and flesh notstaining violet to dull lilac 42 Fruiting body robust pileus 60ndash100 mm diam

yellow-brown to orange-brown hairy especiallynear margin lamellae cream to pale-yellow stipestout cream to pale-yellow often with numerousyellow-brown pits odor spicy-floral flavor mildlyacrid basidiospores 8ndash105 6 6ndash85 mm puta-tively associated with Betula and Picea (possiblyalso Salix) in Rocky Mountain alpine zone andsubalpine and arctic-alpine areas in North Amer-ica and Eurasia L repraesentaneus

2 Fruiting body smaller 20ndash50 mm diam pileus andmargin smooth 3

3 Pileus pale yellow to pale ocher lamellae cream paleyellow or pale orange-ocher stipe cream to pale yel-low odor mild to slightly sweet flavor mild basidios-pores 85ndash115 6 8ndash10 mm in Rocky Mountain alpinezone and other arctic-alpine areas in North Americaand Eurasia with Salix L salicis-reticulatae

3 Pileus blotchy light tan to light brown to gray-brownmargin lighter lamellae white to pale yellow-cream

434 MYCOLOGIA

stipe white to cream odor mild flavor mild basi-

diospores 8ndash10 6 65ndash8 mm in low alpine areas inColorado with Salix planifolia and possibly Wyom-

ing with Salix glauca L pallidomarginatus4 Pileus bright orange to orange-brown 10ndash45

mm diam margin smooth lamellae pale creamto pale orange stipe pale orange odor mildflavor mild in Rocky Mountain alpine zonearctic-alpine Alaska (possibly also subalpine)to Svalbard with Salix L lanceolatus

4 Pileus pinkish buff gray-brown brown ormauve 5

5 With odor of coconuts pileus 15ndash50 mm diam palepinkish buff to pale gray-brown with glaucous coat-ing lamellae cream to pale yellow-orange stipe buffor pale salmon flavor mild basidiospores 7ndash9 6 5ndash7mm in Rocky Mountain alpine zone and subalpinearctic-alpine areas in North America and Eurasiawith Betula L glyciosmus

5 Without odor of coconuts pileus 10ndash50 mm diamgray-brown to liver-brown with whitish glaucous coat-ing lamellae cream to light tan sometimes with pink-ish tinge discoloring to dingy tan where damagedStipe pale apricot to dingy cream-tan flavor mild toslightly acrid basidiospores 7ndash105 6 5ndash8 mm inRocky Mountain alpine zone and arctic-alpine areasin Eurasia and likely other arctic-alpine areas in NorthAmerica with Salix L nanus

DISCUSSION

The number of Lactarius species reported from theRocky Mountain alpine zone represents only a fractionof the total number of Lactarius species reported else-where from arctic-alpine areas however nearly halfof the core arctic-alpine species are now confirmedfor continental North America Core species oftencited as being restricted to arctic-alpine areas withdwarf and shrubby Salix andor Dryas species includeL brunneoviolaceus L dryadophilus L lanceolatus Lnanus L pseudouvidus L salicis-herbaceae and L salicis-reticulatae (Knudsen and Borgen 1982 Heilmann-Clau-sen et al 1998 Basso 1999 Knudsen and Vesterholt2008) Phylogenetic analyses and morphological exam-ination confirm three out of seven of these species forthe Rocky Mountain alpine zone (L lanceolatus Lnanus L salicis-reticulatae) and support the hypothesisthat these species are largely restricted to arctic-alpineareas with Salix One definitive new species L pallido-marginatus (with Salix) was revealed along with a possi-ble cryptic species L aff salicis-reticulatae (also withSalix) Many Lactarius species previously reportedfrom arctic-alpine areas are also commonly reportedfrom subalpine areas with Betulaceae andor Pinaceae

as well as with other plant families (Heilmann-Clausenet al 1998) From this set of species we report L gly-ciosmus which associates with Betula glandulosa in theRocky Mountain alpine zone and Betula spp acrossthe rest of its distribution and L repraesentaneus whichis commonly reported with Betula and Picea (Heslerand Smith 1979 Heilmann-Clausen et al 1998) andappears to associate with Salix glauca andor perhapskrummholz Picea in the Rocky Mountain alpine zone

Several taxa of interest were not molecularly distinctIn dataset A L lanceolatus did not form a monophyleticgroup but was paraphyletic with respect to Lactarius sp2 the L luculentus group and L aurantiacus In datasetB L nanus and L hysginoides were not clearly distinctIn dataset C L salicis-reticulatae did not form a clearmonophyletic group and L pseudouvidus L brunneovio-laceus and L luridus were not clearly distinguishedfrom each other especially at the RPB2 locus poormolecular resolution also has been encountered forthe latter three taxa by Das et al (2015)

Overall there was little genetic variation distinguish-ing closely related species in this study These findingsare similar to reports from other researchers who sug-gest that there is in general relatively little molecularvariation yet high morphological variation in genusLactarius (Verbeken and Nuytinck 2013) While manyof the species could be morphologically distinguishedall of the Rocky Mountain alpine taxa delineated inthis study are 97 or more similar at the ITS regionto one or more species For example L repraesentaneusand L dryadophilus are as much as 994 similar L sal-icis-reticulatae is approximately 98 similar to L salicis-herbaceae and L flavopalustris L pallidomarginatus isapproximately 97ndash98 similar to L aff brunneoviola-ceus L luridus L montanus and members of the Laff pseudouvidus group and approximately 99 similarto L aspideus L glyciosmus is approximately 98 simi-lar to L mammosus L nanus is approximately 98similar to L hysginoides and L lanceolatus is approxi-mately 97 similar to L substriatussubflammeus L aur-antiacus L luculentus v laetus and Lactarius sp 1 and 2and approximately 98 similar to L luculentus Thishigh degree of similarity could be problematic forenvironmental sequencing studies that use a strict97 ITS region similarity cutoff for recognizing spe-cies (eg Geml et al 2012 Timling et al 2012 2014)In addition Tedersoo et al (2014) suggested that theITS region in the sebacina lineage evolves moreslowly in high latitude areas which could be true forother genera The RPB2 locus proved better for differ-entiating some species and overall phylogenetic ana-lyses revealed several taxonomic issues that we werenot able to resolve A search for additional phylogen-etically informative loci for Lactarius should beinitiated along with more species-level phylogenetic

BARGE ET AL ROCKY MOUNTAIN ALPINE LACTARIUS 435

studies to provide a high quality molecular databasefor ecological studies Furthermore improvement ofmethods for sequencing older material such as typespecimens is critical

Most of the Rocky Mountain alpine Lactarius speciesdelineated in this study (except L pallidomarginatusand L aff salicis-reticulatae for which further studycould reveal broader ranges) appear to have broadintercontinental distributions in North America andEurasia The current distributions of these specieslikely were shaped by a number of biotic and abioticfactors that include host dispersal and distribution cli-mate glaciation and geography Because the genusLactarius is obligately ectomycorrhizal it is likely thatthe distribution of each fungal species was shaped lar-gely by the distribution of suitable host plants manyof which also have broad intercontinental distribu-tions Long-distance dispersal is hypothesized to haveplayed an important role in structuring present distri-bution patterns of arctic-alpine plants although manyappear clonal (Abbott and Brochmann 2003 Alsoset al 2007) and ectomycorrhizal fungi (Geml et al2012 Timling et al 2014) It also has been suggestedthat long-distance dispersal mechanisms have beenselected for in arctic flora due to repeated glaciationsand the need to track potential niches (Alsos et al2007) Pollen data from Blacktail Pond YellowstoneNational Park suggest that montane mid-elevationsin the northern greater Yellowstone ecosystem (Mon-tana Wyoming) were occupied by alpine tundra(Betula Salix many herbaceous taxa) similar to thatof the present Rocky Mountain alpine vegetation dur-ing the last glacial period (14 000ndash11 500 calibratedyears before present) (Krause and Whitlock 2013)Alpine vegetation also appears to have dominatedareas in the southern Rocky Mountains (WyomingColorado) 17 500ndash12 600 calibrated years before pre-sent (Minckley 2014) Both studies suggest the pre-sence of broad low-elevation glacial refugia for arctic-alpine life in the Rocky Mountains along the glacialfront and along with this study lend support to theidea that arctic-alpine Lactarius species (and manyother ectomycorrhizal fungi) may have occupied tun-dra areas south of the icesheets during cooler weatherduring the last glacial maximum and then dispersedupward in elevation and northward as the glaciersand ectomycorrhizal host plants retreated Broaderecological tolerances of taxa such as L glyciosmus andL repraesentaneus which are not restricted to arctic-alpine habitats likely let these species persist in tundraand non-tundra areas during the last glacial maximumas well as to track arctic-alpine vegetation as it movedupward in elevation and northward following theretreat of the icesheets For these species to haveachieved and maintained such broad distributions

gene flow must have been possible over long distancesduring the Pleistocene perhaps mediated by long-dis-tance dispersal (Geml et al 2012) range shifts due toclimate change and glacial expansion and retreat(Birks 2008) glacial and interglacial refugia (Billings1974) and the Bering land bridge (Shapiro et al2004 Geml et al 2006)

ACKNOWLEDGMENTS

We thank Dr Ellen Larsson Dr Pierre-Arthur Moreau JukkaVauras Dr Ursula Peintner Dr Egon Horak and herbaria GIB TU O GB TURA C LIP MICH DBG VPI and NY forloan of specimens Carol Johnson and Dr Matt Lavin areacknowledged for reviewing early stages of the manuscriptWe thank Don Bachman Marcy Barge and Charissa Bujakfor assistance with field work We also gratefully acknowledgesupport from the Montana Institute on Ecosystems graduateenhancement fund

LITERATURE CITED

Abbott RJ Brochmann C 2003 History and evolution of thearctic flora in the footsteps of Eric Hulteacuten Mol Ecol12299ndash313 doi101046j1365-294X200301731x

Alsos IG Eidesen PB Ehrich D Skrede I Westergaard KJacobsen GH Landvik JY Taberlet P Brochmann C2007 Frequent long-distance plant colonization in thechanging arctic Science 3161606ndash1609 doi101126science1139178

Ballaraacute J 1997 Nou estudi drsquoespegravecies fuacutengiques interessantsdels estatges alpiacute i subalpiacute dels Pirineus catalans RevCatalana Micol 201ndash24

Barge E 2015 Systematics of Lactarius in the Rocky Moun-tain alpine zone (masterrsquos thesis) Bozeman MontanaMontana State University 265 p

Basso MT 1999 Lactarius Pers Fungi Europaei 7 AlassioMykoflora (Italy) 845 p

Beker HJ Eberhardt U Vesterholt J 2010 Hebeloma hiemaleBres In arcticalpine habitats N Am Fungi 551ndash65

Bessette AE Harris DB Bessette AR 2009 Milk mushroomsof North America a field identification guide to thegenus Lactarius Syracuse New York Syracuse UniversityPress 297 p

Billings WD 1974 Adaptations and origins of alpine plantsArctic Alpine Res 6129ndash142 doi1023071550081

mdashmdashmdash 1988 Alpine vegetation In Barbour MG BillingsWD eds North American terrestrial vegetation NewYork Cambridge University Press p 392ndash420

Birks HH 2008 The late-quaternary history of arctic andalpine plants Plant Ecol Divers 1135ndash146 doi10108017550870802328652

Bogar LM Kennedy PG 2013 New wrinkles in an old para-digm neighborhood effects can modify the structureand specificity of Alnus-associated ectomycorrhizal fun-gal communities FEMS Microbiol Ecol 83767ndash777doi1011111574-694112032

Bon M 1985a Stage mycologie alpine Lanslebourg (Savoie)du 1 aug 3 sep 1984 Bull Feacuted Mycol Daupineacute-Savoie9619ndash25

436 MYCOLOGIA

pseudouvidus holotype was unsuccessful and cryptictaxa may be present Collections matching the mor-phological concept of the similar arctic-alpine taxonL brunneoviolaceus (syn L robertianus Bon) occur in a

moderately supported clade (labeled L aff brunneovio-laceus)in the combined (FIG 5) and ITS analyses (SUPPLEMEN-

TARY FIG 9) but these are paraphyletic with the L

FIG 4 Combined (ITS+RPB2) Bayesian 50 majority-rule consensus tree of dataset B (Lactarius subgenus Piperites pp)Support values (PAUP bootstrapRAxML bootstrapBayesian posterior probability) $ 60 (bootstrap) and $ 080 (posteriorprobability) are indicated above or below branches leading up to clades Thickened branches lead to clades receiving $ 75bootstrap support and Bayesian posterior probabilities $ 095 Boldface tip labels represent Rocky Mountain alpine specimensAA 5 arctic-alpine

BARGE ET AL ROCKY MOUNTAIN ALPINE LACTARIUS 423

FIG 5 Combined (ITS+RPB2) Bayesian 50 majority-rule consensus tree of dataset C (Lactarius subgenus Piperites sectionUvidi) Support values (PAUP bootstrapRAxML bootstrapBayesian posterior probability) $ 60 (bootstrap) and $ 080(posterior probability) are indicated above or below branches leading up to clades Thickened branches lead to clades receiving

424 MYCOLOGIA

pseudouvidus group in RPB2 analyses (SUPPLEMENTARY

FIG 10) Collections of Lactarius pallidomarginatus Lac-tarius sp 3 L aspideus L montanus L luridus L affpseudouvidus and L aff brunneoviolaceus for which ITSdata are available all have a similar 268ndash269 base-pairinsertion in the ITS1 region There is variation withinthe base-pair content of the insertion between collec-tions however inclusion of this insertion did notincrease phylogenetic resolution in preliminary ana-lyses and it therefore was omitted The insertion itselfis highly similar to an insertion in Russula olivacea (eg GenBank accessions AF418634 AY061699) as tolength sequence (approximately 90 similar) andplacement within the ITS1 region

Also in dataset C Rocky Mountain alpine collectionsinitially identified as L repraesentaneus form a well-supported clade with a collection of L repraesentaneusfrom Germany (the lectotype country) as well as withnumerous collections from subalpine and arctic-alpineareas elsewhere in Eurasia and Alaska (FIG 5) Thisclade is sister to a poorly supported L dryadophilusKuumlhner clade The two GenBank sequences JX630967from arctic Canada with Dryas and JF394416 from arcticSvalbard which occur just outside the L dryadophilusclade are likely conspecific with L dryadophilusalthough this cannot be confirmed based on presentresults Lactarius repraesentaneus and L dryadophilus con-sistently differ at two nucleotide positions in the ITSlocus and at four positions in the RPB2 locus and haveconsistent morphological and host differences

In dataset C collections originally identified as L sal-icis-reticulatae from alpine areas in the Rocky Mountainsare paraphyletic with regard to L salicis-herbaceaeKuumlhner L aspideoides BurlL flavopalustris Kytoumlvand L aff flavopalustris (FIG 5) Collections of Lsalicis-reticulatae from alpine areas on the BeartoothPlateau (central Rocky Mountains) form a poorlysupported clade with an alpine collection of L salicis-reticulatae from Mt Laringktatjaringkka Sweden the type local-ity (sequencing of the holotype of L salicis-reticulataewas not successful) as well as with other arctic-alpinecollections of L salicis-reticulatae from Finland andAlaska and an unidentified collection from Svalbard(FIG 5) this clade is hypothesized to represent Lsalicis-reticulatae Collections from southern RockyMountain alpine areas in Colorado are larger thanreported for L salicis-reticulatae and form an unre-solved group that consistently differs at one nucleotideposition in the ITS region but no positions in the RPB2

locus they are tentatively referred to as L aff salicis-reticulatae pending data from additional loci

Overall phylogenetic analyses support the recogni-tion of six to seven Lactarius species from the RockyMountain alpine zone Lactarius lanceolatus (FIG 3)L nanus L glyciosmus (FIG 4) L pallidomarginatus spnov L repraesentaneus L salicis-reticulatae and possiblyL aff salicis-reticulatae (FIG 5) Violet-staining taxa Lpallidomarginatus L repraesentaneus L salicis-reticulataeand L aff salicis-reticulatae are resolved as a poorly sup-ported clade exclusively containing other members ofthe violet-staining section Uvidi supporting the mono-phyly of this section (FIG 2)

TAXONOMY

All descriptions are based on Rocky Mountain alpinematerial

Lactarius lanceolatus OK Mill amp Laursen FIGS 6 7Short description Lactarius lanceolatus is recognized by

its small to medium size and bright light to deeporange pileus pale cream to pale orange lamellae dis-coloring brownish in age or where damaged paleorange stipe watery unchanging latex and mild flavorgrowing in arctic-alpine (possibly also subalpine) areaswith Salix species

Pileus 10ndash45 mm diam convex at first becomingplane to infundibuliform with a depressed centerwith or without a small papilla smooth sometimesfaintly scaly toward center viscid deep orange-brownto deep orange when immature becoming light todeep orange often blotchy often darker toward cen-ter margin incurved to straight when immaturebecoming straight to upturned and often slightlywavy when mature Lamellae adnate to subdecur-rent slightly crowded pale cream to pale yellow topale orange discoloring brownish orange in age orwhere damaged Stipe 10ndash20 6 25ndash75 mm equalto clavate central smooth dry at first covered byfaint whitish pubescence pale orange discoloringdingy orange to dingy light brown where damagedhollow Latex scarce watery unchanging Contextof pileus and stipe pale orange Odor mild Flavormild Basidiospore deposit not observed Basidios-pores 8ndash10 6 6ndash8 mm average 5 86 6 7 mmQ 5 114ndash138 Q5 124 hyaline in 25 KOHbroadly ellipsoid ornamentation amyloid of isolatedwarts connected by fine lines up to 05 mm highBasidia 381ndash660 6 89ndash102 mm four-spored Pleur-omacrocystidia 737ndash127 6 64ndash102 mm narrowly

r$75 bootstrap support and Bayesian posterior probabilities $ 095 Boldface tip labels represent Rocky Mountain alpinespecimens AA 5 arctic-alpine See TABLE I for original identifications

BARGE ET AL ROCKY MOUNTAIN ALPINE LACTARIUS 425

fusiform to lanceolate with acute to moniliformapex thin-walled scattered to abundant Cheiloma-crocystidia 356ndash89 6 51ndash102 mm conical to fusi-form with acute to moniliform apex thin-walledsparse to abundant Pileipellis an ixotrichodermwith well-developed gelatinous layer

Distribution and habitat Lactarius lanceolatus origin-ally was described from arctic-alpine Alaska (Millerand Laursen 1973) and phylogenetic analyses(including the holotype) further confirm its pre-sence in the central Rocky Mountain alpine zoneand Svalbard In alpine areas on the Beartooth Pla-teau it occurs with Salix reticulata and possibly otherSalix species (S arctica S planifolia) it is notreported from the southern Rocky Mountains Over-all L lanceolatus appears largely confined to arctic-alpine habitats however an ITS sequence from amaritime forest in Alaska (GAL18572) clusters withthe arctic-alpine collections in phylogenetic analyses(FIG 3)

Previous arctic-alpine reports include Canada (Ohe-noja and Ohenoja 1993 2010) Alaska (Miller andLaursen 1973 Laursen and Ammirati 1982 Heil-mann-Clausen et al 1998) Beartooth Plateau in theRocky Mountain alpine zone (Cripps and Barge2013) Greenland (Heilmann-Clausen et al 1998 Bor-gen et al 2006) Fennoscandia (Bon 1998 Heilmann-Clausen et al 1998 Knudsen and Vesterholt 2008)Svalbard (Gulden and Jenssen 1988 Geml et al2012) the British Isles (Watling et al 2001) andSiberia (Nezdoiminogo 1997 Heilmann-Clausen et al1998 Karatygin et al 1999 Knudsen and Mukhin1998) however phylogenetic analyses (FIG 3) suggestthat some of these reports may represent L aurantiacus(Pers Fr) Gray

Specimens examined USA ALASKA North Slope CountyBeaufort Lagoon 30 Jul 1971 F4239 (Holotype VPI) MON-TANA Carbon County Beartooth Plateau Highline Trail 1Aug 1997 ZT6214 (MONT) 7 Aug 1998 ZT6412 (MONT)20 Aug 1999 CLC 1389 (MONT) 5 Aug 2008 CLC 2358(MONT) 27 Jul 1997 CLC 1139 (MONT) WYOMINGPark County Beartooth Plateau Solifluction Terraces 20July 2001 CLC 1578 (MONT) 13 Aug 2007 CLC 2319(MONT) 17 Aug 2013 EB 105-13 (MONT)

Other specimens examined DENMARK Broby Vesterskov 16Oct 1997 JHC97-251 (L aurantiacus C) NORWAY Leikan-ger 11 Sept 1999 JHC00-057 (L aurantiacus C) Nordland14 Aug 1999 JV15112F (L cf aurantiacus originally identi-fied as L lanceolatus TURA) Svalbard Longyearbyen 18Aug 2003 CLC 1885 (L cf aurantiacus originally identifiedas L lanceolatus MONT) SWEDEN Uppland 8 Sept 1994JV94-422 (L aurantiacus C) USA ALASKA Kenai PeninsulaCounty Palmer Creek Road 7 Aug 2011 CLC 2743 (L cfaurantiacus originally identified as L lanceolatus MONT)COLORADO El Paso County 27 Aug 2011 DBG-F-022653(L luculentus v laetus DBG) Gilpin County Perigo 13 Aug1974 AH Smith 84923 (Paratype of L luculentus v laetus

MICH) Larimer County 23 Aug 2008 DBG-F-024643 (Lluculentus v laetus DBG) OREGON Polk County 14 Nov1970 AH Smith 79943 (L luculentus MICH) WASHING-TON Lewis County 30 Oct 1972 AH Smith 83066 (Paratypeof L subviscidus MICH) 1 Nov 1972 AH Smith 83331 (Para-type of L subviscidus MICH) Tucannon River 11 Oct1980 AH Smith 90905 (L luculentus MICH)

Comments Lactarius lanceolatus belongs to a taxono-mically problematic group of species with orangeorange-brown or orange-red pilei including L auran-tiacus L luculentus L luculentus v laetus L subflammeusand L substriatus

Traditionally L lanceolatus and L aurantiacus havebeen separated mainly by ecology with L aurantiacusbeing restricted to a variety of subalpine habitats(although it has been reported from arctic-alpine habi-tats) and L lanceolatus to arctic-alpine habitats withSalix spp however it is clear from phylogenetic ana-lyses (FIG 3) that L aurantiacus also may occur in arc-tic-alpine habitats likely associating with Salix as well asKobresia and that the ranges of the two taxa overlap atleast in arctic-alpine areas of Alaska and Svalbard Ithas been suggested that L lanceolatus has longermacrocystidia and larger basidiospores than L auran-tiacus (eg Heilmann-Clausen et al 1998) howevercollections designated as L aurantiacus measured inthis study have pleuromacrocystidia that overlap andactually exceed the upper limit measured for L lanceo-latus including the holotype (see Barge 2015) Basi-diospore sizes for L aurantiacus also overlap andactually exceed the upper limit measured for L lanceo-latus in the collections examined (Barge 2015) Overallsolid morphological differences between the two taxawere not detected and further study may reveal thatthey are conspecific however provisionally L lanceola-tus may tend to have a thicker gelatinous layer over thepileipellis as well as a smooth pileus margin in contrastto the crenulate pileus margin reported for L aurantia-cus Lactarius aurantiacus is not reported from theRocky Mountains

For North American taxa Lactarius luculentus has abrowner pileus a slowly acrid flavor shorter cheilo-macrocystidia (23ndash45 6 4ndash6 mm) and exsiccati arepaler and more yellow-tan (Hesler and Smith 1979)Lactarius luculentus v laetus is similar in appearanceto L lanceolatus but also has shorter cheilomacrocysti-dia (15ndash30 6 4ndash8 mm) and possibly more crowdedlamellae (Hesler and Smith 1979) Lactarius substria-tus has a redder pileus that is glutinous when wetwith a translucent-striate margin latex that slowlybecomes straw yellow shorter cheilomacrocystidiaand an acrid flavor (Hesler and Smith 1979) Lactariussubflammeus also has a redder pileus (at least whenyoung) short translucent striations along its pileusmargin shorter cheilomacrocystidia and a slowly

426 MYCOLOGIA

FIG 6 A L lanceolatus (EB105-13) B L nanus (EB106-13) C L nanus (EB138) D L glyciosmus (ZT12723) E Lpallidomarginatus Barge amp Cripps sp nov (EB0041 HOLOTYPE) F L repraesentaneus (EB107-13) G L salicis-reticulatae(EB0057-14) H L aff salicis-reticulatae (EB0036) Photographs by E Barge (A-C E-H) E Horak (D) Bars 5 2 cm

BARGE ET AL ROCKY MOUNTAIN ALPINE LACTARIUS 427

acrid taste (pers obs of holotype AH Smith 83602Hesler and Smith 1979) Molecular phylogenetic ana-lyses (FIG 3) suggest that L subflammeus is conspecificwith L substriatus however sequencing of the holo-type of L substriatus is needed to confirm this observa-tion Lactarius luculentus L luculentus v laetus Lsubflammeus and L substriatus have been reportedonly from subalpine areas with conifers in westernNorth America (Hesler and Smith 1979)

Lactarius duplicatus AH Sm (syn L lapponicus Har-maja) a North American and European Betula associ-ate is also morphologically close to L lanceolatusHowever it can be distinguished by the productionof strongly yellowing latex We did not procure anymolecular data from material representing this taxonand no molecular data is publicly available on Gen-Bank or UNITE However L duplicatus should beincluded in future phylogenetic analyses investigatingthe L lanceolatusL aurantiacus group

Lactarius nanus J Favre FIGS 6 7Short description Lactarius nanus is recognized by its

typically small size gray-brown to liver brown pileustypically with a glaucous coating at least when youngcream to pinkish tan lamellae pale cream-tan to paleapricot stipe watery unchanging latex mild odormild to slightly acrid flavor and occurrence with Salixspecies in arctic-alpine areas

Pileus 10ndash50 mm diam shallowly convex to plano-convex becoming plane to infundibuliform with orwithout a depressed center and small papilla smoothslightly viscid to dry with whitish glaucous coatingwhen immature easily rubbing away or fading in ageoften in patches deep brown to gray-brown to liverbrown with lighter and darker areas present oftenlighter toward margin becoming lighter overall upondessication margin straight when immature becomingupturned and wavy to convoluted when matureLamellae adnate to subdecurrent subdistant to slightlycrowded cream when immature becoming dingycream to light tan in age sometimes with a faint pink-ish tinge discoloring to dingy tan where damagedStipe 5ndash30 6 3ndash15 mm equal to clavate centralsmooth dry at first covered with whitish glaucous coat-ing as in pileus pale apricot to dingy cream-tan hol-low Latex scarce watery unchanging Context ofpileus and stipe dingy cream to brownish Odor mildFlavor mild to slightly acrid Basidiospore deposit notobserved Basidiospores 7ndash105(ndash12) 6 5ndash8 mm av 584 6 66 mm Q 5 107ndash160 Q 5 127 hyaline in25 KOH subglobose to ellipsoid ornamentationof amyloid scattered ridges and warts forming anincomplete reticulum with some completely reticulateareas to 07 mm high Basidia 406ndash762 6 89ndash152 mm mostly four-spored some two-spored

Pleuromacrocystidia 533ndash965(ndash1143) 6 64ndash114mm lanceolate with acute to moniliform apexscattered to abundant Cheilomacrocystidia 260ndash9146 76ndash102 mm subulate to linear with undulatingmargin with acute to rounded apex scattered toabundant Pileipellis an (ixo-) cutis with some ascend-ing hyphae embedded in a very faint gelatinouslayer

Distribution and habitat Lactarius nanus originally wasdescribed by Jules Favre from the Swiss Alps with Salixherbacea L (Favre 1955) Phylogenetic analyses includ-ing a specimen from near the type locality confirmthis species in the Rocky Mountain alpine zone whereit is one of the most commonly encountered Lactariusspecies it associates with Salix arctica S reticulata andS planifolia Overall L nanus appears to be largelyrestricted to arctic-alpine areas with Salix

Previous arctic-alpine reports include Canada (Ohe-noja and Ohenoja 1993 2010) the Rocky Mountainalpine zone (Moser and McKnight 1987 Cripps andHorak 2008 Cripps and Barge 2013) Greenland(Knudsen and Borgen 1982 Lamoure et al 1982 Heil-mann-Clausen et al 1998 Borgen et al 2006) Iceland(Eyjolfsdottir pers comm Knudsen and Vesterholt2008) the European Alps (Favre 1955 Kuumlhner 1975Kuumlhner and Lamoure 1986 Senn-Irlet 1993 Bon1985a 1998 Heilmann-Clausen et al 1998 Jamoni1995) the Pyrenees (Bon and Ballaraacute 1996) the Car-pathians (Ronikier 2008) Fennoscandia (Kuumlhner1975 Gulden et al 1985 Heilmann-Clausen et al1998 Gulden 2005 Knudsen and Vesterholt 2008)Svalbard (Gulden et al 1985) Siberia (Nezdoiminogo1997) and alpine areas in the Altai Mountains (Karaty-gin et al 1999)

Specimens examined FRANCE Savoie 24 Aug 1989 Bon89093 (LIP) NORWAY Finse 5 Aug 2010 CLC 2134(MONT) Svalbard Longyearbyen 20 Aug 2002 CLC 1896(MONT) SWEDEN Torne Lappmark 15 Aug 1999JV15148 (TURA) SWITZERLAND Grisons 20 Aug 1943ZA192c (Holotype G) USA COLORADO Pitkin CountySawatch Range Independence Pass 6 Aug 2000 CLC 1471(MONT) San Juan County San Juan Range Black BearPass 11 Aug 2001 CLC 1716 (MONT) 11 Aug 2001ZT9529 (MONT) San Juan County San Juan Range Cinna-mon Pass 27 July 2002 CLC 1801 (MONT) 15 Aug 2012EB138 (MONT) San Miguel County San Juan Range Imo-gene Pass 29 July 2002 CLC 1829 (MONT) MONTANACarbon County Beartooth Plateau Highline Trail 7 Aug1998 CLC 1221 (MONT) 8 Aug 1998 ZT6422 (MONT) 18Aug 2013 EB106-13 (MONT) WYOMING Park CountyBeartooth Plateau Frozen Lakes 21 Aug 1999 CLC 1403(MONT) Park County Beartooth Plateau Solifluction Ter-races 11 Aug 2012 EB125 (MONT)

Other specimens examined FINLAND Koillismaa 28 Aug2011 JV28432 (L hysginoides TURA)

428 MYCOLOGIA

Comments Lactarius nanus is morphologically andphylogenetically close to L hysginoides however thelatter has a more cinnamon-toned pileus smaller basi-diospores (6ndash8 6 5ndash7 mm av 68 6 57 mm) and isgenerally reported from boreal areas with Betula Piceaand Salix in Europe (Heilmann-Clausen et al 1998)although phylogenetic placement of environmentalsequences extend its range into boreal Alaska (FIG4) Its range likely overlaps that of L nanus in alpine-subalpine transition areas It is not reported from theRocky Mountains Lactarius mammosus is a more robustspecies with a coconut odor (Heilmann-Clausen et al1998) that associates with members of the Pinaceaeit has not been reported from the Rocky MountainsDifferences between L glyciosmus and L nanus are dis-cussed in the taxonomy section for L glyciosmus

Lactarius glyciosmus (Fr) Fr FIGS 6 7Short description Lactarius glyciosmus is recognized by

its small to medium size pale gray-brown to mauvepileus typically with a glaucous coating at least whenyoung cream pale pinkish or pale yellow-orangelamellae buff or pale salmon stipe watery unchan-ging latex odor of coconuts mild flavor and associa-tion with Betula

Pileus 15ndash50 mm diam broadly convex later becom-ing plane with or without a depressed center and smallpapilla smooth dry pale gray-brown to mauve (violet-brown) with whitish glaucous coating when immaturefading in age azonate to lightly zoned especially nearmargin margin incurved when young and remainingso or becoming straight when mature Lamellaeadnate to subdecurrent slightly crowded to crowdedcream to pale pinkish to pale yellow-orange Stipe10ndash40 6 3ndash12 mm equal to slightly clavate oftencurved central to eccentric smooth dry buff or palesalmon with faint glaucous coating at first as in pileusstuffed or hollow often white-mycelioid toward baseLatex scarce watery unchanging Context of pileusand stipe pale cream Odor of coconut Flavor mildBasidiospore deposit not observed Basidiospores 7ndash96 5ndash7 mm av 5 77 6 60 mm Q 5 117ndash142 Q 5

128 hyaline in 25 KOH broadly ellipsoid to ellip-soid ornamentation of amyloid scattered ridges andwarts forming an incomplete to nearly complete reticu-lum to 05 mm high Basidia 381ndash635 6 76ndash102 mmmostly four-spored some two-spored Pleuromacrocys-tidia 457ndash635 6 64ndash76 mm subclavate to lanceolatewith rounded to occasionally mucronate apex scarceto scattered Cheilomacrocystidia 33ndash66 6 51ndash89mm cylindrical to subclavate with rounded apex scat-tered to abundant Pileipellis a cutis with tufts ofascending hyphae

Distribution and habitat Lactarius glyciosmus was firstdescribed by Fries from a hemiboreal to boreal habitat

in the Femsjouml region of Sweden and the species lacks atype specimen Phylogenetic analyses including speci-mens from the type locality confirm this species inthe central and southern Rocky Mountain alpinezone and suggest it has a broad ecological and geogra-phical distribution confirmed from subalpine and arc-tic-alpine areas in Alaska western Canada IdahoWisconsin New Hampshire Svalbard and Fennoscan-dia In subalpine and alpine areas of the Rocky Moun-tains L glyciosmus associates with Betula glandulosaPhylogenetic placement (FIG 4) of ectomycorrhizalroot tip sequences confirm Betula nana L as a host inAlaska (GU998223 GU998695) and likely B occidenta-lis Hook in Idaho (JX198526 Bogar amp Kennedy2013) Elsewhere it is reported with B pubescens Ehrh(eg Elborne amp Knudsen 1990) and B papyrifera Mar-shall (eg Durall et al 2006)

Previous reports from arctic-alpine areas includeCanada (Ohenoja and Ohenoja 1993 2010) theRocky Mountain alpine zone (Cripps and Horak2008 Cripps and Barge 2013) Greenland (Lange1957 Knudsen and Borgen 1982 Lamoure et al1982 Elbourne and Knudsen 1990 Borgen 2006 Bor-gen et al 2006) Iceland (Eyjolfsdottir pers comm) theAlps (Bon 1998) Fennoscandia (Kallio and Kankainen1964 Kuumlhner 1975 Bon 1998) Svalbard (Ohenoja1971 Geml et al 2012) and Siberia (Karatygin et al1999) At least some reports might be L aff glyciosmusas defined here

Specimens examined FINLAND Fjell Ailigas 27 Aug 2012CLC 2874 (MONT) SWEDEN Femsjouml 13 Aug 1978 MMoser 19780191 (Type locality IB) 16 Aug 1978 M Moser19780234 (Type locality IB) 9 Aug 1981 M Moser19810148 (Type locality IB) USA COLORADO BoulderCounty Front Range Blue Lake 2 Aug 2001 CLC 1624(MONT) Boulder County Front Range Brainard Lake 13Aug 2000 20923 (DBG) Ouray County San Juan RangeIronton Park 13 Aug 2012 EB133 (MONT) MONTANACarbon County Beartooth Plateau Birch Site 29 July 1997CLC 1134 5 ZT6096 (MONT) 8 Aug 1998 CLC 1217(MONT) 10 Aug 1999 TWO 269 (MONT) 19 Aug 1999CLC 1380 (MONT) 13 Aug 2007 ZT12723 (MONT)

Other specimens examined ESTONIA Saare CountyLuumlmanda Commune 17 Sep 2012 TU118535 (L aff glycios-mus TU) Tartu County Votildennu Commune 12 Sept 2010TU106737 (L aff glyciosmus TU) 13 Sept 2010 TU106724(L aff glyciosmus TU)

Comments Lactarius glyciosmus is morphologically andphylogenetically similar to L mammosus however thelatter has a browner pileus stouter stature and moreelongate more reticulate basidiospores (Heilmann-Clausen et al 1998) Phylogenetic placement ofsequences isolated from ectomycorrhizal root tips ofPinus sylvestris L (AJ633116 Heinonsalo et al 2007)and Picea abies (L) Karst (FM992937 Kjoslashller andClemmensen 2009) (FIG 4) confirm that L mammosus

BARGE ET AL ROCKY MOUNTAIN ALPINE LACTARIUS 429

FIG 7 Comparison of micromorphological features of Rocky Mountain alpine specimens and Holotypes (where available)Basidiospores pleuromacrocystidia (left) and cheilomacrocystidia (right) of A Lactarius lanceolatus (CLC2319) B L lanceolatus(F 4239 HOLOTYPE) C L nanus (CLC1471) D L nanus (ZA 192c HOLOTYPE) E L pallidomarginatus Barge amp Cripps spnov (EB0041 HOLOTYPE) F L pseudouvidus (Va 71971 HOLOTYPE) G L salicis-reticulatae (CLC2776) H L salicis-reticulatae

430 MYCOLOGIA

associates with members of the Pinaceae however italso has been reported with Betula mainly in subalpineareas (Heilmann-Clausen et al 1998) Lactarius vietus(Fr) Fr is also similar but has a distinct pale zonenear the stipe apex latex that dries greenish graylonger pleuromacrocystidia and a mild to slightly fruityodor (Heilmann-Clausen et al 1998) It has not beenreported from alpine areas in the Rocky Mountainsalthough it has been reported from elsewhere (mostlynorthern areas) in North America with Betula (Heslerand Smith 1979) The similar-looking L nanus alsooccurs in alpine areas of the Rocky Mountains andhas larger basidiospores larger and differently shapedmacrocystidia lacks an odor of coconut and associateswith Salix Lactarius spinosulus is phylogenetically closeto L glyciosmus (as well as L nanus) (FIG 4) howevermorphologically they are very different Lactarius spino-sulus has a distinctly zoned coarsely squamulose pink-ish salmon- or brick-colored pileus with a finely hairymargin and fruity odor (Heilmann-Clausen et al1998) it has not been reported from North AmericaNo definitive morphological differences were detectedbetween L glyciosmus and L aff glyciosmus and the lat-ter is not reported from the Rocky Mountainalpine zone

Lactarius pallidomarginatus Barge amp CL Cripps spnov FIGS 6 7Mycobank MB812246

Typification USA COLORADO San Juan CountySan Juan Range Stony Pass scattered solitary onsoil under Salix planifolia 13 Aug 2011 EB0041 (holo-type MONT) GenBank KR090940 (ITS) andKR091010 (RPB2)

Etymology Referring to the pale pileus marginDiagnosis Lactarius pallidomarginatus is recognized by

its small to medium size blotchy light tan to lightbrown to gray-brown pileus which is typically lightertoward the margin white to pale yellow-cream lamel-lae pale cream stipe violet staining tissue mild odorand flavor and association with Salix Morphologicallyit is close to L pseudouvidus Kuumlhner but differs by hav-ing smaller basidiospores with an ornamentation ofthinner ridges as well as macrocystidia that becomemore lanceolate (as opposed to subfusiform) and atte-nuated Phylogenetically it is close to L aspideus (Fr)Fr but differs consistently in two nucleotide positionsin the rDNA ITS locus and at eight nucleotide posi-tions in the RPB2 locus morphologically L aspideushas larger yellow basidiomata

Pileus 20ndash50 mm diam convex to broadly convexbecoming broadly convex to plane with or without adepressed center smooth dry to slightly viscid azo-nate blotchy light tan to light brown to gray-browndeveloping violet stains lighter (to cream) toward mar-gin margin incurved to downturned when youngremaining downturned or becoming nearly straightwhen mature Lamellae adnate to subdecurrent subdi-stant to slightly crowded white to pale yellow-creamstaining violet where damaged Stipe 10ndash40 6 5ndash10mm equal to slightly clavate central smooth drywhite to cream staining violet where damaged hollowLatex scarce watery staining tissue violet Context ofpileus and stipe white to cream staining violet wheredamaged Odor mild Flavor mild Basidiosporedeposit not observed Basidiospores 8ndash10 6 65ndash8mm av 5 87 6 71 mm Q 5 114ndash136 Q5 123 hya-line in 25 KOH broadly ellipsoid to ellipsoid orna-mentation amyloid of thin ridges and a few isolatedwarts forming an incomplete to dense reticulum to05 mm high Basidia 508ndash711 6 102ndash127 mm four-spored Pleuromacrocystidia 813ndash1118 6 89ndash102mm cylindrical to lanceolate with acute to moniliformapex scarce to scattered Cheilomacrocystidia 483ndash1016 6 76ndash127 mm cylindrical to lanceolate withacute to moniliform apex scattered Pileipellis an(ixo-)cutis with some ascending hyphae embedded ina faint gelatinous layer

Distribution and habitat Rare in low alpine areas withSalix planifolia reported from Independence Pass inthe Sawatch Range and Stony Pass in the San JuanRange in Colorado An additional collection(ZT5229) from Union Peak in the Wind River Rangeof Wyoming with Salix glauca is likely conspecific how-ever this hypothesis has not been confirmed withmolecular data Due to confusion with L pseudouvidusit is possible that L pallidomarginatus is more wide-spread than confirmed here

Specimens examined USA COLORADO Pitkin CountySawatch Range Independence Pass 6 Aug 2000 CLC 1470(MONT) 9 Aug 2000 ZT9093 (MONT) San Juan CountySan Juan Range Stony Pass 13 Aug 2011 EB0041 (HolotypeMONT) WYOMING Sublette County Wind River RangeUnion Peak 22 Aug 1994 ZT5229 (MONT)

Other specimens examined AUSTRIA Tyrol 3 July 2007 UPeintner 20070035 (L aff pseudouvidus from near type local-ity IB) FINLAND Inarin Lappi 16 Aug 1995 JV10468 (Laff pseudouvidus TURA) Koillismaa JV28448F 30 Aug 2011(L aff pseudouvidus originally identified as L brunneoviola-ceus TURA) FRANCE Isegravere 13 Aug 1971 Va 7 1971 (Holo-type of L pseudouvidus G) ITALY Trentino 20 Aug 2004 U

r(K72-104 HOLOTYPE no cheilocystidia drawn) I L glyciosmus (TWO269) J L repraesentaneus (CLC1747) Bars 5 10 mm forbasidiospores and 20 mm for cystidia Drawings by E Barge

BARGE ET AL ROCKY MOUNTAIN ALPINE LACTARIUS 431

Peintner 20040156 (L aff pseudouvidus from near type local-ity originally identified as L robertianus IB) NORWAYFinse 10 Aug 2005 CLC 2133 (Lactarius aff brunneoviolaceusMONT) Stordal 26 Aug 2006 P Larsen 361395 (L aff brun-neoviolaceus originally identified as L pseudouvidus O) Sval-bard 18 Aug 2002 TWO809 (L aff pseudouvidus MONT)20 Aug 2002 CLC 1910 (Lactarius sp 5 MONT) Ulvik 11Aug 2011 E Soslashyland 73867 (L aff pseudouvidus O) SWE-DEN Latnjajaure 4 Aug 2011 EL101-11 (L aff pseudouvidusGB) Latnjavagge 17 Aug 2010 EL63-10 (L aff pseudouvi-dus GB)

Comments Morphological features of L pallidomar-ginatus fit the holotype description of L pseudouvidusand both are described as lighter near the pileus mar-gin (Kuumlhner 1975 Basso 1999) however basidios-pores of L pallidomarginatus are slightly smaller (8ndash10 6 65ndash8 mm as opposed to 75ndash12 6 65ndash8(85)mm) and have narrower ridges that form a less densereticulum and macrocystidia take on a more lanceo-late form with greater attenuation of the apex (FIG7) In phylogenetic analyses exsiccati identified as Lpseudouvidus from Europe (including specimensfrom near the type locality) appear as distinct fromRocky Mountain alpine material and may representa species complex Lactarius sp 3 (CLC1910) col-lected in Svalbard has darker more vinaceous pileithan L pallidomarginatus and may represent anotherundescribed species although only one collectionwas morphologically examined Members of the Laff brunneoviolaceus clade (FIG 5) that were exam-ined differ from L pallidomarginatus in having dar-ker more vinaceous pilei larger more elongatebasidiospores (to 13 mm) and green staining of exsic-cati in 25 KOH a feature not mentioned previouslyfor L brunneoviolaceus but which could be of taxo-nomic interest Based on taxa included in the phylo-genetic analyses (FIG 5) Lactarius aspideus appearsclosely related to L pallidomarginatus however theformer has more yellow coloration and typically lar-ger sporocarps (pileus 10ndash70 mm diam) and smallerbasidiospores (67ndash95 6 56ndash78 mm) traditionallyL aspideus has been placed with other yellow violet-staining taxa such as L flavidus Boud L salicis-reticu-latae and L salicis-herbaceae (Heilmann-Clausen et al1998) but it is separated here

Lactarius montanus another violet-staining speciesoriginally described from the Rocky Mountains dif-fers from L pallidomarginatus by its larger staturedarker grayish vinaceous to vinaceous-brown pileusand green staining flesh in 25 KOH it is typicallyfound in subalpine habitats with conifers but alsooccurs in the krummholz zone The violet-stainingLactarius pallescens Hesler amp AH Sm occurs in theRocky Mountains but its sporocarps are larger andlighter in color and it is reported from subalpineconifer forests (Hesler and Smith 1979) Other

gray-brown to brownish violet-staining species suchas L luridus L uvidus (Fr) Fr L violascens (JOtto) Fr L pallescens v palmerensis Hesler amp AHSm and L cordovaensis Hesler amp AH Sm are alsolarger and have a subalpine ecology none havebeen reported from the central or southern RockyMountains (Hesler and Smith 1979 Heilmann-Clau-sen et al 1998) Lactarius cistophilus Bon amp Trimbachtypically has larger basidiomata larger basidiospores(10ndash14 6 72ndash9 mm) and occurs under Cistus inthe Mediterranean region (Basso 1999) The smallpale gray-brown European alpine taxon L uvidus falpigines Jamoni amp Bon has microscopic featuresthat match those of L uvidus (Jamoni and Bon1991) but not L pallidomarginatus The small non-violet staining L nanus has basidiospores withthicker more jagged ridges and differently shapedmacrocystidia (often with more rounded apices)(FIG 7)

Lactarius repraesentaneus Britzelm FIGS 6 7Short description Lactarius repraesentaneus is recog-

nized by its medium to large size orange-brown to yel-low-brown hairy pileus with a heavily bearded margincream to pale-yellow lamellae stout cream to pale-yel-low often pitted stipe violet staining latex and tissuespicy-floral odor slightly acrid flavor and associationwith Betula Picea and possibly shrubby Salix speciessuch as S glauca

Pileus 60ndash100 mm diam convex to plano-convexusually with a depressed center and sometimes with apapilla smooth at center becoming increasingly hairyto bearded toward margin dry to viscid azonate sur-face orange-brown at center becoming pale yellow-brown to cream toward margin hairs pale yellow-brown to red-brown margin strongly incurvedremaining so or becoming nearly straight whenmature Lamellae adnate to decurrent crowdedcream to pale-yellow staining violet where damagedStipe 30ndash70 6 15ndash35 mm stout equal to clavate cen-tral dry to viscid cream to pale yellow surface oftenwith numerous dingy yellow golden yellow yellow-brown or light orange-brown pits hollow Latex scarceto abundant white becoming violet Context of pileusand stipe white staining violet where damaged Odorspicy-floral Flavor slightly acrid Basidiospore depositnot observed Basidiospores 8ndash105 6 6ndash85 mm av5 93 6 73 mm Q 5 113ndash143 Q5 127 hyaline in25 KOH broadly ellipsoid to ellipsoid ornamenta-tion amyloid of narrow to broad ridges and isolatedwarts forming an incomplete to dense reticulum to1 mm high Basidia 610ndash762 6 102ndash114 mm four-spored Pleuromacrocystidia 787ndash1448 6 89ndash127 mm subfusiform to lanceolate thin-walled withacute to moniliform apex scattered to abundant

432 MYCOLOGIA

Cheilomacrocystidia 559ndash1397 6 76ndash127 mm subfu-siform to lanceolate thin-walled with acute to monili-form apex scattered to abundant Pileipellis anixotrichoderm with well-developed gelatinous layer

Distribution and habitat Lactarius repraesentaneus firstwas described from Germany (Britzelmayr 1885) Phy-logenetic analyses including material from Germanyconfirm L repraesentaneus from alpine areas of theRocky Mountains and overall reinforce the traditionalconcept that this species has a broad ecological andgeographic range in arctic-alpine and subalpine habi-tats in North America northern central and southernEurope In alpine areas of the Rocky Mountains itassociates with Salix glauca however krummholz Piceaalso is typically (maybe always) present it is reportedwith Picea and Betula elsewhere

Previous reports from arctic-alpine areas mainlywith Betula include Alaska (Laursen and Ammirati1982 Geml et al 2009) the Rocky Mountain alpinezone (Cripps and Horak 2008 Cripps and Barge2013) Greenland (Knudsen and Borgen 1982 Watling1983 Elbourne and Knudsen 1990 Borgen 2006 Bor-gen et al 2006) Iceland (Eyjolfsdottir pers comm)Fennoscandia (Kallio and Kankainen 1964 Kuumlhner1975 Bon 1998) Siberia (Karatygin et al 1999) andthe Altai Mountains (Gorbunova 2010)

Specimens examined FINLAND Enontakiouml 16 Aug 2004JV21671 (TURA) Varsinais-Suomi 9 Aug 1998 JV13837F(TURA) SWEDEN Latnjavagge 12 Aug 2007 EL92-07(GB) USA COLORADO Chaffee County Sawatch RangeCottonwood Pass 4 Aug 2001 CLC 1643 (MONT) 16 Aug2011 EB0048 (MONT) Pitkin County Sawatch Range Inde-pendence Pass 14 Aug 2001 CLC 1747 (MONT) ZT9837(MONT) 15 Aug 2001 ZT9537 (MONT) MONTANA Car-bon County Beartooth Plateau Birch Site 12 Aug 2002CLC 1971 (MONT) 13 Aug 2007 CLC 2318 (MONT) 18Aug 2013 EB107-13 (MONT) WYOMING Park CountyBeartooth Plateau Frozen Lakes 21 Aug 1999 CLC1394 (MONT)

Other specimens examined SWEDEN Latnjavagge 14 Aug2010 EL57-10 (L dryadophilus GB) USA ALASKA 5 Aug2011 CLC 2729 (L dryadophilus MONT) Kenai PeninsulaCounty Palmer Creek Road 7 Aug 2011 CLC 2744 (L drya-dophilus MONT)

Comments The combination of a large pale yellow-brown to orange-brown pileus with a bearded marginand violet staining latex clearly separates L repraesenta-neus from other Lactarius species of the central andsouthern Rocky Mountain alpine zone Phylogeneti-cally close the arctic-alpine taxon L dryadophilus ismorphologically similar but has a cream to yellowsmoother pileus with hairs restricted to near the mar-gin basidiospore ornamentation consisting of sparsethinner ridges and ecological associations with Dryasand Salix It has not been reported from the RockyMountains

Lactarius salicis-reticulatae Kuumlhner FIGS 6 7Short description Lactarius salicis-reticulatae is recog-

nized by its small to medium cream pale yellow orpale ocher sporocarps with pale yellowish well-spacedlamellae often with a pinkish buff tint cream to pale yel-low stipe violet staining latex and tissue mild to slightlysweet odor mild flavor and association with dwarf (Sreticulata) and possibly also shrubby Salix species

Pileus 20ndash40 mm diam convex to broadly convexusually with a depressed center smooth dry to viscidazonate cream pale yellow or pale ocher sometimesdarker toward center staining violet where damagedmargin invcurved remaining so or becoming down-turned when mature Lamellae adnate to subdecur-rent fairly well-spaced cream pale yellow or paleorange-ocher often with a pinkish-buff tint stainingviolet where damaged Stipe 15ndash20 6 10ndash15 mmequal to clavate central dry to viscid cream to pale-yellow staining violet where damaged hollow Latexscarce watery-white becoming violet Context ofpileus and stipe white staining violet where damagedOdor mild to slightly sweet Flavor mild Basidiosporedeposit not observed Basidiospores (7ndash)85ndash115 6(7ndash)8ndash10 mm av 5 100 6 88 mm Q 5 (100ndash)113ndash143 Q 5 125 hyaline in 25 KOH sublgobose toellipsoid ornamentation amyloid of narrow ridgesand a few isolated warts forming an incomplete reticu-lum to 03 mm high Basidia 737ndash889 6 102ndash127 mm four-spored Pleuromacrocystidia 762ndash10166 76ndash114 mm subfusiform to fusiform thin-walledwith rounded to acute to moniliform apex scarceCheilomacrocystidia 686ndash9146 76ndash102 mm subfusi-form to fusiform thin-walled with acute to rounded tomoniliform apex scattered to abundant Pileipellis anixocutis

Distribution and habitat Lactarius salicis-reticulatae firstwas described from alpine tundra with Salix on MtLaringktatjaringkka Sweden (Kuumlhner 1975) Phylogenetic ana-lyses including material from the type locality confirmthis species from alpine areas on the Beartooth Plateauwith Salix reticulata and shrubby Salix spp and in arctic-alpine areas of Alaska Svalbard and Finland OverallL salicis-reticulatae appears largely confined to arctic-alpine habitats Colorado collections with S reticulataand S planifolia may represent a distinct taxonalthough support is low

Previous arctic-alpine reports include Greenland(Lamoure et al 1982 Heilmann-Clausen et al1998 Borgen et al 2006) the Rocky Mountainalpine zone (Cripps and Horak 2008 Cripps andBarge 2013) the Alps (Kuumlhner 1975 Kuumlhner andLamoure 1986 Jamoni 1995 2008 Bon 1998 Heil-mann-Clausen et al 1998) the Pyrenees (Ballaraacute1997) Fennoscandia (Kuumlhner 1975 Heilmann-Clau-sen et al 1998 Gulden 2005 Knudsen and

BARGE ET AL ROCKY MOUNTAIN ALPINE LACTARIUS 433

Vesterholt 2008) and Siberia (Nezdoiminogo 1997Karatygin et al 1999) previous reports of L aspi-deoides Burl from arctic-alpine areas in Alaska (Laur-sen and Ammirati 1982) Greenland (Knudsen andBorgen 1982) and the British Isles (Watling 1987)may refer to this species

Specimens examined FINLAND Near Utsjoki 28 Aug 2012CLC 2885 (MONT) SWEDEN Mt Laringktatjaringkka 12 Aug1972 K72-104 (Holotype G) 15 Aug 1999 JV15133 (Typelocality TURA) USA ALASKA Kenai Peninsula County Pal-mer Creek Road 7 Aug 2011 CLC 2745 (MONT) MON-TANA Carbon County Beartooth Plateau Birch Site 17Aug 2011 CLC 2776 (MONT) Carbon County BeartoothPlateau Highline Trail 8 Aug 1998 CLC 1211 (MONT)WYOMING Park County Beartooth Plateau GardnerLake 16 Aug 2014 EB0057-14 (MONT)

Other specimens examined FINLAND Koillismaa 31 Aug2005 JV23334 (L flavopalustris TURA) GREENLAND Sis-miut 19 Aug 2000 CLC 1536 (L salicis-herbaceae MONT)USA COLORADO Pitkin County Sawatch Range Indepen-dence Pass 13 Aug 2001 CLC 1741 (L aff salicis-reticulataeMONT) San Juan County San Juan Range CinnamonPass 10 Aug 2001 CLC 1710 (L aff salicis-reticulataeMONT) San Juan County San Juan Range Maggie Gulch11 Aug 2011 EB0036 (L aff salicis-reticulatae MONT)San Juan County San Juan Range Stony Pass 9 Aug 2001CLC 1689 (L aff salicis-reticulatae MONT) 13 Aug 2011EB0039 (L aff salicis-reticulatae MONT) MICHIGANEmmet County 10 Aug 1973 RL Shaffer 6957 (L aspi-deoides MICH)

Comments Some taxonomic confusion has beencaused by the fact that Kuumlhner (1975) originallynamed this species L aspideoides Kuumlhner but subse-quently renamed it because the name had been pre-viously applied L aspideoides Burl (1907) is generallylarger with a somewhat zonate pileus more crowdedlamellae that lack salmon-colored hues slightly smallerbasidiospores with broader ridges a bitter to slightlyacrid flavor and a subalpine ecology apparently ineastern and western North America (pers obs of RLShaffer 6957 Hesler and Smith 1979) Lactarius fla-vopalustris is also similar however it has a largermore brightly yellow pileus with faint zonationmore crowded lamellae a weakly scrobiculate stipe(sometimes) smaller basidiospores with thickerridges and a subalpine ecology (Kytoumlvuori 2009)Phylogenetic analyses (FIG 5) reveal three collec-tions of L flavopalustris (two from near the typelocality in Finland) nested in a well-supported cladewith L aspideoides from a subalpine forest in north-ern Michigan suggesting the possible conspecificityof these taxa Neither is reported from the RockyMountains Two other collections identified as L fla-vopalustris (from Estonia) were resolved as distinctfrom the L aspideoidesflavopalustris clade (FIG 5)their identity is unclear

Lactarius aspideus is differentiated from L salicis-reti-culate by its smaller basidiospores with denser reticula-tion more crowded creamier-colored lamellae andhabitat mainly in subalpine areas (Heilmann-Clausenet al 1998) although an environmental sequence iso-lated from an S polaris root tip from arctic Svalbardclusters with L aspideus in phylogenetic analyses (FIG5) Lactarius dryadophilus is often larger with a cream-colored pileus that has a tomentose to bearded margin(Heilmann-Clausen et al 1998) Lactarius flavidus is lar-ger with a sparsely zonate pileus more crowdedcream-colored lamellae darker violet-staining contextbasidiospores with more prominent ornamentationand a distally amyloid plage and subalpine ecology(Heilmann-Clausen et al 1998) Lactarius flavoaspideusKytoumlv has slightly smaller basidiospores on averagewith a denser reticulum and subalpine ecology withSalix (Kytoumlvuori 2009) it has been reported onlyfrom northern Europe Lactarius salicis-herbaceae asimilar arctic-alpine Salix associate has smaller (typi-cally) darker yellow to yellow-brown sporocarpspale cream to grayish buff lamellae and more denselyreticulate basidiospores (pers obs Heilmann-Clausenet al 1998) It is fairly widespread in arctic-alpineareas but is not reported from alpine areas in the cen-tral and southern Rocky Mountains Lactarius aff sali-cis-reticulatae from Colorado may be a distinct taxonand sporocarps can obtain a larger size (pileus to 90mm diam) but otherwise morphological featuresstrongly overlap

KEY TO ROCKY MOUNTAIN ALPINE LACTARIUS SPECIES

1 Latex white turning violet to dull lilac or flesh stain-ing violet to dull lilac 2

1 Latex watery to white unchanging and flesh notstaining violet to dull lilac 42 Fruiting body robust pileus 60ndash100 mm diam

yellow-brown to orange-brown hairy especiallynear margin lamellae cream to pale-yellow stipestout cream to pale-yellow often with numerousyellow-brown pits odor spicy-floral flavor mildlyacrid basidiospores 8ndash105 6 6ndash85 mm puta-tively associated with Betula and Picea (possiblyalso Salix) in Rocky Mountain alpine zone andsubalpine and arctic-alpine areas in North Amer-ica and Eurasia L repraesentaneus

2 Fruiting body smaller 20ndash50 mm diam pileus andmargin smooth 3

3 Pileus pale yellow to pale ocher lamellae cream paleyellow or pale orange-ocher stipe cream to pale yel-low odor mild to slightly sweet flavor mild basidios-pores 85ndash115 6 8ndash10 mm in Rocky Mountain alpinezone and other arctic-alpine areas in North Americaand Eurasia with Salix L salicis-reticulatae

3 Pileus blotchy light tan to light brown to gray-brownmargin lighter lamellae white to pale yellow-cream

434 MYCOLOGIA

stipe white to cream odor mild flavor mild basi-

diospores 8ndash10 6 65ndash8 mm in low alpine areas inColorado with Salix planifolia and possibly Wyom-

ing with Salix glauca L pallidomarginatus4 Pileus bright orange to orange-brown 10ndash45

mm diam margin smooth lamellae pale creamto pale orange stipe pale orange odor mildflavor mild in Rocky Mountain alpine zonearctic-alpine Alaska (possibly also subalpine)to Svalbard with Salix L lanceolatus

4 Pileus pinkish buff gray-brown brown ormauve 5

5 With odor of coconuts pileus 15ndash50 mm diam palepinkish buff to pale gray-brown with glaucous coat-ing lamellae cream to pale yellow-orange stipe buffor pale salmon flavor mild basidiospores 7ndash9 6 5ndash7mm in Rocky Mountain alpine zone and subalpinearctic-alpine areas in North America and Eurasiawith Betula L glyciosmus

5 Without odor of coconuts pileus 10ndash50 mm diamgray-brown to liver-brown with whitish glaucous coat-ing lamellae cream to light tan sometimes with pink-ish tinge discoloring to dingy tan where damagedStipe pale apricot to dingy cream-tan flavor mild toslightly acrid basidiospores 7ndash105 6 5ndash8 mm inRocky Mountain alpine zone and arctic-alpine areasin Eurasia and likely other arctic-alpine areas in NorthAmerica with Salix L nanus

DISCUSSION

The number of Lactarius species reported from theRocky Mountain alpine zone represents only a fractionof the total number of Lactarius species reported else-where from arctic-alpine areas however nearly halfof the core arctic-alpine species are now confirmedfor continental North America Core species oftencited as being restricted to arctic-alpine areas withdwarf and shrubby Salix andor Dryas species includeL brunneoviolaceus L dryadophilus L lanceolatus Lnanus L pseudouvidus L salicis-herbaceae and L salicis-reticulatae (Knudsen and Borgen 1982 Heilmann-Clau-sen et al 1998 Basso 1999 Knudsen and Vesterholt2008) Phylogenetic analyses and morphological exam-ination confirm three out of seven of these species forthe Rocky Mountain alpine zone (L lanceolatus Lnanus L salicis-reticulatae) and support the hypothesisthat these species are largely restricted to arctic-alpineareas with Salix One definitive new species L pallido-marginatus (with Salix) was revealed along with a possi-ble cryptic species L aff salicis-reticulatae (also withSalix) Many Lactarius species previously reportedfrom arctic-alpine areas are also commonly reportedfrom subalpine areas with Betulaceae andor Pinaceae

as well as with other plant families (Heilmann-Clausenet al 1998) From this set of species we report L gly-ciosmus which associates with Betula glandulosa in theRocky Mountain alpine zone and Betula spp acrossthe rest of its distribution and L repraesentaneus whichis commonly reported with Betula and Picea (Heslerand Smith 1979 Heilmann-Clausen et al 1998) andappears to associate with Salix glauca andor perhapskrummholz Picea in the Rocky Mountain alpine zone

Several taxa of interest were not molecularly distinctIn dataset A L lanceolatus did not form a monophyleticgroup but was paraphyletic with respect to Lactarius sp2 the L luculentus group and L aurantiacus In datasetB L nanus and L hysginoides were not clearly distinctIn dataset C L salicis-reticulatae did not form a clearmonophyletic group and L pseudouvidus L brunneovio-laceus and L luridus were not clearly distinguishedfrom each other especially at the RPB2 locus poormolecular resolution also has been encountered forthe latter three taxa by Das et al (2015)

Overall there was little genetic variation distinguish-ing closely related species in this study These findingsare similar to reports from other researchers who sug-gest that there is in general relatively little molecularvariation yet high morphological variation in genusLactarius (Verbeken and Nuytinck 2013) While manyof the species could be morphologically distinguishedall of the Rocky Mountain alpine taxa delineated inthis study are 97 or more similar at the ITS regionto one or more species For example L repraesentaneusand L dryadophilus are as much as 994 similar L sal-icis-reticulatae is approximately 98 similar to L salicis-herbaceae and L flavopalustris L pallidomarginatus isapproximately 97ndash98 similar to L aff brunneoviola-ceus L luridus L montanus and members of the Laff pseudouvidus group and approximately 99 similarto L aspideus L glyciosmus is approximately 98 simi-lar to L mammosus L nanus is approximately 98similar to L hysginoides and L lanceolatus is approxi-mately 97 similar to L substriatussubflammeus L aur-antiacus L luculentus v laetus and Lactarius sp 1 and 2and approximately 98 similar to L luculentus Thishigh degree of similarity could be problematic forenvironmental sequencing studies that use a strict97 ITS region similarity cutoff for recognizing spe-cies (eg Geml et al 2012 Timling et al 2012 2014)In addition Tedersoo et al (2014) suggested that theITS region in the sebacina lineage evolves moreslowly in high latitude areas which could be true forother genera The RPB2 locus proved better for differ-entiating some species and overall phylogenetic ana-lyses revealed several taxonomic issues that we werenot able to resolve A search for additional phylogen-etically informative loci for Lactarius should beinitiated along with more species-level phylogenetic

BARGE ET AL ROCKY MOUNTAIN ALPINE LACTARIUS 435

studies to provide a high quality molecular databasefor ecological studies Furthermore improvement ofmethods for sequencing older material such as typespecimens is critical

Most of the Rocky Mountain alpine Lactarius speciesdelineated in this study (except L pallidomarginatusand L aff salicis-reticulatae for which further studycould reveal broader ranges) appear to have broadintercontinental distributions in North America andEurasia The current distributions of these specieslikely were shaped by a number of biotic and abioticfactors that include host dispersal and distribution cli-mate glaciation and geography Because the genusLactarius is obligately ectomycorrhizal it is likely thatthe distribution of each fungal species was shaped lar-gely by the distribution of suitable host plants manyof which also have broad intercontinental distribu-tions Long-distance dispersal is hypothesized to haveplayed an important role in structuring present distri-bution patterns of arctic-alpine plants although manyappear clonal (Abbott and Brochmann 2003 Alsoset al 2007) and ectomycorrhizal fungi (Geml et al2012 Timling et al 2014) It also has been suggestedthat long-distance dispersal mechanisms have beenselected for in arctic flora due to repeated glaciationsand the need to track potential niches (Alsos et al2007) Pollen data from Blacktail Pond YellowstoneNational Park suggest that montane mid-elevationsin the northern greater Yellowstone ecosystem (Mon-tana Wyoming) were occupied by alpine tundra(Betula Salix many herbaceous taxa) similar to thatof the present Rocky Mountain alpine vegetation dur-ing the last glacial period (14 000ndash11 500 calibratedyears before present) (Krause and Whitlock 2013)Alpine vegetation also appears to have dominatedareas in the southern Rocky Mountains (WyomingColorado) 17 500ndash12 600 calibrated years before pre-sent (Minckley 2014) Both studies suggest the pre-sence of broad low-elevation glacial refugia for arctic-alpine life in the Rocky Mountains along the glacialfront and along with this study lend support to theidea that arctic-alpine Lactarius species (and manyother ectomycorrhizal fungi) may have occupied tun-dra areas south of the icesheets during cooler weatherduring the last glacial maximum and then dispersedupward in elevation and northward as the glaciersand ectomycorrhizal host plants retreated Broaderecological tolerances of taxa such as L glyciosmus andL repraesentaneus which are not restricted to arctic-alpine habitats likely let these species persist in tundraand non-tundra areas during the last glacial maximumas well as to track arctic-alpine vegetation as it movedupward in elevation and northward following theretreat of the icesheets For these species to haveachieved and maintained such broad distributions

gene flow must have been possible over long distancesduring the Pleistocene perhaps mediated by long-dis-tance dispersal (Geml et al 2012) range shifts due toclimate change and glacial expansion and retreat(Birks 2008) glacial and interglacial refugia (Billings1974) and the Bering land bridge (Shapiro et al2004 Geml et al 2006)

ACKNOWLEDGMENTS

We thank Dr Ellen Larsson Dr Pierre-Arthur Moreau JukkaVauras Dr Ursula Peintner Dr Egon Horak and herbaria GIB TU O GB TURA C LIP MICH DBG VPI and NY forloan of specimens Carol Johnson and Dr Matt Lavin areacknowledged for reviewing early stages of the manuscriptWe thank Don Bachman Marcy Barge and Charissa Bujakfor assistance with field work We also gratefully acknowledgesupport from the Montana Institute on Ecosystems graduateenhancement fund

LITERATURE CITED

Abbott RJ Brochmann C 2003 History and evolution of thearctic flora in the footsteps of Eric Hulteacuten Mol Ecol12299ndash313 doi101046j1365-294X200301731x

Alsos IG Eidesen PB Ehrich D Skrede I Westergaard KJacobsen GH Landvik JY Taberlet P Brochmann C2007 Frequent long-distance plant colonization in thechanging arctic Science 3161606ndash1609 doi101126science1139178

Ballaraacute J 1997 Nou estudi drsquoespegravecies fuacutengiques interessantsdels estatges alpiacute i subalpiacute dels Pirineus catalans RevCatalana Micol 201ndash24

Barge E 2015 Systematics of Lactarius in the Rocky Moun-tain alpine zone (masterrsquos thesis) Bozeman MontanaMontana State University 265 p

Basso MT 1999 Lactarius Pers Fungi Europaei 7 AlassioMykoflora (Italy) 845 p

Beker HJ Eberhardt U Vesterholt J 2010 Hebeloma hiemaleBres In arcticalpine habitats N Am Fungi 551ndash65

Bessette AE Harris DB Bessette AR 2009 Milk mushroomsof North America a field identification guide to thegenus Lactarius Syracuse New York Syracuse UniversityPress 297 p

Billings WD 1974 Adaptations and origins of alpine plantsArctic Alpine Res 6129ndash142 doi1023071550081

mdashmdashmdash 1988 Alpine vegetation In Barbour MG BillingsWD eds North American terrestrial vegetation NewYork Cambridge University Press p 392ndash420

Birks HH 2008 The late-quaternary history of arctic andalpine plants Plant Ecol Divers 1135ndash146 doi10108017550870802328652

Bogar LM Kennedy PG 2013 New wrinkles in an old para-digm neighborhood effects can modify the structureand specificity of Alnus-associated ectomycorrhizal fun-gal communities FEMS Microbiol Ecol 83767ndash777doi1011111574-694112032

Bon M 1985a Stage mycologie alpine Lanslebourg (Savoie)du 1 aug 3 sep 1984 Bull Feacuted Mycol Daupineacute-Savoie9619ndash25

436 MYCOLOGIA

FIG 5 Combined (ITS+RPB2) Bayesian 50 majority-rule consensus tree of dataset C (Lactarius subgenus Piperites sectionUvidi) Support values (PAUP bootstrapRAxML bootstrapBayesian posterior probability) $ 60 (bootstrap) and $ 080(posterior probability) are indicated above or below branches leading up to clades Thickened branches lead to clades receiving

424 MYCOLOGIA

pseudouvidus group in RPB2 analyses (SUPPLEMENTARY

FIG 10) Collections of Lactarius pallidomarginatus Lac-tarius sp 3 L aspideus L montanus L luridus L affpseudouvidus and L aff brunneoviolaceus for which ITSdata are available all have a similar 268ndash269 base-pairinsertion in the ITS1 region There is variation withinthe base-pair content of the insertion between collec-tions however inclusion of this insertion did notincrease phylogenetic resolution in preliminary ana-lyses and it therefore was omitted The insertion itselfis highly similar to an insertion in Russula olivacea (eg GenBank accessions AF418634 AY061699) as tolength sequence (approximately 90 similar) andplacement within the ITS1 region

Also in dataset C Rocky Mountain alpine collectionsinitially identified as L repraesentaneus form a well-supported clade with a collection of L repraesentaneusfrom Germany (the lectotype country) as well as withnumerous collections from subalpine and arctic-alpineareas elsewhere in Eurasia and Alaska (FIG 5) Thisclade is sister to a poorly supported L dryadophilusKuumlhner clade The two GenBank sequences JX630967from arctic Canada with Dryas and JF394416 from arcticSvalbard which occur just outside the L dryadophilusclade are likely conspecific with L dryadophilusalthough this cannot be confirmed based on presentresults Lactarius repraesentaneus and L dryadophilus con-sistently differ at two nucleotide positions in the ITSlocus and at four positions in the RPB2 locus and haveconsistent morphological and host differences

In dataset C collections originally identified as L sal-icis-reticulatae from alpine areas in the Rocky Mountainsare paraphyletic with regard to L salicis-herbaceaeKuumlhner L aspideoides BurlL flavopalustris Kytoumlvand L aff flavopalustris (FIG 5) Collections of Lsalicis-reticulatae from alpine areas on the BeartoothPlateau (central Rocky Mountains) form a poorlysupported clade with an alpine collection of L salicis-reticulatae from Mt Laringktatjaringkka Sweden the type local-ity (sequencing of the holotype of L salicis-reticulataewas not successful) as well as with other arctic-alpinecollections of L salicis-reticulatae from Finland andAlaska and an unidentified collection from Svalbard(FIG 5) this clade is hypothesized to represent Lsalicis-reticulatae Collections from southern RockyMountain alpine areas in Colorado are larger thanreported for L salicis-reticulatae and form an unre-solved group that consistently differs at one nucleotideposition in the ITS region but no positions in the RPB2

locus they are tentatively referred to as L aff salicis-reticulatae pending data from additional loci

Overall phylogenetic analyses support the recogni-tion of six to seven Lactarius species from the RockyMountain alpine zone Lactarius lanceolatus (FIG 3)L nanus L glyciosmus (FIG 4) L pallidomarginatus spnov L repraesentaneus L salicis-reticulatae and possiblyL aff salicis-reticulatae (FIG 5) Violet-staining taxa Lpallidomarginatus L repraesentaneus L salicis-reticulataeand L aff salicis-reticulatae are resolved as a poorly sup-ported clade exclusively containing other members ofthe violet-staining section Uvidi supporting the mono-phyly of this section (FIG 2)

TAXONOMY

All descriptions are based on Rocky Mountain alpinematerial

Lactarius lanceolatus OK Mill amp Laursen FIGS 6 7Short description Lactarius lanceolatus is recognized by

its small to medium size and bright light to deeporange pileus pale cream to pale orange lamellae dis-coloring brownish in age or where damaged paleorange stipe watery unchanging latex and mild flavorgrowing in arctic-alpine (possibly also subalpine) areaswith Salix species

Pileus 10ndash45 mm diam convex at first becomingplane to infundibuliform with a depressed centerwith or without a small papilla smooth sometimesfaintly scaly toward center viscid deep orange-brownto deep orange when immature becoming light todeep orange often blotchy often darker toward cen-ter margin incurved to straight when immaturebecoming straight to upturned and often slightlywavy when mature Lamellae adnate to subdecur-rent slightly crowded pale cream to pale yellow topale orange discoloring brownish orange in age orwhere damaged Stipe 10ndash20 6 25ndash75 mm equalto clavate central smooth dry at first covered byfaint whitish pubescence pale orange discoloringdingy orange to dingy light brown where damagedhollow Latex scarce watery unchanging Contextof pileus and stipe pale orange Odor mild Flavormild Basidiospore deposit not observed Basidios-pores 8ndash10 6 6ndash8 mm average 5 86 6 7 mmQ 5 114ndash138 Q5 124 hyaline in 25 KOHbroadly ellipsoid ornamentation amyloid of isolatedwarts connected by fine lines up to 05 mm highBasidia 381ndash660 6 89ndash102 mm four-spored Pleur-omacrocystidia 737ndash127 6 64ndash102 mm narrowly

r$75 bootstrap support and Bayesian posterior probabilities $ 095 Boldface tip labels represent Rocky Mountain alpinespecimens AA 5 arctic-alpine See TABLE I for original identifications

BARGE ET AL ROCKY MOUNTAIN ALPINE LACTARIUS 425

fusiform to lanceolate with acute to moniliformapex thin-walled scattered to abundant Cheiloma-crocystidia 356ndash89 6 51ndash102 mm conical to fusi-form with acute to moniliform apex thin-walledsparse to abundant Pileipellis an ixotrichodermwith well-developed gelatinous layer

Distribution and habitat Lactarius lanceolatus origin-ally was described from arctic-alpine Alaska (Millerand Laursen 1973) and phylogenetic analyses(including the holotype) further confirm its pre-sence in the central Rocky Mountain alpine zoneand Svalbard In alpine areas on the Beartooth Pla-teau it occurs with Salix reticulata and possibly otherSalix species (S arctica S planifolia) it is notreported from the southern Rocky Mountains Over-all L lanceolatus appears largely confined to arctic-alpine habitats however an ITS sequence from amaritime forest in Alaska (GAL18572) clusters withthe arctic-alpine collections in phylogenetic analyses(FIG 3)

Previous arctic-alpine reports include Canada (Ohe-noja and Ohenoja 1993 2010) Alaska (Miller andLaursen 1973 Laursen and Ammirati 1982 Heil-mann-Clausen et al 1998) Beartooth Plateau in theRocky Mountain alpine zone (Cripps and Barge2013) Greenland (Heilmann-Clausen et al 1998 Bor-gen et al 2006) Fennoscandia (Bon 1998 Heilmann-Clausen et al 1998 Knudsen and Vesterholt 2008)Svalbard (Gulden and Jenssen 1988 Geml et al2012) the British Isles (Watling et al 2001) andSiberia (Nezdoiminogo 1997 Heilmann-Clausen et al1998 Karatygin et al 1999 Knudsen and Mukhin1998) however phylogenetic analyses (FIG 3) suggestthat some of these reports may represent L aurantiacus(Pers Fr) Gray

Specimens examined USA ALASKA North Slope CountyBeaufort Lagoon 30 Jul 1971 F4239 (Holotype VPI) MON-TANA Carbon County Beartooth Plateau Highline Trail 1Aug 1997 ZT6214 (MONT) 7 Aug 1998 ZT6412 (MONT)20 Aug 1999 CLC 1389 (MONT) 5 Aug 2008 CLC 2358(MONT) 27 Jul 1997 CLC 1139 (MONT) WYOMINGPark County Beartooth Plateau Solifluction Terraces 20July 2001 CLC 1578 (MONT) 13 Aug 2007 CLC 2319(MONT) 17 Aug 2013 EB 105-13 (MONT)

Other specimens examined DENMARK Broby Vesterskov 16Oct 1997 JHC97-251 (L aurantiacus C) NORWAY Leikan-ger 11 Sept 1999 JHC00-057 (L aurantiacus C) Nordland14 Aug 1999 JV15112F (L cf aurantiacus originally identi-fied as L lanceolatus TURA) Svalbard Longyearbyen 18Aug 2003 CLC 1885 (L cf aurantiacus originally identifiedas L lanceolatus MONT) SWEDEN Uppland 8 Sept 1994JV94-422 (L aurantiacus C) USA ALASKA Kenai PeninsulaCounty Palmer Creek Road 7 Aug 2011 CLC 2743 (L cfaurantiacus originally identified as L lanceolatus MONT)COLORADO El Paso County 27 Aug 2011 DBG-F-022653(L luculentus v laetus DBG) Gilpin County Perigo 13 Aug1974 AH Smith 84923 (Paratype of L luculentus v laetus

MICH) Larimer County 23 Aug 2008 DBG-F-024643 (Lluculentus v laetus DBG) OREGON Polk County 14 Nov1970 AH Smith 79943 (L luculentus MICH) WASHING-TON Lewis County 30 Oct 1972 AH Smith 83066 (Paratypeof L subviscidus MICH) 1 Nov 1972 AH Smith 83331 (Para-type of L subviscidus MICH) Tucannon River 11 Oct1980 AH Smith 90905 (L luculentus MICH)

Comments Lactarius lanceolatus belongs to a taxono-mically problematic group of species with orangeorange-brown or orange-red pilei including L auran-tiacus L luculentus L luculentus v laetus L subflammeusand L substriatus

Traditionally L lanceolatus and L aurantiacus havebeen separated mainly by ecology with L aurantiacusbeing restricted to a variety of subalpine habitats(although it has been reported from arctic-alpine habi-tats) and L lanceolatus to arctic-alpine habitats withSalix spp however it is clear from phylogenetic ana-lyses (FIG 3) that L aurantiacus also may occur in arc-tic-alpine habitats likely associating with Salix as well asKobresia and that the ranges of the two taxa overlap atleast in arctic-alpine areas of Alaska and Svalbard Ithas been suggested that L lanceolatus has longermacrocystidia and larger basidiospores than L auran-tiacus (eg Heilmann-Clausen et al 1998) howevercollections designated as L aurantiacus measured inthis study have pleuromacrocystidia that overlap andactually exceed the upper limit measured for L lanceo-latus including the holotype (see Barge 2015) Basi-diospore sizes for L aurantiacus also overlap andactually exceed the upper limit measured for L lanceo-latus in the collections examined (Barge 2015) Overallsolid morphological differences between the two taxawere not detected and further study may reveal thatthey are conspecific however provisionally L lanceola-tus may tend to have a thicker gelatinous layer over thepileipellis as well as a smooth pileus margin in contrastto the crenulate pileus margin reported for L aurantia-cus Lactarius aurantiacus is not reported from theRocky Mountains

For North American taxa Lactarius luculentus has abrowner pileus a slowly acrid flavor shorter cheilo-macrocystidia (23ndash45 6 4ndash6 mm) and exsiccati arepaler and more yellow-tan (Hesler and Smith 1979)Lactarius luculentus v laetus is similar in appearanceto L lanceolatus but also has shorter cheilomacrocysti-dia (15ndash30 6 4ndash8 mm) and possibly more crowdedlamellae (Hesler and Smith 1979) Lactarius substria-tus has a redder pileus that is glutinous when wetwith a translucent-striate margin latex that slowlybecomes straw yellow shorter cheilomacrocystidiaand an acrid flavor (Hesler and Smith 1979) Lactariussubflammeus also has a redder pileus (at least whenyoung) short translucent striations along its pileusmargin shorter cheilomacrocystidia and a slowly

426 MYCOLOGIA

FIG 6 A L lanceolatus (EB105-13) B L nanus (EB106-13) C L nanus (EB138) D L glyciosmus (ZT12723) E Lpallidomarginatus Barge amp Cripps sp nov (EB0041 HOLOTYPE) F L repraesentaneus (EB107-13) G L salicis-reticulatae(EB0057-14) H L aff salicis-reticulatae (EB0036) Photographs by E Barge (A-C E-H) E Horak (D) Bars 5 2 cm

BARGE ET AL ROCKY MOUNTAIN ALPINE LACTARIUS 427

acrid taste (pers obs of holotype AH Smith 83602Hesler and Smith 1979) Molecular phylogenetic ana-lyses (FIG 3) suggest that L subflammeus is conspecificwith L substriatus however sequencing of the holo-type of L substriatus is needed to confirm this observa-tion Lactarius luculentus L luculentus v laetus Lsubflammeus and L substriatus have been reportedonly from subalpine areas with conifers in westernNorth America (Hesler and Smith 1979)

Lactarius duplicatus AH Sm (syn L lapponicus Har-maja) a North American and European Betula associ-ate is also morphologically close to L lanceolatusHowever it can be distinguished by the productionof strongly yellowing latex We did not procure anymolecular data from material representing this taxonand no molecular data is publicly available on Gen-Bank or UNITE However L duplicatus should beincluded in future phylogenetic analyses investigatingthe L lanceolatusL aurantiacus group

Lactarius nanus J Favre FIGS 6 7Short description Lactarius nanus is recognized by its

typically small size gray-brown to liver brown pileustypically with a glaucous coating at least when youngcream to pinkish tan lamellae pale cream-tan to paleapricot stipe watery unchanging latex mild odormild to slightly acrid flavor and occurrence with Salixspecies in arctic-alpine areas

Pileus 10ndash50 mm diam shallowly convex to plano-convex becoming plane to infundibuliform with orwithout a depressed center and small papilla smoothslightly viscid to dry with whitish glaucous coatingwhen immature easily rubbing away or fading in ageoften in patches deep brown to gray-brown to liverbrown with lighter and darker areas present oftenlighter toward margin becoming lighter overall upondessication margin straight when immature becomingupturned and wavy to convoluted when matureLamellae adnate to subdecurrent subdistant to slightlycrowded cream when immature becoming dingycream to light tan in age sometimes with a faint pink-ish tinge discoloring to dingy tan where damagedStipe 5ndash30 6 3ndash15 mm equal to clavate centralsmooth dry at first covered with whitish glaucous coat-ing as in pileus pale apricot to dingy cream-tan hol-low Latex scarce watery unchanging Context ofpileus and stipe dingy cream to brownish Odor mildFlavor mild to slightly acrid Basidiospore deposit notobserved Basidiospores 7ndash105(ndash12) 6 5ndash8 mm av 584 6 66 mm Q 5 107ndash160 Q 5 127 hyaline in25 KOH subglobose to ellipsoid ornamentationof amyloid scattered ridges and warts forming anincomplete reticulum with some completely reticulateareas to 07 mm high Basidia 406ndash762 6 89ndash152 mm mostly four-spored some two-spored

Pleuromacrocystidia 533ndash965(ndash1143) 6 64ndash114mm lanceolate with acute to moniliform apexscattered to abundant Cheilomacrocystidia 260ndash9146 76ndash102 mm subulate to linear with undulatingmargin with acute to rounded apex scattered toabundant Pileipellis an (ixo-) cutis with some ascend-ing hyphae embedded in a very faint gelatinouslayer

Distribution and habitat Lactarius nanus originally wasdescribed by Jules Favre from the Swiss Alps with Salixherbacea L (Favre 1955) Phylogenetic analyses includ-ing a specimen from near the type locality confirmthis species in the Rocky Mountain alpine zone whereit is one of the most commonly encountered Lactariusspecies it associates with Salix arctica S reticulata andS planifolia Overall L nanus appears to be largelyrestricted to arctic-alpine areas with Salix

Previous arctic-alpine reports include Canada (Ohe-noja and Ohenoja 1993 2010) the Rocky Mountainalpine zone (Moser and McKnight 1987 Cripps andHorak 2008 Cripps and Barge 2013) Greenland(Knudsen and Borgen 1982 Lamoure et al 1982 Heil-mann-Clausen et al 1998 Borgen et al 2006) Iceland(Eyjolfsdottir pers comm Knudsen and Vesterholt2008) the European Alps (Favre 1955 Kuumlhner 1975Kuumlhner and Lamoure 1986 Senn-Irlet 1993 Bon1985a 1998 Heilmann-Clausen et al 1998 Jamoni1995) the Pyrenees (Bon and Ballaraacute 1996) the Car-pathians (Ronikier 2008) Fennoscandia (Kuumlhner1975 Gulden et al 1985 Heilmann-Clausen et al1998 Gulden 2005 Knudsen and Vesterholt 2008)Svalbard (Gulden et al 1985) Siberia (Nezdoiminogo1997) and alpine areas in the Altai Mountains (Karaty-gin et al 1999)

Specimens examined FRANCE Savoie 24 Aug 1989 Bon89093 (LIP) NORWAY Finse 5 Aug 2010 CLC 2134(MONT) Svalbard Longyearbyen 20 Aug 2002 CLC 1896(MONT) SWEDEN Torne Lappmark 15 Aug 1999JV15148 (TURA) SWITZERLAND Grisons 20 Aug 1943ZA192c (Holotype G) USA COLORADO Pitkin CountySawatch Range Independence Pass 6 Aug 2000 CLC 1471(MONT) San Juan County San Juan Range Black BearPass 11 Aug 2001 CLC 1716 (MONT) 11 Aug 2001ZT9529 (MONT) San Juan County San Juan Range Cinna-mon Pass 27 July 2002 CLC 1801 (MONT) 15 Aug 2012EB138 (MONT) San Miguel County San Juan Range Imo-gene Pass 29 July 2002 CLC 1829 (MONT) MONTANACarbon County Beartooth Plateau Highline Trail 7 Aug1998 CLC 1221 (MONT) 8 Aug 1998 ZT6422 (MONT) 18Aug 2013 EB106-13 (MONT) WYOMING Park CountyBeartooth Plateau Frozen Lakes 21 Aug 1999 CLC 1403(MONT) Park County Beartooth Plateau Solifluction Ter-races 11 Aug 2012 EB125 (MONT)

Other specimens examined FINLAND Koillismaa 28 Aug2011 JV28432 (L hysginoides TURA)

428 MYCOLOGIA

Comments Lactarius nanus is morphologically andphylogenetically close to L hysginoides however thelatter has a more cinnamon-toned pileus smaller basi-diospores (6ndash8 6 5ndash7 mm av 68 6 57 mm) and isgenerally reported from boreal areas with Betula Piceaand Salix in Europe (Heilmann-Clausen et al 1998)although phylogenetic placement of environmentalsequences extend its range into boreal Alaska (FIG4) Its range likely overlaps that of L nanus in alpine-subalpine transition areas It is not reported from theRocky Mountains Lactarius mammosus is a more robustspecies with a coconut odor (Heilmann-Clausen et al1998) that associates with members of the Pinaceaeit has not been reported from the Rocky MountainsDifferences between L glyciosmus and L nanus are dis-cussed in the taxonomy section for L glyciosmus

Lactarius glyciosmus (Fr) Fr FIGS 6 7Short description Lactarius glyciosmus is recognized by

its small to medium size pale gray-brown to mauvepileus typically with a glaucous coating at least whenyoung cream pale pinkish or pale yellow-orangelamellae buff or pale salmon stipe watery unchan-ging latex odor of coconuts mild flavor and associa-tion with Betula

Pileus 15ndash50 mm diam broadly convex later becom-ing plane with or without a depressed center and smallpapilla smooth dry pale gray-brown to mauve (violet-brown) with whitish glaucous coating when immaturefading in age azonate to lightly zoned especially nearmargin margin incurved when young and remainingso or becoming straight when mature Lamellaeadnate to subdecurrent slightly crowded to crowdedcream to pale pinkish to pale yellow-orange Stipe10ndash40 6 3ndash12 mm equal to slightly clavate oftencurved central to eccentric smooth dry buff or palesalmon with faint glaucous coating at first as in pileusstuffed or hollow often white-mycelioid toward baseLatex scarce watery unchanging Context of pileusand stipe pale cream Odor of coconut Flavor mildBasidiospore deposit not observed Basidiospores 7ndash96 5ndash7 mm av 5 77 6 60 mm Q 5 117ndash142 Q 5

128 hyaline in 25 KOH broadly ellipsoid to ellip-soid ornamentation of amyloid scattered ridges andwarts forming an incomplete to nearly complete reticu-lum to 05 mm high Basidia 381ndash635 6 76ndash102 mmmostly four-spored some two-spored Pleuromacrocys-tidia 457ndash635 6 64ndash76 mm subclavate to lanceolatewith rounded to occasionally mucronate apex scarceto scattered Cheilomacrocystidia 33ndash66 6 51ndash89mm cylindrical to subclavate with rounded apex scat-tered to abundant Pileipellis a cutis with tufts ofascending hyphae

Distribution and habitat Lactarius glyciosmus was firstdescribed by Fries from a hemiboreal to boreal habitat

in the Femsjouml region of Sweden and the species lacks atype specimen Phylogenetic analyses including speci-mens from the type locality confirm this species inthe central and southern Rocky Mountain alpinezone and suggest it has a broad ecological and geogra-phical distribution confirmed from subalpine and arc-tic-alpine areas in Alaska western Canada IdahoWisconsin New Hampshire Svalbard and Fennoscan-dia In subalpine and alpine areas of the Rocky Moun-tains L glyciosmus associates with Betula glandulosaPhylogenetic placement (FIG 4) of ectomycorrhizalroot tip sequences confirm Betula nana L as a host inAlaska (GU998223 GU998695) and likely B occidenta-lis Hook in Idaho (JX198526 Bogar amp Kennedy2013) Elsewhere it is reported with B pubescens Ehrh(eg Elborne amp Knudsen 1990) and B papyrifera Mar-shall (eg Durall et al 2006)

Previous reports from arctic-alpine areas includeCanada (Ohenoja and Ohenoja 1993 2010) theRocky Mountain alpine zone (Cripps and Horak2008 Cripps and Barge 2013) Greenland (Lange1957 Knudsen and Borgen 1982 Lamoure et al1982 Elbourne and Knudsen 1990 Borgen 2006 Bor-gen et al 2006) Iceland (Eyjolfsdottir pers comm) theAlps (Bon 1998) Fennoscandia (Kallio and Kankainen1964 Kuumlhner 1975 Bon 1998) Svalbard (Ohenoja1971 Geml et al 2012) and Siberia (Karatygin et al1999) At least some reports might be L aff glyciosmusas defined here

Specimens examined FINLAND Fjell Ailigas 27 Aug 2012CLC 2874 (MONT) SWEDEN Femsjouml 13 Aug 1978 MMoser 19780191 (Type locality IB) 16 Aug 1978 M Moser19780234 (Type locality IB) 9 Aug 1981 M Moser19810148 (Type locality IB) USA COLORADO BoulderCounty Front Range Blue Lake 2 Aug 2001 CLC 1624(MONT) Boulder County Front Range Brainard Lake 13Aug 2000 20923 (DBG) Ouray County San Juan RangeIronton Park 13 Aug 2012 EB133 (MONT) MONTANACarbon County Beartooth Plateau Birch Site 29 July 1997CLC 1134 5 ZT6096 (MONT) 8 Aug 1998 CLC 1217(MONT) 10 Aug 1999 TWO 269 (MONT) 19 Aug 1999CLC 1380 (MONT) 13 Aug 2007 ZT12723 (MONT)

Other specimens examined ESTONIA Saare CountyLuumlmanda Commune 17 Sep 2012 TU118535 (L aff glycios-mus TU) Tartu County Votildennu Commune 12 Sept 2010TU106737 (L aff glyciosmus TU) 13 Sept 2010 TU106724(L aff glyciosmus TU)

Comments Lactarius glyciosmus is morphologically andphylogenetically similar to L mammosus however thelatter has a browner pileus stouter stature and moreelongate more reticulate basidiospores (Heilmann-Clausen et al 1998) Phylogenetic placement ofsequences isolated from ectomycorrhizal root tips ofPinus sylvestris L (AJ633116 Heinonsalo et al 2007)and Picea abies (L) Karst (FM992937 Kjoslashller andClemmensen 2009) (FIG 4) confirm that L mammosus

BARGE ET AL ROCKY MOUNTAIN ALPINE LACTARIUS 429

FIG 7 Comparison of micromorphological features of Rocky Mountain alpine specimens and Holotypes (where available)Basidiospores pleuromacrocystidia (left) and cheilomacrocystidia (right) of A Lactarius lanceolatus (CLC2319) B L lanceolatus(F 4239 HOLOTYPE) C L nanus (CLC1471) D L nanus (ZA 192c HOLOTYPE) E L pallidomarginatus Barge amp Cripps spnov (EB0041 HOLOTYPE) F L pseudouvidus (Va 71971 HOLOTYPE) G L salicis-reticulatae (CLC2776) H L salicis-reticulatae

430 MYCOLOGIA

associates with members of the Pinaceae however italso has been reported with Betula mainly in subalpineareas (Heilmann-Clausen et al 1998) Lactarius vietus(Fr) Fr is also similar but has a distinct pale zonenear the stipe apex latex that dries greenish graylonger pleuromacrocystidia and a mild to slightly fruityodor (Heilmann-Clausen et al 1998) It has not beenreported from alpine areas in the Rocky Mountainsalthough it has been reported from elsewhere (mostlynorthern areas) in North America with Betula (Heslerand Smith 1979) The similar-looking L nanus alsooccurs in alpine areas of the Rocky Mountains andhas larger basidiospores larger and differently shapedmacrocystidia lacks an odor of coconut and associateswith Salix Lactarius spinosulus is phylogenetically closeto L glyciosmus (as well as L nanus) (FIG 4) howevermorphologically they are very different Lactarius spino-sulus has a distinctly zoned coarsely squamulose pink-ish salmon- or brick-colored pileus with a finely hairymargin and fruity odor (Heilmann-Clausen et al1998) it has not been reported from North AmericaNo definitive morphological differences were detectedbetween L glyciosmus and L aff glyciosmus and the lat-ter is not reported from the Rocky Mountainalpine zone

Lactarius pallidomarginatus Barge amp CL Cripps spnov FIGS 6 7Mycobank MB812246

Typification USA COLORADO San Juan CountySan Juan Range Stony Pass scattered solitary onsoil under Salix planifolia 13 Aug 2011 EB0041 (holo-type MONT) GenBank KR090940 (ITS) andKR091010 (RPB2)

Etymology Referring to the pale pileus marginDiagnosis Lactarius pallidomarginatus is recognized by

its small to medium size blotchy light tan to lightbrown to gray-brown pileus which is typically lightertoward the margin white to pale yellow-cream lamel-lae pale cream stipe violet staining tissue mild odorand flavor and association with Salix Morphologicallyit is close to L pseudouvidus Kuumlhner but differs by hav-ing smaller basidiospores with an ornamentation ofthinner ridges as well as macrocystidia that becomemore lanceolate (as opposed to subfusiform) and atte-nuated Phylogenetically it is close to L aspideus (Fr)Fr but differs consistently in two nucleotide positionsin the rDNA ITS locus and at eight nucleotide posi-tions in the RPB2 locus morphologically L aspideushas larger yellow basidiomata

Pileus 20ndash50 mm diam convex to broadly convexbecoming broadly convex to plane with or without adepressed center smooth dry to slightly viscid azo-nate blotchy light tan to light brown to gray-browndeveloping violet stains lighter (to cream) toward mar-gin margin incurved to downturned when youngremaining downturned or becoming nearly straightwhen mature Lamellae adnate to subdecurrent subdi-stant to slightly crowded white to pale yellow-creamstaining violet where damaged Stipe 10ndash40 6 5ndash10mm equal to slightly clavate central smooth drywhite to cream staining violet where damaged hollowLatex scarce watery staining tissue violet Context ofpileus and stipe white to cream staining violet wheredamaged Odor mild Flavor mild Basidiosporedeposit not observed Basidiospores 8ndash10 6 65ndash8mm av 5 87 6 71 mm Q 5 114ndash136 Q5 123 hya-line in 25 KOH broadly ellipsoid to ellipsoid orna-mentation amyloid of thin ridges and a few isolatedwarts forming an incomplete to dense reticulum to05 mm high Basidia 508ndash711 6 102ndash127 mm four-spored Pleuromacrocystidia 813ndash1118 6 89ndash102mm cylindrical to lanceolate with acute to moniliformapex scarce to scattered Cheilomacrocystidia 483ndash1016 6 76ndash127 mm cylindrical to lanceolate withacute to moniliform apex scattered Pileipellis an(ixo-)cutis with some ascending hyphae embedded ina faint gelatinous layer

Distribution and habitat Rare in low alpine areas withSalix planifolia reported from Independence Pass inthe Sawatch Range and Stony Pass in the San JuanRange in Colorado An additional collection(ZT5229) from Union Peak in the Wind River Rangeof Wyoming with Salix glauca is likely conspecific how-ever this hypothesis has not been confirmed withmolecular data Due to confusion with L pseudouvidusit is possible that L pallidomarginatus is more wide-spread than confirmed here

Specimens examined USA COLORADO Pitkin CountySawatch Range Independence Pass 6 Aug 2000 CLC 1470(MONT) 9 Aug 2000 ZT9093 (MONT) San Juan CountySan Juan Range Stony Pass 13 Aug 2011 EB0041 (HolotypeMONT) WYOMING Sublette County Wind River RangeUnion Peak 22 Aug 1994 ZT5229 (MONT)

Other specimens examined AUSTRIA Tyrol 3 July 2007 UPeintner 20070035 (L aff pseudouvidus from near type local-ity IB) FINLAND Inarin Lappi 16 Aug 1995 JV10468 (Laff pseudouvidus TURA) Koillismaa JV28448F 30 Aug 2011(L aff pseudouvidus originally identified as L brunneoviola-ceus TURA) FRANCE Isegravere 13 Aug 1971 Va 7 1971 (Holo-type of L pseudouvidus G) ITALY Trentino 20 Aug 2004 U

r(K72-104 HOLOTYPE no cheilocystidia drawn) I L glyciosmus (TWO269) J L repraesentaneus (CLC1747) Bars 5 10 mm forbasidiospores and 20 mm for cystidia Drawings by E Barge

BARGE ET AL ROCKY MOUNTAIN ALPINE LACTARIUS 431

Peintner 20040156 (L aff pseudouvidus from near type local-ity originally identified as L robertianus IB) NORWAYFinse 10 Aug 2005 CLC 2133 (Lactarius aff brunneoviolaceusMONT) Stordal 26 Aug 2006 P Larsen 361395 (L aff brun-neoviolaceus originally identified as L pseudouvidus O) Sval-bard 18 Aug 2002 TWO809 (L aff pseudouvidus MONT)20 Aug 2002 CLC 1910 (Lactarius sp 5 MONT) Ulvik 11Aug 2011 E Soslashyland 73867 (L aff pseudouvidus O) SWE-DEN Latnjajaure 4 Aug 2011 EL101-11 (L aff pseudouvidusGB) Latnjavagge 17 Aug 2010 EL63-10 (L aff pseudouvi-dus GB)

Comments Morphological features of L pallidomar-ginatus fit the holotype description of L pseudouvidusand both are described as lighter near the pileus mar-gin (Kuumlhner 1975 Basso 1999) however basidios-pores of L pallidomarginatus are slightly smaller (8ndash10 6 65ndash8 mm as opposed to 75ndash12 6 65ndash8(85)mm) and have narrower ridges that form a less densereticulum and macrocystidia take on a more lanceo-late form with greater attenuation of the apex (FIG7) In phylogenetic analyses exsiccati identified as Lpseudouvidus from Europe (including specimensfrom near the type locality) appear as distinct fromRocky Mountain alpine material and may representa species complex Lactarius sp 3 (CLC1910) col-lected in Svalbard has darker more vinaceous pileithan L pallidomarginatus and may represent anotherundescribed species although only one collectionwas morphologically examined Members of the Laff brunneoviolaceus clade (FIG 5) that were exam-ined differ from L pallidomarginatus in having dar-ker more vinaceous pilei larger more elongatebasidiospores (to 13 mm) and green staining of exsic-cati in 25 KOH a feature not mentioned previouslyfor L brunneoviolaceus but which could be of taxo-nomic interest Based on taxa included in the phylo-genetic analyses (FIG 5) Lactarius aspideus appearsclosely related to L pallidomarginatus however theformer has more yellow coloration and typically lar-ger sporocarps (pileus 10ndash70 mm diam) and smallerbasidiospores (67ndash95 6 56ndash78 mm) traditionallyL aspideus has been placed with other yellow violet-staining taxa such as L flavidus Boud L salicis-reticu-latae and L salicis-herbaceae (Heilmann-Clausen et al1998) but it is separated here

Lactarius montanus another violet-staining speciesoriginally described from the Rocky Mountains dif-fers from L pallidomarginatus by its larger staturedarker grayish vinaceous to vinaceous-brown pileusand green staining flesh in 25 KOH it is typicallyfound in subalpine habitats with conifers but alsooccurs in the krummholz zone The violet-stainingLactarius pallescens Hesler amp AH Sm occurs in theRocky Mountains but its sporocarps are larger andlighter in color and it is reported from subalpineconifer forests (Hesler and Smith 1979) Other

gray-brown to brownish violet-staining species suchas L luridus L uvidus (Fr) Fr L violascens (JOtto) Fr L pallescens v palmerensis Hesler amp AHSm and L cordovaensis Hesler amp AH Sm are alsolarger and have a subalpine ecology none havebeen reported from the central or southern RockyMountains (Hesler and Smith 1979 Heilmann-Clau-sen et al 1998) Lactarius cistophilus Bon amp Trimbachtypically has larger basidiomata larger basidiospores(10ndash14 6 72ndash9 mm) and occurs under Cistus inthe Mediterranean region (Basso 1999) The smallpale gray-brown European alpine taxon L uvidus falpigines Jamoni amp Bon has microscopic featuresthat match those of L uvidus (Jamoni and Bon1991) but not L pallidomarginatus The small non-violet staining L nanus has basidiospores withthicker more jagged ridges and differently shapedmacrocystidia (often with more rounded apices)(FIG 7)

Lactarius repraesentaneus Britzelm FIGS 6 7Short description Lactarius repraesentaneus is recog-

nized by its medium to large size orange-brown to yel-low-brown hairy pileus with a heavily bearded margincream to pale-yellow lamellae stout cream to pale-yel-low often pitted stipe violet staining latex and tissuespicy-floral odor slightly acrid flavor and associationwith Betula Picea and possibly shrubby Salix speciessuch as S glauca

Pileus 60ndash100 mm diam convex to plano-convexusually with a depressed center and sometimes with apapilla smooth at center becoming increasingly hairyto bearded toward margin dry to viscid azonate sur-face orange-brown at center becoming pale yellow-brown to cream toward margin hairs pale yellow-brown to red-brown margin strongly incurvedremaining so or becoming nearly straight whenmature Lamellae adnate to decurrent crowdedcream to pale-yellow staining violet where damagedStipe 30ndash70 6 15ndash35 mm stout equal to clavate cen-tral dry to viscid cream to pale yellow surface oftenwith numerous dingy yellow golden yellow yellow-brown or light orange-brown pits hollow Latex scarceto abundant white becoming violet Context of pileusand stipe white staining violet where damaged Odorspicy-floral Flavor slightly acrid Basidiospore depositnot observed Basidiospores 8ndash105 6 6ndash85 mm av5 93 6 73 mm Q 5 113ndash143 Q5 127 hyaline in25 KOH broadly ellipsoid to ellipsoid ornamenta-tion amyloid of narrow to broad ridges and isolatedwarts forming an incomplete to dense reticulum to1 mm high Basidia 610ndash762 6 102ndash114 mm four-spored Pleuromacrocystidia 787ndash1448 6 89ndash127 mm subfusiform to lanceolate thin-walled withacute to moniliform apex scattered to abundant

432 MYCOLOGIA

Cheilomacrocystidia 559ndash1397 6 76ndash127 mm subfu-siform to lanceolate thin-walled with acute to monili-form apex scattered to abundant Pileipellis anixotrichoderm with well-developed gelatinous layer

Distribution and habitat Lactarius repraesentaneus firstwas described from Germany (Britzelmayr 1885) Phy-logenetic analyses including material from Germanyconfirm L repraesentaneus from alpine areas of theRocky Mountains and overall reinforce the traditionalconcept that this species has a broad ecological andgeographic range in arctic-alpine and subalpine habi-tats in North America northern central and southernEurope In alpine areas of the Rocky Mountains itassociates with Salix glauca however krummholz Piceaalso is typically (maybe always) present it is reportedwith Picea and Betula elsewhere

Previous reports from arctic-alpine areas mainlywith Betula include Alaska (Laursen and Ammirati1982 Geml et al 2009) the Rocky Mountain alpinezone (Cripps and Horak 2008 Cripps and Barge2013) Greenland (Knudsen and Borgen 1982 Watling1983 Elbourne and Knudsen 1990 Borgen 2006 Bor-gen et al 2006) Iceland (Eyjolfsdottir pers comm)Fennoscandia (Kallio and Kankainen 1964 Kuumlhner1975 Bon 1998) Siberia (Karatygin et al 1999) andthe Altai Mountains (Gorbunova 2010)

Specimens examined FINLAND Enontakiouml 16 Aug 2004JV21671 (TURA) Varsinais-Suomi 9 Aug 1998 JV13837F(TURA) SWEDEN Latnjavagge 12 Aug 2007 EL92-07(GB) USA COLORADO Chaffee County Sawatch RangeCottonwood Pass 4 Aug 2001 CLC 1643 (MONT) 16 Aug2011 EB0048 (MONT) Pitkin County Sawatch Range Inde-pendence Pass 14 Aug 2001 CLC 1747 (MONT) ZT9837(MONT) 15 Aug 2001 ZT9537 (MONT) MONTANA Car-bon County Beartooth Plateau Birch Site 12 Aug 2002CLC 1971 (MONT) 13 Aug 2007 CLC 2318 (MONT) 18Aug 2013 EB107-13 (MONT) WYOMING Park CountyBeartooth Plateau Frozen Lakes 21 Aug 1999 CLC1394 (MONT)

Other specimens examined SWEDEN Latnjavagge 14 Aug2010 EL57-10 (L dryadophilus GB) USA ALASKA 5 Aug2011 CLC 2729 (L dryadophilus MONT) Kenai PeninsulaCounty Palmer Creek Road 7 Aug 2011 CLC 2744 (L drya-dophilus MONT)

Comments The combination of a large pale yellow-brown to orange-brown pileus with a bearded marginand violet staining latex clearly separates L repraesenta-neus from other Lactarius species of the central andsouthern Rocky Mountain alpine zone Phylogeneti-cally close the arctic-alpine taxon L dryadophilus ismorphologically similar but has a cream to yellowsmoother pileus with hairs restricted to near the mar-gin basidiospore ornamentation consisting of sparsethinner ridges and ecological associations with Dryasand Salix It has not been reported from the RockyMountains

Lactarius salicis-reticulatae Kuumlhner FIGS 6 7Short description Lactarius salicis-reticulatae is recog-

nized by its small to medium cream pale yellow orpale ocher sporocarps with pale yellowish well-spacedlamellae often with a pinkish buff tint cream to pale yel-low stipe violet staining latex and tissue mild to slightlysweet odor mild flavor and association with dwarf (Sreticulata) and possibly also shrubby Salix species

Pileus 20ndash40 mm diam convex to broadly convexusually with a depressed center smooth dry to viscidazonate cream pale yellow or pale ocher sometimesdarker toward center staining violet where damagedmargin invcurved remaining so or becoming down-turned when mature Lamellae adnate to subdecur-rent fairly well-spaced cream pale yellow or paleorange-ocher often with a pinkish-buff tint stainingviolet where damaged Stipe 15ndash20 6 10ndash15 mmequal to clavate central dry to viscid cream to pale-yellow staining violet where damaged hollow Latexscarce watery-white becoming violet Context ofpileus and stipe white staining violet where damagedOdor mild to slightly sweet Flavor mild Basidiosporedeposit not observed Basidiospores (7ndash)85ndash115 6(7ndash)8ndash10 mm av 5 100 6 88 mm Q 5 (100ndash)113ndash143 Q 5 125 hyaline in 25 KOH sublgobose toellipsoid ornamentation amyloid of narrow ridgesand a few isolated warts forming an incomplete reticu-lum to 03 mm high Basidia 737ndash889 6 102ndash127 mm four-spored Pleuromacrocystidia 762ndash10166 76ndash114 mm subfusiform to fusiform thin-walledwith rounded to acute to moniliform apex scarceCheilomacrocystidia 686ndash9146 76ndash102 mm subfusi-form to fusiform thin-walled with acute to rounded tomoniliform apex scattered to abundant Pileipellis anixocutis

Distribution and habitat Lactarius salicis-reticulatae firstwas described from alpine tundra with Salix on MtLaringktatjaringkka Sweden (Kuumlhner 1975) Phylogenetic ana-lyses including material from the type locality confirmthis species from alpine areas on the Beartooth Plateauwith Salix reticulata and shrubby Salix spp and in arctic-alpine areas of Alaska Svalbard and Finland OverallL salicis-reticulatae appears largely confined to arctic-alpine habitats Colorado collections with S reticulataand S planifolia may represent a distinct taxonalthough support is low

Previous arctic-alpine reports include Greenland(Lamoure et al 1982 Heilmann-Clausen et al1998 Borgen et al 2006) the Rocky Mountainalpine zone (Cripps and Horak 2008 Cripps andBarge 2013) the Alps (Kuumlhner 1975 Kuumlhner andLamoure 1986 Jamoni 1995 2008 Bon 1998 Heil-mann-Clausen et al 1998) the Pyrenees (Ballaraacute1997) Fennoscandia (Kuumlhner 1975 Heilmann-Clau-sen et al 1998 Gulden 2005 Knudsen and

BARGE ET AL ROCKY MOUNTAIN ALPINE LACTARIUS 433

Vesterholt 2008) and Siberia (Nezdoiminogo 1997Karatygin et al 1999) previous reports of L aspi-deoides Burl from arctic-alpine areas in Alaska (Laur-sen and Ammirati 1982) Greenland (Knudsen andBorgen 1982) and the British Isles (Watling 1987)may refer to this species

Specimens examined FINLAND Near Utsjoki 28 Aug 2012CLC 2885 (MONT) SWEDEN Mt Laringktatjaringkka 12 Aug1972 K72-104 (Holotype G) 15 Aug 1999 JV15133 (Typelocality TURA) USA ALASKA Kenai Peninsula County Pal-mer Creek Road 7 Aug 2011 CLC 2745 (MONT) MON-TANA Carbon County Beartooth Plateau Birch Site 17Aug 2011 CLC 2776 (MONT) Carbon County BeartoothPlateau Highline Trail 8 Aug 1998 CLC 1211 (MONT)WYOMING Park County Beartooth Plateau GardnerLake 16 Aug 2014 EB0057-14 (MONT)

Other specimens examined FINLAND Koillismaa 31 Aug2005 JV23334 (L flavopalustris TURA) GREENLAND Sis-miut 19 Aug 2000 CLC 1536 (L salicis-herbaceae MONT)USA COLORADO Pitkin County Sawatch Range Indepen-dence Pass 13 Aug 2001 CLC 1741 (L aff salicis-reticulataeMONT) San Juan County San Juan Range CinnamonPass 10 Aug 2001 CLC 1710 (L aff salicis-reticulataeMONT) San Juan County San Juan Range Maggie Gulch11 Aug 2011 EB0036 (L aff salicis-reticulatae MONT)San Juan County San Juan Range Stony Pass 9 Aug 2001CLC 1689 (L aff salicis-reticulatae MONT) 13 Aug 2011EB0039 (L aff salicis-reticulatae MONT) MICHIGANEmmet County 10 Aug 1973 RL Shaffer 6957 (L aspi-deoides MICH)

Comments Some taxonomic confusion has beencaused by the fact that Kuumlhner (1975) originallynamed this species L aspideoides Kuumlhner but subse-quently renamed it because the name had been pre-viously applied L aspideoides Burl (1907) is generallylarger with a somewhat zonate pileus more crowdedlamellae that lack salmon-colored hues slightly smallerbasidiospores with broader ridges a bitter to slightlyacrid flavor and a subalpine ecology apparently ineastern and western North America (pers obs of RLShaffer 6957 Hesler and Smith 1979) Lactarius fla-vopalustris is also similar however it has a largermore brightly yellow pileus with faint zonationmore crowded lamellae a weakly scrobiculate stipe(sometimes) smaller basidiospores with thickerridges and a subalpine ecology (Kytoumlvuori 2009)Phylogenetic analyses (FIG 5) reveal three collec-tions of L flavopalustris (two from near the typelocality in Finland) nested in a well-supported cladewith L aspideoides from a subalpine forest in north-ern Michigan suggesting the possible conspecificityof these taxa Neither is reported from the RockyMountains Two other collections identified as L fla-vopalustris (from Estonia) were resolved as distinctfrom the L aspideoidesflavopalustris clade (FIG 5)their identity is unclear

Lactarius aspideus is differentiated from L salicis-reti-culate by its smaller basidiospores with denser reticula-tion more crowded creamier-colored lamellae andhabitat mainly in subalpine areas (Heilmann-Clausenet al 1998) although an environmental sequence iso-lated from an S polaris root tip from arctic Svalbardclusters with L aspideus in phylogenetic analyses (FIG5) Lactarius dryadophilus is often larger with a cream-colored pileus that has a tomentose to bearded margin(Heilmann-Clausen et al 1998) Lactarius flavidus is lar-ger with a sparsely zonate pileus more crowdedcream-colored lamellae darker violet-staining contextbasidiospores with more prominent ornamentationand a distally amyloid plage and subalpine ecology(Heilmann-Clausen et al 1998) Lactarius flavoaspideusKytoumlv has slightly smaller basidiospores on averagewith a denser reticulum and subalpine ecology withSalix (Kytoumlvuori 2009) it has been reported onlyfrom northern Europe Lactarius salicis-herbaceae asimilar arctic-alpine Salix associate has smaller (typi-cally) darker yellow to yellow-brown sporocarpspale cream to grayish buff lamellae and more denselyreticulate basidiospores (pers obs Heilmann-Clausenet al 1998) It is fairly widespread in arctic-alpineareas but is not reported from alpine areas in the cen-tral and southern Rocky Mountains Lactarius aff sali-cis-reticulatae from Colorado may be a distinct taxonand sporocarps can obtain a larger size (pileus to 90mm diam) but otherwise morphological featuresstrongly overlap

KEY TO ROCKY MOUNTAIN ALPINE LACTARIUS SPECIES

1 Latex white turning violet to dull lilac or flesh stain-ing violet to dull lilac 2

1 Latex watery to white unchanging and flesh notstaining violet to dull lilac 42 Fruiting body robust pileus 60ndash100 mm diam

yellow-brown to orange-brown hairy especiallynear margin lamellae cream to pale-yellow stipestout cream to pale-yellow often with numerousyellow-brown pits odor spicy-floral flavor mildlyacrid basidiospores 8ndash105 6 6ndash85 mm puta-tively associated with Betula and Picea (possiblyalso Salix) in Rocky Mountain alpine zone andsubalpine and arctic-alpine areas in North Amer-ica and Eurasia L repraesentaneus

2 Fruiting body smaller 20ndash50 mm diam pileus andmargin smooth 3

3 Pileus pale yellow to pale ocher lamellae cream paleyellow or pale orange-ocher stipe cream to pale yel-low odor mild to slightly sweet flavor mild basidios-pores 85ndash115 6 8ndash10 mm in Rocky Mountain alpinezone and other arctic-alpine areas in North Americaand Eurasia with Salix L salicis-reticulatae

3 Pileus blotchy light tan to light brown to gray-brownmargin lighter lamellae white to pale yellow-cream

434 MYCOLOGIA

stipe white to cream odor mild flavor mild basi-

diospores 8ndash10 6 65ndash8 mm in low alpine areas inColorado with Salix planifolia and possibly Wyom-

ing with Salix glauca L pallidomarginatus4 Pileus bright orange to orange-brown 10ndash45

mm diam margin smooth lamellae pale creamto pale orange stipe pale orange odor mildflavor mild in Rocky Mountain alpine zonearctic-alpine Alaska (possibly also subalpine)to Svalbard with Salix L lanceolatus

4 Pileus pinkish buff gray-brown brown ormauve 5

5 With odor of coconuts pileus 15ndash50 mm diam palepinkish buff to pale gray-brown with glaucous coat-ing lamellae cream to pale yellow-orange stipe buffor pale salmon flavor mild basidiospores 7ndash9 6 5ndash7mm in Rocky Mountain alpine zone and subalpinearctic-alpine areas in North America and Eurasiawith Betula L glyciosmus

5 Without odor of coconuts pileus 10ndash50 mm diamgray-brown to liver-brown with whitish glaucous coat-ing lamellae cream to light tan sometimes with pink-ish tinge discoloring to dingy tan where damagedStipe pale apricot to dingy cream-tan flavor mild toslightly acrid basidiospores 7ndash105 6 5ndash8 mm inRocky Mountain alpine zone and arctic-alpine areasin Eurasia and likely other arctic-alpine areas in NorthAmerica with Salix L nanus

DISCUSSION

The number of Lactarius species reported from theRocky Mountain alpine zone represents only a fractionof the total number of Lactarius species reported else-where from arctic-alpine areas however nearly halfof the core arctic-alpine species are now confirmedfor continental North America Core species oftencited as being restricted to arctic-alpine areas withdwarf and shrubby Salix andor Dryas species includeL brunneoviolaceus L dryadophilus L lanceolatus Lnanus L pseudouvidus L salicis-herbaceae and L salicis-reticulatae (Knudsen and Borgen 1982 Heilmann-Clau-sen et al 1998 Basso 1999 Knudsen and Vesterholt2008) Phylogenetic analyses and morphological exam-ination confirm three out of seven of these species forthe Rocky Mountain alpine zone (L lanceolatus Lnanus L salicis-reticulatae) and support the hypothesisthat these species are largely restricted to arctic-alpineareas with Salix One definitive new species L pallido-marginatus (with Salix) was revealed along with a possi-ble cryptic species L aff salicis-reticulatae (also withSalix) Many Lactarius species previously reportedfrom arctic-alpine areas are also commonly reportedfrom subalpine areas with Betulaceae andor Pinaceae

as well as with other plant families (Heilmann-Clausenet al 1998) From this set of species we report L gly-ciosmus which associates with Betula glandulosa in theRocky Mountain alpine zone and Betula spp acrossthe rest of its distribution and L repraesentaneus whichis commonly reported with Betula and Picea (Heslerand Smith 1979 Heilmann-Clausen et al 1998) andappears to associate with Salix glauca andor perhapskrummholz Picea in the Rocky Mountain alpine zone

Several taxa of interest were not molecularly distinctIn dataset A L lanceolatus did not form a monophyleticgroup but was paraphyletic with respect to Lactarius sp2 the L luculentus group and L aurantiacus In datasetB L nanus and L hysginoides were not clearly distinctIn dataset C L salicis-reticulatae did not form a clearmonophyletic group and L pseudouvidus L brunneovio-laceus and L luridus were not clearly distinguishedfrom each other especially at the RPB2 locus poormolecular resolution also has been encountered forthe latter three taxa by Das et al (2015)

Overall there was little genetic variation distinguish-ing closely related species in this study These findingsare similar to reports from other researchers who sug-gest that there is in general relatively little molecularvariation yet high morphological variation in genusLactarius (Verbeken and Nuytinck 2013) While manyof the species could be morphologically distinguishedall of the Rocky Mountain alpine taxa delineated inthis study are 97 or more similar at the ITS regionto one or more species For example L repraesentaneusand L dryadophilus are as much as 994 similar L sal-icis-reticulatae is approximately 98 similar to L salicis-herbaceae and L flavopalustris L pallidomarginatus isapproximately 97ndash98 similar to L aff brunneoviola-ceus L luridus L montanus and members of the Laff pseudouvidus group and approximately 99 similarto L aspideus L glyciosmus is approximately 98 simi-lar to L mammosus L nanus is approximately 98similar to L hysginoides and L lanceolatus is approxi-mately 97 similar to L substriatussubflammeus L aur-antiacus L luculentus v laetus and Lactarius sp 1 and 2and approximately 98 similar to L luculentus Thishigh degree of similarity could be problematic forenvironmental sequencing studies that use a strict97 ITS region similarity cutoff for recognizing spe-cies (eg Geml et al 2012 Timling et al 2012 2014)In addition Tedersoo et al (2014) suggested that theITS region in the sebacina lineage evolves moreslowly in high latitude areas which could be true forother genera The RPB2 locus proved better for differ-entiating some species and overall phylogenetic ana-lyses revealed several taxonomic issues that we werenot able to resolve A search for additional phylogen-etically informative loci for Lactarius should beinitiated along with more species-level phylogenetic

BARGE ET AL ROCKY MOUNTAIN ALPINE LACTARIUS 435

studies to provide a high quality molecular databasefor ecological studies Furthermore improvement ofmethods for sequencing older material such as typespecimens is critical

Most of the Rocky Mountain alpine Lactarius speciesdelineated in this study (except L pallidomarginatusand L aff salicis-reticulatae for which further studycould reveal broader ranges) appear to have broadintercontinental distributions in North America andEurasia The current distributions of these specieslikely were shaped by a number of biotic and abioticfactors that include host dispersal and distribution cli-mate glaciation and geography Because the genusLactarius is obligately ectomycorrhizal it is likely thatthe distribution of each fungal species was shaped lar-gely by the distribution of suitable host plants manyof which also have broad intercontinental distribu-tions Long-distance dispersal is hypothesized to haveplayed an important role in structuring present distri-bution patterns of arctic-alpine plants although manyappear clonal (Abbott and Brochmann 2003 Alsoset al 2007) and ectomycorrhizal fungi (Geml et al2012 Timling et al 2014) It also has been suggestedthat long-distance dispersal mechanisms have beenselected for in arctic flora due to repeated glaciationsand the need to track potential niches (Alsos et al2007) Pollen data from Blacktail Pond YellowstoneNational Park suggest that montane mid-elevationsin the northern greater Yellowstone ecosystem (Mon-tana Wyoming) were occupied by alpine tundra(Betula Salix many herbaceous taxa) similar to thatof the present Rocky Mountain alpine vegetation dur-ing the last glacial period (14 000ndash11 500 calibratedyears before present) (Krause and Whitlock 2013)Alpine vegetation also appears to have dominatedareas in the southern Rocky Mountains (WyomingColorado) 17 500ndash12 600 calibrated years before pre-sent (Minckley 2014) Both studies suggest the pre-sence of broad low-elevation glacial refugia for arctic-alpine life in the Rocky Mountains along the glacialfront and along with this study lend support to theidea that arctic-alpine Lactarius species (and manyother ectomycorrhizal fungi) may have occupied tun-dra areas south of the icesheets during cooler weatherduring the last glacial maximum and then dispersedupward in elevation and northward as the glaciersand ectomycorrhizal host plants retreated Broaderecological tolerances of taxa such as L glyciosmus andL repraesentaneus which are not restricted to arctic-alpine habitats likely let these species persist in tundraand non-tundra areas during the last glacial maximumas well as to track arctic-alpine vegetation as it movedupward in elevation and northward following theretreat of the icesheets For these species to haveachieved and maintained such broad distributions

gene flow must have been possible over long distancesduring the Pleistocene perhaps mediated by long-dis-tance dispersal (Geml et al 2012) range shifts due toclimate change and glacial expansion and retreat(Birks 2008) glacial and interglacial refugia (Billings1974) and the Bering land bridge (Shapiro et al2004 Geml et al 2006)

ACKNOWLEDGMENTS

We thank Dr Ellen Larsson Dr Pierre-Arthur Moreau JukkaVauras Dr Ursula Peintner Dr Egon Horak and herbaria GIB TU O GB TURA C LIP MICH DBG VPI and NY forloan of specimens Carol Johnson and Dr Matt Lavin areacknowledged for reviewing early stages of the manuscriptWe thank Don Bachman Marcy Barge and Charissa Bujakfor assistance with field work We also gratefully acknowledgesupport from the Montana Institute on Ecosystems graduateenhancement fund

LITERATURE CITED

Abbott RJ Brochmann C 2003 History and evolution of thearctic flora in the footsteps of Eric Hulteacuten Mol Ecol12299ndash313 doi101046j1365-294X200301731x

Alsos IG Eidesen PB Ehrich D Skrede I Westergaard KJacobsen GH Landvik JY Taberlet P Brochmann C2007 Frequent long-distance plant colonization in thechanging arctic Science 3161606ndash1609 doi101126science1139178

Ballaraacute J 1997 Nou estudi drsquoespegravecies fuacutengiques interessantsdels estatges alpiacute i subalpiacute dels Pirineus catalans RevCatalana Micol 201ndash24

Barge E 2015 Systematics of Lactarius in the Rocky Moun-tain alpine zone (masterrsquos thesis) Bozeman MontanaMontana State University 265 p

Basso MT 1999 Lactarius Pers Fungi Europaei 7 AlassioMykoflora (Italy) 845 p

Beker HJ Eberhardt U Vesterholt J 2010 Hebeloma hiemaleBres In arcticalpine habitats N Am Fungi 551ndash65

Bessette AE Harris DB Bessette AR 2009 Milk mushroomsof North America a field identification guide to thegenus Lactarius Syracuse New York Syracuse UniversityPress 297 p

Billings WD 1974 Adaptations and origins of alpine plantsArctic Alpine Res 6129ndash142 doi1023071550081

mdashmdashmdash 1988 Alpine vegetation In Barbour MG BillingsWD eds North American terrestrial vegetation NewYork Cambridge University Press p 392ndash420

Birks HH 2008 The late-quaternary history of arctic andalpine plants Plant Ecol Divers 1135ndash146 doi10108017550870802328652

Bogar LM Kennedy PG 2013 New wrinkles in an old para-digm neighborhood effects can modify the structureand specificity of Alnus-associated ectomycorrhizal fun-gal communities FEMS Microbiol Ecol 83767ndash777doi1011111574-694112032

Bon M 1985a Stage mycologie alpine Lanslebourg (Savoie)du 1 aug 3 sep 1984 Bull Feacuted Mycol Daupineacute-Savoie9619ndash25

436 MYCOLOGIA

pseudouvidus group in RPB2 analyses (SUPPLEMENTARY

FIG 10) Collections of Lactarius pallidomarginatus Lac-tarius sp 3 L aspideus L montanus L luridus L affpseudouvidus and L aff brunneoviolaceus for which ITSdata are available all have a similar 268ndash269 base-pairinsertion in the ITS1 region There is variation withinthe base-pair content of the insertion between collec-tions however inclusion of this insertion did notincrease phylogenetic resolution in preliminary ana-lyses and it therefore was omitted The insertion itselfis highly similar to an insertion in Russula olivacea (eg GenBank accessions AF418634 AY061699) as tolength sequence (approximately 90 similar) andplacement within the ITS1 region

Also in dataset C Rocky Mountain alpine collectionsinitially identified as L repraesentaneus form a well-supported clade with a collection of L repraesentaneusfrom Germany (the lectotype country) as well as withnumerous collections from subalpine and arctic-alpineareas elsewhere in Eurasia and Alaska (FIG 5) Thisclade is sister to a poorly supported L dryadophilusKuumlhner clade The two GenBank sequences JX630967from arctic Canada with Dryas and JF394416 from arcticSvalbard which occur just outside the L dryadophilusclade are likely conspecific with L dryadophilusalthough this cannot be confirmed based on presentresults Lactarius repraesentaneus and L dryadophilus con-sistently differ at two nucleotide positions in the ITSlocus and at four positions in the RPB2 locus and haveconsistent morphological and host differences

In dataset C collections originally identified as L sal-icis-reticulatae from alpine areas in the Rocky Mountainsare paraphyletic with regard to L salicis-herbaceaeKuumlhner L aspideoides BurlL flavopalustris Kytoumlvand L aff flavopalustris (FIG 5) Collections of Lsalicis-reticulatae from alpine areas on the BeartoothPlateau (central Rocky Mountains) form a poorlysupported clade with an alpine collection of L salicis-reticulatae from Mt Laringktatjaringkka Sweden the type local-ity (sequencing of the holotype of L salicis-reticulataewas not successful) as well as with other arctic-alpinecollections of L salicis-reticulatae from Finland andAlaska and an unidentified collection from Svalbard(FIG 5) this clade is hypothesized to represent Lsalicis-reticulatae Collections from southern RockyMountain alpine areas in Colorado are larger thanreported for L salicis-reticulatae and form an unre-solved group that consistently differs at one nucleotideposition in the ITS region but no positions in the RPB2

locus they are tentatively referred to as L aff salicis-reticulatae pending data from additional loci

Overall phylogenetic analyses support the recogni-tion of six to seven Lactarius species from the RockyMountain alpine zone Lactarius lanceolatus (FIG 3)L nanus L glyciosmus (FIG 4) L pallidomarginatus spnov L repraesentaneus L salicis-reticulatae and possiblyL aff salicis-reticulatae (FIG 5) Violet-staining taxa Lpallidomarginatus L repraesentaneus L salicis-reticulataeand L aff salicis-reticulatae are resolved as a poorly sup-ported clade exclusively containing other members ofthe violet-staining section Uvidi supporting the mono-phyly of this section (FIG 2)

TAXONOMY

All descriptions are based on Rocky Mountain alpinematerial

Lactarius lanceolatus OK Mill amp Laursen FIGS 6 7Short description Lactarius lanceolatus is recognized by

its small to medium size and bright light to deeporange pileus pale cream to pale orange lamellae dis-coloring brownish in age or where damaged paleorange stipe watery unchanging latex and mild flavorgrowing in arctic-alpine (possibly also subalpine) areaswith Salix species

Pileus 10ndash45 mm diam convex at first becomingplane to infundibuliform with a depressed centerwith or without a small papilla smooth sometimesfaintly scaly toward center viscid deep orange-brownto deep orange when immature becoming light todeep orange often blotchy often darker toward cen-ter margin incurved to straight when immaturebecoming straight to upturned and often slightlywavy when mature Lamellae adnate to subdecur-rent slightly crowded pale cream to pale yellow topale orange discoloring brownish orange in age orwhere damaged Stipe 10ndash20 6 25ndash75 mm equalto clavate central smooth dry at first covered byfaint whitish pubescence pale orange discoloringdingy orange to dingy light brown where damagedhollow Latex scarce watery unchanging Contextof pileus and stipe pale orange Odor mild Flavormild Basidiospore deposit not observed Basidios-pores 8ndash10 6 6ndash8 mm average 5 86 6 7 mmQ 5 114ndash138 Q5 124 hyaline in 25 KOHbroadly ellipsoid ornamentation amyloid of isolatedwarts connected by fine lines up to 05 mm highBasidia 381ndash660 6 89ndash102 mm four-spored Pleur-omacrocystidia 737ndash127 6 64ndash102 mm narrowly

r$75 bootstrap support and Bayesian posterior probabilities $ 095 Boldface tip labels represent Rocky Mountain alpinespecimens AA 5 arctic-alpine See TABLE I for original identifications

BARGE ET AL ROCKY MOUNTAIN ALPINE LACTARIUS 425

fusiform to lanceolate with acute to moniliformapex thin-walled scattered to abundant Cheiloma-crocystidia 356ndash89 6 51ndash102 mm conical to fusi-form with acute to moniliform apex thin-walledsparse to abundant Pileipellis an ixotrichodermwith well-developed gelatinous layer

Distribution and habitat Lactarius lanceolatus origin-ally was described from arctic-alpine Alaska (Millerand Laursen 1973) and phylogenetic analyses(including the holotype) further confirm its pre-sence in the central Rocky Mountain alpine zoneand Svalbard In alpine areas on the Beartooth Pla-teau it occurs with Salix reticulata and possibly otherSalix species (S arctica S planifolia) it is notreported from the southern Rocky Mountains Over-all L lanceolatus appears largely confined to arctic-alpine habitats however an ITS sequence from amaritime forest in Alaska (GAL18572) clusters withthe arctic-alpine collections in phylogenetic analyses(FIG 3)

Previous arctic-alpine reports include Canada (Ohe-noja and Ohenoja 1993 2010) Alaska (Miller andLaursen 1973 Laursen and Ammirati 1982 Heil-mann-Clausen et al 1998) Beartooth Plateau in theRocky Mountain alpine zone (Cripps and Barge2013) Greenland (Heilmann-Clausen et al 1998 Bor-gen et al 2006) Fennoscandia (Bon 1998 Heilmann-Clausen et al 1998 Knudsen and Vesterholt 2008)Svalbard (Gulden and Jenssen 1988 Geml et al2012) the British Isles (Watling et al 2001) andSiberia (Nezdoiminogo 1997 Heilmann-Clausen et al1998 Karatygin et al 1999 Knudsen and Mukhin1998) however phylogenetic analyses (FIG 3) suggestthat some of these reports may represent L aurantiacus(Pers Fr) Gray

Specimens examined USA ALASKA North Slope CountyBeaufort Lagoon 30 Jul 1971 F4239 (Holotype VPI) MON-TANA Carbon County Beartooth Plateau Highline Trail 1Aug 1997 ZT6214 (MONT) 7 Aug 1998 ZT6412 (MONT)20 Aug 1999 CLC 1389 (MONT) 5 Aug 2008 CLC 2358(MONT) 27 Jul 1997 CLC 1139 (MONT) WYOMINGPark County Beartooth Plateau Solifluction Terraces 20July 2001 CLC 1578 (MONT) 13 Aug 2007 CLC 2319(MONT) 17 Aug 2013 EB 105-13 (MONT)

Other specimens examined DENMARK Broby Vesterskov 16Oct 1997 JHC97-251 (L aurantiacus C) NORWAY Leikan-ger 11 Sept 1999 JHC00-057 (L aurantiacus C) Nordland14 Aug 1999 JV15112F (L cf aurantiacus originally identi-fied as L lanceolatus TURA) Svalbard Longyearbyen 18Aug 2003 CLC 1885 (L cf aurantiacus originally identifiedas L lanceolatus MONT) SWEDEN Uppland 8 Sept 1994JV94-422 (L aurantiacus C) USA ALASKA Kenai PeninsulaCounty Palmer Creek Road 7 Aug 2011 CLC 2743 (L cfaurantiacus originally identified as L lanceolatus MONT)COLORADO El Paso County 27 Aug 2011 DBG-F-022653(L luculentus v laetus DBG) Gilpin County Perigo 13 Aug1974 AH Smith 84923 (Paratype of L luculentus v laetus

MICH) Larimer County 23 Aug 2008 DBG-F-024643 (Lluculentus v laetus DBG) OREGON Polk County 14 Nov1970 AH Smith 79943 (L luculentus MICH) WASHING-TON Lewis County 30 Oct 1972 AH Smith 83066 (Paratypeof L subviscidus MICH) 1 Nov 1972 AH Smith 83331 (Para-type of L subviscidus MICH) Tucannon River 11 Oct1980 AH Smith 90905 (L luculentus MICH)

Comments Lactarius lanceolatus belongs to a taxono-mically problematic group of species with orangeorange-brown or orange-red pilei including L auran-tiacus L luculentus L luculentus v laetus L subflammeusand L substriatus

Traditionally L lanceolatus and L aurantiacus havebeen separated mainly by ecology with L aurantiacusbeing restricted to a variety of subalpine habitats(although it has been reported from arctic-alpine habi-tats) and L lanceolatus to arctic-alpine habitats withSalix spp however it is clear from phylogenetic ana-lyses (FIG 3) that L aurantiacus also may occur in arc-tic-alpine habitats likely associating with Salix as well asKobresia and that the ranges of the two taxa overlap atleast in arctic-alpine areas of Alaska and Svalbard Ithas been suggested that L lanceolatus has longermacrocystidia and larger basidiospores than L auran-tiacus (eg Heilmann-Clausen et al 1998) howevercollections designated as L aurantiacus measured inthis study have pleuromacrocystidia that overlap andactually exceed the upper limit measured for L lanceo-latus including the holotype (see Barge 2015) Basi-diospore sizes for L aurantiacus also overlap andactually exceed the upper limit measured for L lanceo-latus in the collections examined (Barge 2015) Overallsolid morphological differences between the two taxawere not detected and further study may reveal thatthey are conspecific however provisionally L lanceola-tus may tend to have a thicker gelatinous layer over thepileipellis as well as a smooth pileus margin in contrastto the crenulate pileus margin reported for L aurantia-cus Lactarius aurantiacus is not reported from theRocky Mountains

For North American taxa Lactarius luculentus has abrowner pileus a slowly acrid flavor shorter cheilo-macrocystidia (23ndash45 6 4ndash6 mm) and exsiccati arepaler and more yellow-tan (Hesler and Smith 1979)Lactarius luculentus v laetus is similar in appearanceto L lanceolatus but also has shorter cheilomacrocysti-dia (15ndash30 6 4ndash8 mm) and possibly more crowdedlamellae (Hesler and Smith 1979) Lactarius substria-tus has a redder pileus that is glutinous when wetwith a translucent-striate margin latex that slowlybecomes straw yellow shorter cheilomacrocystidiaand an acrid flavor (Hesler and Smith 1979) Lactariussubflammeus also has a redder pileus (at least whenyoung) short translucent striations along its pileusmargin shorter cheilomacrocystidia and a slowly

426 MYCOLOGIA

FIG 6 A L lanceolatus (EB105-13) B L nanus (EB106-13) C L nanus (EB138) D L glyciosmus (ZT12723) E Lpallidomarginatus Barge amp Cripps sp nov (EB0041 HOLOTYPE) F L repraesentaneus (EB107-13) G L salicis-reticulatae(EB0057-14) H L aff salicis-reticulatae (EB0036) Photographs by E Barge (A-C E-H) E Horak (D) Bars 5 2 cm

BARGE ET AL ROCKY MOUNTAIN ALPINE LACTARIUS 427

acrid taste (pers obs of holotype AH Smith 83602Hesler and Smith 1979) Molecular phylogenetic ana-lyses (FIG 3) suggest that L subflammeus is conspecificwith L substriatus however sequencing of the holo-type of L substriatus is needed to confirm this observa-tion Lactarius luculentus L luculentus v laetus Lsubflammeus and L substriatus have been reportedonly from subalpine areas with conifers in westernNorth America (Hesler and Smith 1979)

Lactarius duplicatus AH Sm (syn L lapponicus Har-maja) a North American and European Betula associ-ate is also morphologically close to L lanceolatusHowever it can be distinguished by the productionof strongly yellowing latex We did not procure anymolecular data from material representing this taxonand no molecular data is publicly available on Gen-Bank or UNITE However L duplicatus should beincluded in future phylogenetic analyses investigatingthe L lanceolatusL aurantiacus group

Lactarius nanus J Favre FIGS 6 7Short description Lactarius nanus is recognized by its

typically small size gray-brown to liver brown pileustypically with a glaucous coating at least when youngcream to pinkish tan lamellae pale cream-tan to paleapricot stipe watery unchanging latex mild odormild to slightly acrid flavor and occurrence with Salixspecies in arctic-alpine areas

Pileus 10ndash50 mm diam shallowly convex to plano-convex becoming plane to infundibuliform with orwithout a depressed center and small papilla smoothslightly viscid to dry with whitish glaucous coatingwhen immature easily rubbing away or fading in ageoften in patches deep brown to gray-brown to liverbrown with lighter and darker areas present oftenlighter toward margin becoming lighter overall upondessication margin straight when immature becomingupturned and wavy to convoluted when matureLamellae adnate to subdecurrent subdistant to slightlycrowded cream when immature becoming dingycream to light tan in age sometimes with a faint pink-ish tinge discoloring to dingy tan where damagedStipe 5ndash30 6 3ndash15 mm equal to clavate centralsmooth dry at first covered with whitish glaucous coat-ing as in pileus pale apricot to dingy cream-tan hol-low Latex scarce watery unchanging Context ofpileus and stipe dingy cream to brownish Odor mildFlavor mild to slightly acrid Basidiospore deposit notobserved Basidiospores 7ndash105(ndash12) 6 5ndash8 mm av 584 6 66 mm Q 5 107ndash160 Q 5 127 hyaline in25 KOH subglobose to ellipsoid ornamentationof amyloid scattered ridges and warts forming anincomplete reticulum with some completely reticulateareas to 07 mm high Basidia 406ndash762 6 89ndash152 mm mostly four-spored some two-spored

Pleuromacrocystidia 533ndash965(ndash1143) 6 64ndash114mm lanceolate with acute to moniliform apexscattered to abundant Cheilomacrocystidia 260ndash9146 76ndash102 mm subulate to linear with undulatingmargin with acute to rounded apex scattered toabundant Pileipellis an (ixo-) cutis with some ascend-ing hyphae embedded in a very faint gelatinouslayer

Distribution and habitat Lactarius nanus originally wasdescribed by Jules Favre from the Swiss Alps with Salixherbacea L (Favre 1955) Phylogenetic analyses includ-ing a specimen from near the type locality confirmthis species in the Rocky Mountain alpine zone whereit is one of the most commonly encountered Lactariusspecies it associates with Salix arctica S reticulata andS planifolia Overall L nanus appears to be largelyrestricted to arctic-alpine areas with Salix

Previous arctic-alpine reports include Canada (Ohe-noja and Ohenoja 1993 2010) the Rocky Mountainalpine zone (Moser and McKnight 1987 Cripps andHorak 2008 Cripps and Barge 2013) Greenland(Knudsen and Borgen 1982 Lamoure et al 1982 Heil-mann-Clausen et al 1998 Borgen et al 2006) Iceland(Eyjolfsdottir pers comm Knudsen and Vesterholt2008) the European Alps (Favre 1955 Kuumlhner 1975Kuumlhner and Lamoure 1986 Senn-Irlet 1993 Bon1985a 1998 Heilmann-Clausen et al 1998 Jamoni1995) the Pyrenees (Bon and Ballaraacute 1996) the Car-pathians (Ronikier 2008) Fennoscandia (Kuumlhner1975 Gulden et al 1985 Heilmann-Clausen et al1998 Gulden 2005 Knudsen and Vesterholt 2008)Svalbard (Gulden et al 1985) Siberia (Nezdoiminogo1997) and alpine areas in the Altai Mountains (Karaty-gin et al 1999)

Specimens examined FRANCE Savoie 24 Aug 1989 Bon89093 (LIP) NORWAY Finse 5 Aug 2010 CLC 2134(MONT) Svalbard Longyearbyen 20 Aug 2002 CLC 1896(MONT) SWEDEN Torne Lappmark 15 Aug 1999JV15148 (TURA) SWITZERLAND Grisons 20 Aug 1943ZA192c (Holotype G) USA COLORADO Pitkin CountySawatch Range Independence Pass 6 Aug 2000 CLC 1471(MONT) San Juan County San Juan Range Black BearPass 11 Aug 2001 CLC 1716 (MONT) 11 Aug 2001ZT9529 (MONT) San Juan County San Juan Range Cinna-mon Pass 27 July 2002 CLC 1801 (MONT) 15 Aug 2012EB138 (MONT) San Miguel County San Juan Range Imo-gene Pass 29 July 2002 CLC 1829 (MONT) MONTANACarbon County Beartooth Plateau Highline Trail 7 Aug1998 CLC 1221 (MONT) 8 Aug 1998 ZT6422 (MONT) 18Aug 2013 EB106-13 (MONT) WYOMING Park CountyBeartooth Plateau Frozen Lakes 21 Aug 1999 CLC 1403(MONT) Park County Beartooth Plateau Solifluction Ter-races 11 Aug 2012 EB125 (MONT)

Other specimens examined FINLAND Koillismaa 28 Aug2011 JV28432 (L hysginoides TURA)

428 MYCOLOGIA

Comments Lactarius nanus is morphologically andphylogenetically close to L hysginoides however thelatter has a more cinnamon-toned pileus smaller basi-diospores (6ndash8 6 5ndash7 mm av 68 6 57 mm) and isgenerally reported from boreal areas with Betula Piceaand Salix in Europe (Heilmann-Clausen et al 1998)although phylogenetic placement of environmentalsequences extend its range into boreal Alaska (FIG4) Its range likely overlaps that of L nanus in alpine-subalpine transition areas It is not reported from theRocky Mountains Lactarius mammosus is a more robustspecies with a coconut odor (Heilmann-Clausen et al1998) that associates with members of the Pinaceaeit has not been reported from the Rocky MountainsDifferences between L glyciosmus and L nanus are dis-cussed in the taxonomy section for L glyciosmus

Lactarius glyciosmus (Fr) Fr FIGS 6 7Short description Lactarius glyciosmus is recognized by

its small to medium size pale gray-brown to mauvepileus typically with a glaucous coating at least whenyoung cream pale pinkish or pale yellow-orangelamellae buff or pale salmon stipe watery unchan-ging latex odor of coconuts mild flavor and associa-tion with Betula

Pileus 15ndash50 mm diam broadly convex later becom-ing plane with or without a depressed center and smallpapilla smooth dry pale gray-brown to mauve (violet-brown) with whitish glaucous coating when immaturefading in age azonate to lightly zoned especially nearmargin margin incurved when young and remainingso or becoming straight when mature Lamellaeadnate to subdecurrent slightly crowded to crowdedcream to pale pinkish to pale yellow-orange Stipe10ndash40 6 3ndash12 mm equal to slightly clavate oftencurved central to eccentric smooth dry buff or palesalmon with faint glaucous coating at first as in pileusstuffed or hollow often white-mycelioid toward baseLatex scarce watery unchanging Context of pileusand stipe pale cream Odor of coconut Flavor mildBasidiospore deposit not observed Basidiospores 7ndash96 5ndash7 mm av 5 77 6 60 mm Q 5 117ndash142 Q 5

128 hyaline in 25 KOH broadly ellipsoid to ellip-soid ornamentation of amyloid scattered ridges andwarts forming an incomplete to nearly complete reticu-lum to 05 mm high Basidia 381ndash635 6 76ndash102 mmmostly four-spored some two-spored Pleuromacrocys-tidia 457ndash635 6 64ndash76 mm subclavate to lanceolatewith rounded to occasionally mucronate apex scarceto scattered Cheilomacrocystidia 33ndash66 6 51ndash89mm cylindrical to subclavate with rounded apex scat-tered to abundant Pileipellis a cutis with tufts ofascending hyphae

Distribution and habitat Lactarius glyciosmus was firstdescribed by Fries from a hemiboreal to boreal habitat

in the Femsjouml region of Sweden and the species lacks atype specimen Phylogenetic analyses including speci-mens from the type locality confirm this species inthe central and southern Rocky Mountain alpinezone and suggest it has a broad ecological and geogra-phical distribution confirmed from subalpine and arc-tic-alpine areas in Alaska western Canada IdahoWisconsin New Hampshire Svalbard and Fennoscan-dia In subalpine and alpine areas of the Rocky Moun-tains L glyciosmus associates with Betula glandulosaPhylogenetic placement (FIG 4) of ectomycorrhizalroot tip sequences confirm Betula nana L as a host inAlaska (GU998223 GU998695) and likely B occidenta-lis Hook in Idaho (JX198526 Bogar amp Kennedy2013) Elsewhere it is reported with B pubescens Ehrh(eg Elborne amp Knudsen 1990) and B papyrifera Mar-shall (eg Durall et al 2006)

Previous reports from arctic-alpine areas includeCanada (Ohenoja and Ohenoja 1993 2010) theRocky Mountain alpine zone (Cripps and Horak2008 Cripps and Barge 2013) Greenland (Lange1957 Knudsen and Borgen 1982 Lamoure et al1982 Elbourne and Knudsen 1990 Borgen 2006 Bor-gen et al 2006) Iceland (Eyjolfsdottir pers comm) theAlps (Bon 1998) Fennoscandia (Kallio and Kankainen1964 Kuumlhner 1975 Bon 1998) Svalbard (Ohenoja1971 Geml et al 2012) and Siberia (Karatygin et al1999) At least some reports might be L aff glyciosmusas defined here

Specimens examined FINLAND Fjell Ailigas 27 Aug 2012CLC 2874 (MONT) SWEDEN Femsjouml 13 Aug 1978 MMoser 19780191 (Type locality IB) 16 Aug 1978 M Moser19780234 (Type locality IB) 9 Aug 1981 M Moser19810148 (Type locality IB) USA COLORADO BoulderCounty Front Range Blue Lake 2 Aug 2001 CLC 1624(MONT) Boulder County Front Range Brainard Lake 13Aug 2000 20923 (DBG) Ouray County San Juan RangeIronton Park 13 Aug 2012 EB133 (MONT) MONTANACarbon County Beartooth Plateau Birch Site 29 July 1997CLC 1134 5 ZT6096 (MONT) 8 Aug 1998 CLC 1217(MONT) 10 Aug 1999 TWO 269 (MONT) 19 Aug 1999CLC 1380 (MONT) 13 Aug 2007 ZT12723 (MONT)

Other specimens examined ESTONIA Saare CountyLuumlmanda Commune 17 Sep 2012 TU118535 (L aff glycios-mus TU) Tartu County Votildennu Commune 12 Sept 2010TU106737 (L aff glyciosmus TU) 13 Sept 2010 TU106724(L aff glyciosmus TU)

Comments Lactarius glyciosmus is morphologically andphylogenetically similar to L mammosus however thelatter has a browner pileus stouter stature and moreelongate more reticulate basidiospores (Heilmann-Clausen et al 1998) Phylogenetic placement ofsequences isolated from ectomycorrhizal root tips ofPinus sylvestris L (AJ633116 Heinonsalo et al 2007)and Picea abies (L) Karst (FM992937 Kjoslashller andClemmensen 2009) (FIG 4) confirm that L mammosus

BARGE ET AL ROCKY MOUNTAIN ALPINE LACTARIUS 429

FIG 7 Comparison of micromorphological features of Rocky Mountain alpine specimens and Holotypes (where available)Basidiospores pleuromacrocystidia (left) and cheilomacrocystidia (right) of A Lactarius lanceolatus (CLC2319) B L lanceolatus(F 4239 HOLOTYPE) C L nanus (CLC1471) D L nanus (ZA 192c HOLOTYPE) E L pallidomarginatus Barge amp Cripps spnov (EB0041 HOLOTYPE) F L pseudouvidus (Va 71971 HOLOTYPE) G L salicis-reticulatae (CLC2776) H L salicis-reticulatae

430 MYCOLOGIA

associates with members of the Pinaceae however italso has been reported with Betula mainly in subalpineareas (Heilmann-Clausen et al 1998) Lactarius vietus(Fr) Fr is also similar but has a distinct pale zonenear the stipe apex latex that dries greenish graylonger pleuromacrocystidia and a mild to slightly fruityodor (Heilmann-Clausen et al 1998) It has not beenreported from alpine areas in the Rocky Mountainsalthough it has been reported from elsewhere (mostlynorthern areas) in North America with Betula (Heslerand Smith 1979) The similar-looking L nanus alsooccurs in alpine areas of the Rocky Mountains andhas larger basidiospores larger and differently shapedmacrocystidia lacks an odor of coconut and associateswith Salix Lactarius spinosulus is phylogenetically closeto L glyciosmus (as well as L nanus) (FIG 4) howevermorphologically they are very different Lactarius spino-sulus has a distinctly zoned coarsely squamulose pink-ish salmon- or brick-colored pileus with a finely hairymargin and fruity odor (Heilmann-Clausen et al1998) it has not been reported from North AmericaNo definitive morphological differences were detectedbetween L glyciosmus and L aff glyciosmus and the lat-ter is not reported from the Rocky Mountainalpine zone

Lactarius pallidomarginatus Barge amp CL Cripps spnov FIGS 6 7Mycobank MB812246

Typification USA COLORADO San Juan CountySan Juan Range Stony Pass scattered solitary onsoil under Salix planifolia 13 Aug 2011 EB0041 (holo-type MONT) GenBank KR090940 (ITS) andKR091010 (RPB2)

Etymology Referring to the pale pileus marginDiagnosis Lactarius pallidomarginatus is recognized by

its small to medium size blotchy light tan to lightbrown to gray-brown pileus which is typically lightertoward the margin white to pale yellow-cream lamel-lae pale cream stipe violet staining tissue mild odorand flavor and association with Salix Morphologicallyit is close to L pseudouvidus Kuumlhner but differs by hav-ing smaller basidiospores with an ornamentation ofthinner ridges as well as macrocystidia that becomemore lanceolate (as opposed to subfusiform) and atte-nuated Phylogenetically it is close to L aspideus (Fr)Fr but differs consistently in two nucleotide positionsin the rDNA ITS locus and at eight nucleotide posi-tions in the RPB2 locus morphologically L aspideushas larger yellow basidiomata

Pileus 20ndash50 mm diam convex to broadly convexbecoming broadly convex to plane with or without adepressed center smooth dry to slightly viscid azo-nate blotchy light tan to light brown to gray-browndeveloping violet stains lighter (to cream) toward mar-gin margin incurved to downturned when youngremaining downturned or becoming nearly straightwhen mature Lamellae adnate to subdecurrent subdi-stant to slightly crowded white to pale yellow-creamstaining violet where damaged Stipe 10ndash40 6 5ndash10mm equal to slightly clavate central smooth drywhite to cream staining violet where damaged hollowLatex scarce watery staining tissue violet Context ofpileus and stipe white to cream staining violet wheredamaged Odor mild Flavor mild Basidiosporedeposit not observed Basidiospores 8ndash10 6 65ndash8mm av 5 87 6 71 mm Q 5 114ndash136 Q5 123 hya-line in 25 KOH broadly ellipsoid to ellipsoid orna-mentation amyloid of thin ridges and a few isolatedwarts forming an incomplete to dense reticulum to05 mm high Basidia 508ndash711 6 102ndash127 mm four-spored Pleuromacrocystidia 813ndash1118 6 89ndash102mm cylindrical to lanceolate with acute to moniliformapex scarce to scattered Cheilomacrocystidia 483ndash1016 6 76ndash127 mm cylindrical to lanceolate withacute to moniliform apex scattered Pileipellis an(ixo-)cutis with some ascending hyphae embedded ina faint gelatinous layer

Distribution and habitat Rare in low alpine areas withSalix planifolia reported from Independence Pass inthe Sawatch Range and Stony Pass in the San JuanRange in Colorado An additional collection(ZT5229) from Union Peak in the Wind River Rangeof Wyoming with Salix glauca is likely conspecific how-ever this hypothesis has not been confirmed withmolecular data Due to confusion with L pseudouvidusit is possible that L pallidomarginatus is more wide-spread than confirmed here

Specimens examined USA COLORADO Pitkin CountySawatch Range Independence Pass 6 Aug 2000 CLC 1470(MONT) 9 Aug 2000 ZT9093 (MONT) San Juan CountySan Juan Range Stony Pass 13 Aug 2011 EB0041 (HolotypeMONT) WYOMING Sublette County Wind River RangeUnion Peak 22 Aug 1994 ZT5229 (MONT)

Other specimens examined AUSTRIA Tyrol 3 July 2007 UPeintner 20070035 (L aff pseudouvidus from near type local-ity IB) FINLAND Inarin Lappi 16 Aug 1995 JV10468 (Laff pseudouvidus TURA) Koillismaa JV28448F 30 Aug 2011(L aff pseudouvidus originally identified as L brunneoviola-ceus TURA) FRANCE Isegravere 13 Aug 1971 Va 7 1971 (Holo-type of L pseudouvidus G) ITALY Trentino 20 Aug 2004 U

r(K72-104 HOLOTYPE no cheilocystidia drawn) I L glyciosmus (TWO269) J L repraesentaneus (CLC1747) Bars 5 10 mm forbasidiospores and 20 mm for cystidia Drawings by E Barge

BARGE ET AL ROCKY MOUNTAIN ALPINE LACTARIUS 431

Peintner 20040156 (L aff pseudouvidus from near type local-ity originally identified as L robertianus IB) NORWAYFinse 10 Aug 2005 CLC 2133 (Lactarius aff brunneoviolaceusMONT) Stordal 26 Aug 2006 P Larsen 361395 (L aff brun-neoviolaceus originally identified as L pseudouvidus O) Sval-bard 18 Aug 2002 TWO809 (L aff pseudouvidus MONT)20 Aug 2002 CLC 1910 (Lactarius sp 5 MONT) Ulvik 11Aug 2011 E Soslashyland 73867 (L aff pseudouvidus O) SWE-DEN Latnjajaure 4 Aug 2011 EL101-11 (L aff pseudouvidusGB) Latnjavagge 17 Aug 2010 EL63-10 (L aff pseudouvi-dus GB)

Comments Morphological features of L pallidomar-ginatus fit the holotype description of L pseudouvidusand both are described as lighter near the pileus mar-gin (Kuumlhner 1975 Basso 1999) however basidios-pores of L pallidomarginatus are slightly smaller (8ndash10 6 65ndash8 mm as opposed to 75ndash12 6 65ndash8(85)mm) and have narrower ridges that form a less densereticulum and macrocystidia take on a more lanceo-late form with greater attenuation of the apex (FIG7) In phylogenetic analyses exsiccati identified as Lpseudouvidus from Europe (including specimensfrom near the type locality) appear as distinct fromRocky Mountain alpine material and may representa species complex Lactarius sp 3 (CLC1910) col-lected in Svalbard has darker more vinaceous pileithan L pallidomarginatus and may represent anotherundescribed species although only one collectionwas morphologically examined Members of the Laff brunneoviolaceus clade (FIG 5) that were exam-ined differ from L pallidomarginatus in having dar-ker more vinaceous pilei larger more elongatebasidiospores (to 13 mm) and green staining of exsic-cati in 25 KOH a feature not mentioned previouslyfor L brunneoviolaceus but which could be of taxo-nomic interest Based on taxa included in the phylo-genetic analyses (FIG 5) Lactarius aspideus appearsclosely related to L pallidomarginatus however theformer has more yellow coloration and typically lar-ger sporocarps (pileus 10ndash70 mm diam) and smallerbasidiospores (67ndash95 6 56ndash78 mm) traditionallyL aspideus has been placed with other yellow violet-staining taxa such as L flavidus Boud L salicis-reticu-latae and L salicis-herbaceae (Heilmann-Clausen et al1998) but it is separated here

Lactarius montanus another violet-staining speciesoriginally described from the Rocky Mountains dif-fers from L pallidomarginatus by its larger staturedarker grayish vinaceous to vinaceous-brown pileusand green staining flesh in 25 KOH it is typicallyfound in subalpine habitats with conifers but alsooccurs in the krummholz zone The violet-stainingLactarius pallescens Hesler amp AH Sm occurs in theRocky Mountains but its sporocarps are larger andlighter in color and it is reported from subalpineconifer forests (Hesler and Smith 1979) Other

gray-brown to brownish violet-staining species suchas L luridus L uvidus (Fr) Fr L violascens (JOtto) Fr L pallescens v palmerensis Hesler amp AHSm and L cordovaensis Hesler amp AH Sm are alsolarger and have a subalpine ecology none havebeen reported from the central or southern RockyMountains (Hesler and Smith 1979 Heilmann-Clau-sen et al 1998) Lactarius cistophilus Bon amp Trimbachtypically has larger basidiomata larger basidiospores(10ndash14 6 72ndash9 mm) and occurs under Cistus inthe Mediterranean region (Basso 1999) The smallpale gray-brown European alpine taxon L uvidus falpigines Jamoni amp Bon has microscopic featuresthat match those of L uvidus (Jamoni and Bon1991) but not L pallidomarginatus The small non-violet staining L nanus has basidiospores withthicker more jagged ridges and differently shapedmacrocystidia (often with more rounded apices)(FIG 7)

Lactarius repraesentaneus Britzelm FIGS 6 7Short description Lactarius repraesentaneus is recog-

nized by its medium to large size orange-brown to yel-low-brown hairy pileus with a heavily bearded margincream to pale-yellow lamellae stout cream to pale-yel-low often pitted stipe violet staining latex and tissuespicy-floral odor slightly acrid flavor and associationwith Betula Picea and possibly shrubby Salix speciessuch as S glauca

Pileus 60ndash100 mm diam convex to plano-convexusually with a depressed center and sometimes with apapilla smooth at center becoming increasingly hairyto bearded toward margin dry to viscid azonate sur-face orange-brown at center becoming pale yellow-brown to cream toward margin hairs pale yellow-brown to red-brown margin strongly incurvedremaining so or becoming nearly straight whenmature Lamellae adnate to decurrent crowdedcream to pale-yellow staining violet where damagedStipe 30ndash70 6 15ndash35 mm stout equal to clavate cen-tral dry to viscid cream to pale yellow surface oftenwith numerous dingy yellow golden yellow yellow-brown or light orange-brown pits hollow Latex scarceto abundant white becoming violet Context of pileusand stipe white staining violet where damaged Odorspicy-floral Flavor slightly acrid Basidiospore depositnot observed Basidiospores 8ndash105 6 6ndash85 mm av5 93 6 73 mm Q 5 113ndash143 Q5 127 hyaline in25 KOH broadly ellipsoid to ellipsoid ornamenta-tion amyloid of narrow to broad ridges and isolatedwarts forming an incomplete to dense reticulum to1 mm high Basidia 610ndash762 6 102ndash114 mm four-spored Pleuromacrocystidia 787ndash1448 6 89ndash127 mm subfusiform to lanceolate thin-walled withacute to moniliform apex scattered to abundant

432 MYCOLOGIA

Cheilomacrocystidia 559ndash1397 6 76ndash127 mm subfu-siform to lanceolate thin-walled with acute to monili-form apex scattered to abundant Pileipellis anixotrichoderm with well-developed gelatinous layer

Distribution and habitat Lactarius repraesentaneus firstwas described from Germany (Britzelmayr 1885) Phy-logenetic analyses including material from Germanyconfirm L repraesentaneus from alpine areas of theRocky Mountains and overall reinforce the traditionalconcept that this species has a broad ecological andgeographic range in arctic-alpine and subalpine habi-tats in North America northern central and southernEurope In alpine areas of the Rocky Mountains itassociates with Salix glauca however krummholz Piceaalso is typically (maybe always) present it is reportedwith Picea and Betula elsewhere

Previous reports from arctic-alpine areas mainlywith Betula include Alaska (Laursen and Ammirati1982 Geml et al 2009) the Rocky Mountain alpinezone (Cripps and Horak 2008 Cripps and Barge2013) Greenland (Knudsen and Borgen 1982 Watling1983 Elbourne and Knudsen 1990 Borgen 2006 Bor-gen et al 2006) Iceland (Eyjolfsdottir pers comm)Fennoscandia (Kallio and Kankainen 1964 Kuumlhner1975 Bon 1998) Siberia (Karatygin et al 1999) andthe Altai Mountains (Gorbunova 2010)

Specimens examined FINLAND Enontakiouml 16 Aug 2004JV21671 (TURA) Varsinais-Suomi 9 Aug 1998 JV13837F(TURA) SWEDEN Latnjavagge 12 Aug 2007 EL92-07(GB) USA COLORADO Chaffee County Sawatch RangeCottonwood Pass 4 Aug 2001 CLC 1643 (MONT) 16 Aug2011 EB0048 (MONT) Pitkin County Sawatch Range Inde-pendence Pass 14 Aug 2001 CLC 1747 (MONT) ZT9837(MONT) 15 Aug 2001 ZT9537 (MONT) MONTANA Car-bon County Beartooth Plateau Birch Site 12 Aug 2002CLC 1971 (MONT) 13 Aug 2007 CLC 2318 (MONT) 18Aug 2013 EB107-13 (MONT) WYOMING Park CountyBeartooth Plateau Frozen Lakes 21 Aug 1999 CLC1394 (MONT)

Other specimens examined SWEDEN Latnjavagge 14 Aug2010 EL57-10 (L dryadophilus GB) USA ALASKA 5 Aug2011 CLC 2729 (L dryadophilus MONT) Kenai PeninsulaCounty Palmer Creek Road 7 Aug 2011 CLC 2744 (L drya-dophilus MONT)

Comments The combination of a large pale yellow-brown to orange-brown pileus with a bearded marginand violet staining latex clearly separates L repraesenta-neus from other Lactarius species of the central andsouthern Rocky Mountain alpine zone Phylogeneti-cally close the arctic-alpine taxon L dryadophilus ismorphologically similar but has a cream to yellowsmoother pileus with hairs restricted to near the mar-gin basidiospore ornamentation consisting of sparsethinner ridges and ecological associations with Dryasand Salix It has not been reported from the RockyMountains

Lactarius salicis-reticulatae Kuumlhner FIGS 6 7Short description Lactarius salicis-reticulatae is recog-

nized by its small to medium cream pale yellow orpale ocher sporocarps with pale yellowish well-spacedlamellae often with a pinkish buff tint cream to pale yel-low stipe violet staining latex and tissue mild to slightlysweet odor mild flavor and association with dwarf (Sreticulata) and possibly also shrubby Salix species

Pileus 20ndash40 mm diam convex to broadly convexusually with a depressed center smooth dry to viscidazonate cream pale yellow or pale ocher sometimesdarker toward center staining violet where damagedmargin invcurved remaining so or becoming down-turned when mature Lamellae adnate to subdecur-rent fairly well-spaced cream pale yellow or paleorange-ocher often with a pinkish-buff tint stainingviolet where damaged Stipe 15ndash20 6 10ndash15 mmequal to clavate central dry to viscid cream to pale-yellow staining violet where damaged hollow Latexscarce watery-white becoming violet Context ofpileus and stipe white staining violet where damagedOdor mild to slightly sweet Flavor mild Basidiosporedeposit not observed Basidiospores (7ndash)85ndash115 6(7ndash)8ndash10 mm av 5 100 6 88 mm Q 5 (100ndash)113ndash143 Q 5 125 hyaline in 25 KOH sublgobose toellipsoid ornamentation amyloid of narrow ridgesand a few isolated warts forming an incomplete reticu-lum to 03 mm high Basidia 737ndash889 6 102ndash127 mm four-spored Pleuromacrocystidia 762ndash10166 76ndash114 mm subfusiform to fusiform thin-walledwith rounded to acute to moniliform apex scarceCheilomacrocystidia 686ndash9146 76ndash102 mm subfusi-form to fusiform thin-walled with acute to rounded tomoniliform apex scattered to abundant Pileipellis anixocutis

Distribution and habitat Lactarius salicis-reticulatae firstwas described from alpine tundra with Salix on MtLaringktatjaringkka Sweden (Kuumlhner 1975) Phylogenetic ana-lyses including material from the type locality confirmthis species from alpine areas on the Beartooth Plateauwith Salix reticulata and shrubby Salix spp and in arctic-alpine areas of Alaska Svalbard and Finland OverallL salicis-reticulatae appears largely confined to arctic-alpine habitats Colorado collections with S reticulataand S planifolia may represent a distinct taxonalthough support is low

Previous arctic-alpine reports include Greenland(Lamoure et al 1982 Heilmann-Clausen et al1998 Borgen et al 2006) the Rocky Mountainalpine zone (Cripps and Horak 2008 Cripps andBarge 2013) the Alps (Kuumlhner 1975 Kuumlhner andLamoure 1986 Jamoni 1995 2008 Bon 1998 Heil-mann-Clausen et al 1998) the Pyrenees (Ballaraacute1997) Fennoscandia (Kuumlhner 1975 Heilmann-Clau-sen et al 1998 Gulden 2005 Knudsen and

BARGE ET AL ROCKY MOUNTAIN ALPINE LACTARIUS 433

Vesterholt 2008) and Siberia (Nezdoiminogo 1997Karatygin et al 1999) previous reports of L aspi-deoides Burl from arctic-alpine areas in Alaska (Laur-sen and Ammirati 1982) Greenland (Knudsen andBorgen 1982) and the British Isles (Watling 1987)may refer to this species

Specimens examined FINLAND Near Utsjoki 28 Aug 2012CLC 2885 (MONT) SWEDEN Mt Laringktatjaringkka 12 Aug1972 K72-104 (Holotype G) 15 Aug 1999 JV15133 (Typelocality TURA) USA ALASKA Kenai Peninsula County Pal-mer Creek Road 7 Aug 2011 CLC 2745 (MONT) MON-TANA Carbon County Beartooth Plateau Birch Site 17Aug 2011 CLC 2776 (MONT) Carbon County BeartoothPlateau Highline Trail 8 Aug 1998 CLC 1211 (MONT)WYOMING Park County Beartooth Plateau GardnerLake 16 Aug 2014 EB0057-14 (MONT)

Other specimens examined FINLAND Koillismaa 31 Aug2005 JV23334 (L flavopalustris TURA) GREENLAND Sis-miut 19 Aug 2000 CLC 1536 (L salicis-herbaceae MONT)USA COLORADO Pitkin County Sawatch Range Indepen-dence Pass 13 Aug 2001 CLC 1741 (L aff salicis-reticulataeMONT) San Juan County San Juan Range CinnamonPass 10 Aug 2001 CLC 1710 (L aff salicis-reticulataeMONT) San Juan County San Juan Range Maggie Gulch11 Aug 2011 EB0036 (L aff salicis-reticulatae MONT)San Juan County San Juan Range Stony Pass 9 Aug 2001CLC 1689 (L aff salicis-reticulatae MONT) 13 Aug 2011EB0039 (L aff salicis-reticulatae MONT) MICHIGANEmmet County 10 Aug 1973 RL Shaffer 6957 (L aspi-deoides MICH)

Comments Some taxonomic confusion has beencaused by the fact that Kuumlhner (1975) originallynamed this species L aspideoides Kuumlhner but subse-quently renamed it because the name had been pre-viously applied L aspideoides Burl (1907) is generallylarger with a somewhat zonate pileus more crowdedlamellae that lack salmon-colored hues slightly smallerbasidiospores with broader ridges a bitter to slightlyacrid flavor and a subalpine ecology apparently ineastern and western North America (pers obs of RLShaffer 6957 Hesler and Smith 1979) Lactarius fla-vopalustris is also similar however it has a largermore brightly yellow pileus with faint zonationmore crowded lamellae a weakly scrobiculate stipe(sometimes) smaller basidiospores with thickerridges and a subalpine ecology (Kytoumlvuori 2009)Phylogenetic analyses (FIG 5) reveal three collec-tions of L flavopalustris (two from near the typelocality in Finland) nested in a well-supported cladewith L aspideoides from a subalpine forest in north-ern Michigan suggesting the possible conspecificityof these taxa Neither is reported from the RockyMountains Two other collections identified as L fla-vopalustris (from Estonia) were resolved as distinctfrom the L aspideoidesflavopalustris clade (FIG 5)their identity is unclear

Lactarius aspideus is differentiated from L salicis-reti-culate by its smaller basidiospores with denser reticula-tion more crowded creamier-colored lamellae andhabitat mainly in subalpine areas (Heilmann-Clausenet al 1998) although an environmental sequence iso-lated from an S polaris root tip from arctic Svalbardclusters with L aspideus in phylogenetic analyses (FIG5) Lactarius dryadophilus is often larger with a cream-colored pileus that has a tomentose to bearded margin(Heilmann-Clausen et al 1998) Lactarius flavidus is lar-ger with a sparsely zonate pileus more crowdedcream-colored lamellae darker violet-staining contextbasidiospores with more prominent ornamentationand a distally amyloid plage and subalpine ecology(Heilmann-Clausen et al 1998) Lactarius flavoaspideusKytoumlv has slightly smaller basidiospores on averagewith a denser reticulum and subalpine ecology withSalix (Kytoumlvuori 2009) it has been reported onlyfrom northern Europe Lactarius salicis-herbaceae asimilar arctic-alpine Salix associate has smaller (typi-cally) darker yellow to yellow-brown sporocarpspale cream to grayish buff lamellae and more denselyreticulate basidiospores (pers obs Heilmann-Clausenet al 1998) It is fairly widespread in arctic-alpineareas but is not reported from alpine areas in the cen-tral and southern Rocky Mountains Lactarius aff sali-cis-reticulatae from Colorado may be a distinct taxonand sporocarps can obtain a larger size (pileus to 90mm diam) but otherwise morphological featuresstrongly overlap

KEY TO ROCKY MOUNTAIN ALPINE LACTARIUS SPECIES

1 Latex white turning violet to dull lilac or flesh stain-ing violet to dull lilac 2

1 Latex watery to white unchanging and flesh notstaining violet to dull lilac 42 Fruiting body robust pileus 60ndash100 mm diam

yellow-brown to orange-brown hairy especiallynear margin lamellae cream to pale-yellow stipestout cream to pale-yellow often with numerousyellow-brown pits odor spicy-floral flavor mildlyacrid basidiospores 8ndash105 6 6ndash85 mm puta-tively associated with Betula and Picea (possiblyalso Salix) in Rocky Mountain alpine zone andsubalpine and arctic-alpine areas in North Amer-ica and Eurasia L repraesentaneus

2 Fruiting body smaller 20ndash50 mm diam pileus andmargin smooth 3

3 Pileus pale yellow to pale ocher lamellae cream paleyellow or pale orange-ocher stipe cream to pale yel-low odor mild to slightly sweet flavor mild basidios-pores 85ndash115 6 8ndash10 mm in Rocky Mountain alpinezone and other arctic-alpine areas in North Americaand Eurasia with Salix L salicis-reticulatae

3 Pileus blotchy light tan to light brown to gray-brownmargin lighter lamellae white to pale yellow-cream

434 MYCOLOGIA

stipe white to cream odor mild flavor mild basi-

diospores 8ndash10 6 65ndash8 mm in low alpine areas inColorado with Salix planifolia and possibly Wyom-

ing with Salix glauca L pallidomarginatus4 Pileus bright orange to orange-brown 10ndash45

mm diam margin smooth lamellae pale creamto pale orange stipe pale orange odor mildflavor mild in Rocky Mountain alpine zonearctic-alpine Alaska (possibly also subalpine)to Svalbard with Salix L lanceolatus

4 Pileus pinkish buff gray-brown brown ormauve 5

5 With odor of coconuts pileus 15ndash50 mm diam palepinkish buff to pale gray-brown with glaucous coat-ing lamellae cream to pale yellow-orange stipe buffor pale salmon flavor mild basidiospores 7ndash9 6 5ndash7mm in Rocky Mountain alpine zone and subalpinearctic-alpine areas in North America and Eurasiawith Betula L glyciosmus

5 Without odor of coconuts pileus 10ndash50 mm diamgray-brown to liver-brown with whitish glaucous coat-ing lamellae cream to light tan sometimes with pink-ish tinge discoloring to dingy tan where damagedStipe pale apricot to dingy cream-tan flavor mild toslightly acrid basidiospores 7ndash105 6 5ndash8 mm inRocky Mountain alpine zone and arctic-alpine areasin Eurasia and likely other arctic-alpine areas in NorthAmerica with Salix L nanus

DISCUSSION

The number of Lactarius species reported from theRocky Mountain alpine zone represents only a fractionof the total number of Lactarius species reported else-where from arctic-alpine areas however nearly halfof the core arctic-alpine species are now confirmedfor continental North America Core species oftencited as being restricted to arctic-alpine areas withdwarf and shrubby Salix andor Dryas species includeL brunneoviolaceus L dryadophilus L lanceolatus Lnanus L pseudouvidus L salicis-herbaceae and L salicis-reticulatae (Knudsen and Borgen 1982 Heilmann-Clau-sen et al 1998 Basso 1999 Knudsen and Vesterholt2008) Phylogenetic analyses and morphological exam-ination confirm three out of seven of these species forthe Rocky Mountain alpine zone (L lanceolatus Lnanus L salicis-reticulatae) and support the hypothesisthat these species are largely restricted to arctic-alpineareas with Salix One definitive new species L pallido-marginatus (with Salix) was revealed along with a possi-ble cryptic species L aff salicis-reticulatae (also withSalix) Many Lactarius species previously reportedfrom arctic-alpine areas are also commonly reportedfrom subalpine areas with Betulaceae andor Pinaceae

as well as with other plant families (Heilmann-Clausenet al 1998) From this set of species we report L gly-ciosmus which associates with Betula glandulosa in theRocky Mountain alpine zone and Betula spp acrossthe rest of its distribution and L repraesentaneus whichis commonly reported with Betula and Picea (Heslerand Smith 1979 Heilmann-Clausen et al 1998) andappears to associate with Salix glauca andor perhapskrummholz Picea in the Rocky Mountain alpine zone

Several taxa of interest were not molecularly distinctIn dataset A L lanceolatus did not form a monophyleticgroup but was paraphyletic with respect to Lactarius sp2 the L luculentus group and L aurantiacus In datasetB L nanus and L hysginoides were not clearly distinctIn dataset C L salicis-reticulatae did not form a clearmonophyletic group and L pseudouvidus L brunneovio-laceus and L luridus were not clearly distinguishedfrom each other especially at the RPB2 locus poormolecular resolution also has been encountered forthe latter three taxa by Das et al (2015)

Overall there was little genetic variation distinguish-ing closely related species in this study These findingsare similar to reports from other researchers who sug-gest that there is in general relatively little molecularvariation yet high morphological variation in genusLactarius (Verbeken and Nuytinck 2013) While manyof the species could be morphologically distinguishedall of the Rocky Mountain alpine taxa delineated inthis study are 97 or more similar at the ITS regionto one or more species For example L repraesentaneusand L dryadophilus are as much as 994 similar L sal-icis-reticulatae is approximately 98 similar to L salicis-herbaceae and L flavopalustris L pallidomarginatus isapproximately 97ndash98 similar to L aff brunneoviola-ceus L luridus L montanus and members of the Laff pseudouvidus group and approximately 99 similarto L aspideus L glyciosmus is approximately 98 simi-lar to L mammosus L nanus is approximately 98similar to L hysginoides and L lanceolatus is approxi-mately 97 similar to L substriatussubflammeus L aur-antiacus L luculentus v laetus and Lactarius sp 1 and 2and approximately 98 similar to L luculentus Thishigh degree of similarity could be problematic forenvironmental sequencing studies that use a strict97 ITS region similarity cutoff for recognizing spe-cies (eg Geml et al 2012 Timling et al 2012 2014)In addition Tedersoo et al (2014) suggested that theITS region in the sebacina lineage evolves moreslowly in high latitude areas which could be true forother genera The RPB2 locus proved better for differ-entiating some species and overall phylogenetic ana-lyses revealed several taxonomic issues that we werenot able to resolve A search for additional phylogen-etically informative loci for Lactarius should beinitiated along with more species-level phylogenetic

BARGE ET AL ROCKY MOUNTAIN ALPINE LACTARIUS 435

studies to provide a high quality molecular databasefor ecological studies Furthermore improvement ofmethods for sequencing older material such as typespecimens is critical

Most of the Rocky Mountain alpine Lactarius speciesdelineated in this study (except L pallidomarginatusand L aff salicis-reticulatae for which further studycould reveal broader ranges) appear to have broadintercontinental distributions in North America andEurasia The current distributions of these specieslikely were shaped by a number of biotic and abioticfactors that include host dispersal and distribution cli-mate glaciation and geography Because the genusLactarius is obligately ectomycorrhizal it is likely thatthe distribution of each fungal species was shaped lar-gely by the distribution of suitable host plants manyof which also have broad intercontinental distribu-tions Long-distance dispersal is hypothesized to haveplayed an important role in structuring present distri-bution patterns of arctic-alpine plants although manyappear clonal (Abbott and Brochmann 2003 Alsoset al 2007) and ectomycorrhizal fungi (Geml et al2012 Timling et al 2014) It also has been suggestedthat long-distance dispersal mechanisms have beenselected for in arctic flora due to repeated glaciationsand the need to track potential niches (Alsos et al2007) Pollen data from Blacktail Pond YellowstoneNational Park suggest that montane mid-elevationsin the northern greater Yellowstone ecosystem (Mon-tana Wyoming) were occupied by alpine tundra(Betula Salix many herbaceous taxa) similar to thatof the present Rocky Mountain alpine vegetation dur-ing the last glacial period (14 000ndash11 500 calibratedyears before present) (Krause and Whitlock 2013)Alpine vegetation also appears to have dominatedareas in the southern Rocky Mountains (WyomingColorado) 17 500ndash12 600 calibrated years before pre-sent (Minckley 2014) Both studies suggest the pre-sence of broad low-elevation glacial refugia for arctic-alpine life in the Rocky Mountains along the glacialfront and along with this study lend support to theidea that arctic-alpine Lactarius species (and manyother ectomycorrhizal fungi) may have occupied tun-dra areas south of the icesheets during cooler weatherduring the last glacial maximum and then dispersedupward in elevation and northward as the glaciersand ectomycorrhizal host plants retreated Broaderecological tolerances of taxa such as L glyciosmus andL repraesentaneus which are not restricted to arctic-alpine habitats likely let these species persist in tundraand non-tundra areas during the last glacial maximumas well as to track arctic-alpine vegetation as it movedupward in elevation and northward following theretreat of the icesheets For these species to haveachieved and maintained such broad distributions

gene flow must have been possible over long distancesduring the Pleistocene perhaps mediated by long-dis-tance dispersal (Geml et al 2012) range shifts due toclimate change and glacial expansion and retreat(Birks 2008) glacial and interglacial refugia (Billings1974) and the Bering land bridge (Shapiro et al2004 Geml et al 2006)

ACKNOWLEDGMENTS

We thank Dr Ellen Larsson Dr Pierre-Arthur Moreau JukkaVauras Dr Ursula Peintner Dr Egon Horak and herbaria GIB TU O GB TURA C LIP MICH DBG VPI and NY forloan of specimens Carol Johnson and Dr Matt Lavin areacknowledged for reviewing early stages of the manuscriptWe thank Don Bachman Marcy Barge and Charissa Bujakfor assistance with field work We also gratefully acknowledgesupport from the Montana Institute on Ecosystems graduateenhancement fund

LITERATURE CITED

Abbott RJ Brochmann C 2003 History and evolution of thearctic flora in the footsteps of Eric Hulteacuten Mol Ecol12299ndash313 doi101046j1365-294X200301731x

Alsos IG Eidesen PB Ehrich D Skrede I Westergaard KJacobsen GH Landvik JY Taberlet P Brochmann C2007 Frequent long-distance plant colonization in thechanging arctic Science 3161606ndash1609 doi101126science1139178

Ballaraacute J 1997 Nou estudi drsquoespegravecies fuacutengiques interessantsdels estatges alpiacute i subalpiacute dels Pirineus catalans RevCatalana Micol 201ndash24

Barge E 2015 Systematics of Lactarius in the Rocky Moun-tain alpine zone (masterrsquos thesis) Bozeman MontanaMontana State University 265 p

Basso MT 1999 Lactarius Pers Fungi Europaei 7 AlassioMykoflora (Italy) 845 p

Beker HJ Eberhardt U Vesterholt J 2010 Hebeloma hiemaleBres In arcticalpine habitats N Am Fungi 551ndash65

Bessette AE Harris DB Bessette AR 2009 Milk mushroomsof North America a field identification guide to thegenus Lactarius Syracuse New York Syracuse UniversityPress 297 p

Billings WD 1974 Adaptations and origins of alpine plantsArctic Alpine Res 6129ndash142 doi1023071550081

mdashmdashmdash 1988 Alpine vegetation In Barbour MG BillingsWD eds North American terrestrial vegetation NewYork Cambridge University Press p 392ndash420

Birks HH 2008 The late-quaternary history of arctic andalpine plants Plant Ecol Divers 1135ndash146 doi10108017550870802328652

Bogar LM Kennedy PG 2013 New wrinkles in an old para-digm neighborhood effects can modify the structureand specificity of Alnus-associated ectomycorrhizal fun-gal communities FEMS Microbiol Ecol 83767ndash777doi1011111574-694112032

Bon M 1985a Stage mycologie alpine Lanslebourg (Savoie)du 1 aug 3 sep 1984 Bull Feacuted Mycol Daupineacute-Savoie9619ndash25

436 MYCOLOGIA

fusiform to lanceolate with acute to moniliformapex thin-walled scattered to abundant Cheiloma-crocystidia 356ndash89 6 51ndash102 mm conical to fusi-form with acute to moniliform apex thin-walledsparse to abundant Pileipellis an ixotrichodermwith well-developed gelatinous layer

Distribution and habitat Lactarius lanceolatus origin-ally was described from arctic-alpine Alaska (Millerand Laursen 1973) and phylogenetic analyses(including the holotype) further confirm its pre-sence in the central Rocky Mountain alpine zoneand Svalbard In alpine areas on the Beartooth Pla-teau it occurs with Salix reticulata and possibly otherSalix species (S arctica S planifolia) it is notreported from the southern Rocky Mountains Over-all L lanceolatus appears largely confined to arctic-alpine habitats however an ITS sequence from amaritime forest in Alaska (GAL18572) clusters withthe arctic-alpine collections in phylogenetic analyses(FIG 3)

Previous arctic-alpine reports include Canada (Ohe-noja and Ohenoja 1993 2010) Alaska (Miller andLaursen 1973 Laursen and Ammirati 1982 Heil-mann-Clausen et al 1998) Beartooth Plateau in theRocky Mountain alpine zone (Cripps and Barge2013) Greenland (Heilmann-Clausen et al 1998 Bor-gen et al 2006) Fennoscandia (Bon 1998 Heilmann-Clausen et al 1998 Knudsen and Vesterholt 2008)Svalbard (Gulden and Jenssen 1988 Geml et al2012) the British Isles (Watling et al 2001) andSiberia (Nezdoiminogo 1997 Heilmann-Clausen et al1998 Karatygin et al 1999 Knudsen and Mukhin1998) however phylogenetic analyses (FIG 3) suggestthat some of these reports may represent L aurantiacus(Pers Fr) Gray

Specimens examined USA ALASKA North Slope CountyBeaufort Lagoon 30 Jul 1971 F4239 (Holotype VPI) MON-TANA Carbon County Beartooth Plateau Highline Trail 1Aug 1997 ZT6214 (MONT) 7 Aug 1998 ZT6412 (MONT)20 Aug 1999 CLC 1389 (MONT) 5 Aug 2008 CLC 2358(MONT) 27 Jul 1997 CLC 1139 (MONT) WYOMINGPark County Beartooth Plateau Solifluction Terraces 20July 2001 CLC 1578 (MONT) 13 Aug 2007 CLC 2319(MONT) 17 Aug 2013 EB 105-13 (MONT)

Other specimens examined DENMARK Broby Vesterskov 16Oct 1997 JHC97-251 (L aurantiacus C) NORWAY Leikan-ger 11 Sept 1999 JHC00-057 (L aurantiacus C) Nordland14 Aug 1999 JV15112F (L cf aurantiacus originally identi-fied as L lanceolatus TURA) Svalbard Longyearbyen 18Aug 2003 CLC 1885 (L cf aurantiacus originally identifiedas L lanceolatus MONT) SWEDEN Uppland 8 Sept 1994JV94-422 (L aurantiacus C) USA ALASKA Kenai PeninsulaCounty Palmer Creek Road 7 Aug 2011 CLC 2743 (L cfaurantiacus originally identified as L lanceolatus MONT)COLORADO El Paso County 27 Aug 2011 DBG-F-022653(L luculentus v laetus DBG) Gilpin County Perigo 13 Aug1974 AH Smith 84923 (Paratype of L luculentus v laetus

MICH) Larimer County 23 Aug 2008 DBG-F-024643 (Lluculentus v laetus DBG) OREGON Polk County 14 Nov1970 AH Smith 79943 (L luculentus MICH) WASHING-TON Lewis County 30 Oct 1972 AH Smith 83066 (Paratypeof L subviscidus MICH) 1 Nov 1972 AH Smith 83331 (Para-type of L subviscidus MICH) Tucannon River 11 Oct1980 AH Smith 90905 (L luculentus MICH)

Comments Lactarius lanceolatus belongs to a taxono-mically problematic group of species with orangeorange-brown or orange-red pilei including L auran-tiacus L luculentus L luculentus v laetus L subflammeusand L substriatus

Traditionally L lanceolatus and L aurantiacus havebeen separated mainly by ecology with L aurantiacusbeing restricted to a variety of subalpine habitats(although it has been reported from arctic-alpine habi-tats) and L lanceolatus to arctic-alpine habitats withSalix spp however it is clear from phylogenetic ana-lyses (FIG 3) that L aurantiacus also may occur in arc-tic-alpine habitats likely associating with Salix as well asKobresia and that the ranges of the two taxa overlap atleast in arctic-alpine areas of Alaska and Svalbard Ithas been suggested that L lanceolatus has longermacrocystidia and larger basidiospores than L auran-tiacus (eg Heilmann-Clausen et al 1998) howevercollections designated as L aurantiacus measured inthis study have pleuromacrocystidia that overlap andactually exceed the upper limit measured for L lanceo-latus including the holotype (see Barge 2015) Basi-diospore sizes for L aurantiacus also overlap andactually exceed the upper limit measured for L lanceo-latus in the collections examined (Barge 2015) Overallsolid morphological differences between the two taxawere not detected and further study may reveal thatthey are conspecific however provisionally L lanceola-tus may tend to have a thicker gelatinous layer over thepileipellis as well as a smooth pileus margin in contrastto the crenulate pileus margin reported for L aurantia-cus Lactarius aurantiacus is not reported from theRocky Mountains

For North American taxa Lactarius luculentus has abrowner pileus a slowly acrid flavor shorter cheilo-macrocystidia (23ndash45 6 4ndash6 mm) and exsiccati arepaler and more yellow-tan (Hesler and Smith 1979)Lactarius luculentus v laetus is similar in appearanceto L lanceolatus but also has shorter cheilomacrocysti-dia (15ndash30 6 4ndash8 mm) and possibly more crowdedlamellae (Hesler and Smith 1979) Lactarius substria-tus has a redder pileus that is glutinous when wetwith a translucent-striate margin latex that slowlybecomes straw yellow shorter cheilomacrocystidiaand an acrid flavor (Hesler and Smith 1979) Lactariussubflammeus also has a redder pileus (at least whenyoung) short translucent striations along its pileusmargin shorter cheilomacrocystidia and a slowly

426 MYCOLOGIA

FIG 6 A L lanceolatus (EB105-13) B L nanus (EB106-13) C L nanus (EB138) D L glyciosmus (ZT12723) E Lpallidomarginatus Barge amp Cripps sp nov (EB0041 HOLOTYPE) F L repraesentaneus (EB107-13) G L salicis-reticulatae(EB0057-14) H L aff salicis-reticulatae (EB0036) Photographs by E Barge (A-C E-H) E Horak (D) Bars 5 2 cm

BARGE ET AL ROCKY MOUNTAIN ALPINE LACTARIUS 427

acrid taste (pers obs of holotype AH Smith 83602Hesler and Smith 1979) Molecular phylogenetic ana-lyses (FIG 3) suggest that L subflammeus is conspecificwith L substriatus however sequencing of the holo-type of L substriatus is needed to confirm this observa-tion Lactarius luculentus L luculentus v laetus Lsubflammeus and L substriatus have been reportedonly from subalpine areas with conifers in westernNorth America (Hesler and Smith 1979)

Lactarius duplicatus AH Sm (syn L lapponicus Har-maja) a North American and European Betula associ-ate is also morphologically close to L lanceolatusHowever it can be distinguished by the productionof strongly yellowing latex We did not procure anymolecular data from material representing this taxonand no molecular data is publicly available on Gen-Bank or UNITE However L duplicatus should beincluded in future phylogenetic analyses investigatingthe L lanceolatusL aurantiacus group

Lactarius nanus J Favre FIGS 6 7Short description Lactarius nanus is recognized by its

typically small size gray-brown to liver brown pileustypically with a glaucous coating at least when youngcream to pinkish tan lamellae pale cream-tan to paleapricot stipe watery unchanging latex mild odormild to slightly acrid flavor and occurrence with Salixspecies in arctic-alpine areas

Pileus 10ndash50 mm diam shallowly convex to plano-convex becoming plane to infundibuliform with orwithout a depressed center and small papilla smoothslightly viscid to dry with whitish glaucous coatingwhen immature easily rubbing away or fading in ageoften in patches deep brown to gray-brown to liverbrown with lighter and darker areas present oftenlighter toward margin becoming lighter overall upondessication margin straight when immature becomingupturned and wavy to convoluted when matureLamellae adnate to subdecurrent subdistant to slightlycrowded cream when immature becoming dingycream to light tan in age sometimes with a faint pink-ish tinge discoloring to dingy tan where damagedStipe 5ndash30 6 3ndash15 mm equal to clavate centralsmooth dry at first covered with whitish glaucous coat-ing as in pileus pale apricot to dingy cream-tan hol-low Latex scarce watery unchanging Context ofpileus and stipe dingy cream to brownish Odor mildFlavor mild to slightly acrid Basidiospore deposit notobserved Basidiospores 7ndash105(ndash12) 6 5ndash8 mm av 584 6 66 mm Q 5 107ndash160 Q 5 127 hyaline in25 KOH subglobose to ellipsoid ornamentationof amyloid scattered ridges and warts forming anincomplete reticulum with some completely reticulateareas to 07 mm high Basidia 406ndash762 6 89ndash152 mm mostly four-spored some two-spored

Pleuromacrocystidia 533ndash965(ndash1143) 6 64ndash114mm lanceolate with acute to moniliform apexscattered to abundant Cheilomacrocystidia 260ndash9146 76ndash102 mm subulate to linear with undulatingmargin with acute to rounded apex scattered toabundant Pileipellis an (ixo-) cutis with some ascend-ing hyphae embedded in a very faint gelatinouslayer

Distribution and habitat Lactarius nanus originally wasdescribed by Jules Favre from the Swiss Alps with Salixherbacea L (Favre 1955) Phylogenetic analyses includ-ing a specimen from near the type locality confirmthis species in the Rocky Mountain alpine zone whereit is one of the most commonly encountered Lactariusspecies it associates with Salix arctica S reticulata andS planifolia Overall L nanus appears to be largelyrestricted to arctic-alpine areas with Salix

Previous arctic-alpine reports include Canada (Ohe-noja and Ohenoja 1993 2010) the Rocky Mountainalpine zone (Moser and McKnight 1987 Cripps andHorak 2008 Cripps and Barge 2013) Greenland(Knudsen and Borgen 1982 Lamoure et al 1982 Heil-mann-Clausen et al 1998 Borgen et al 2006) Iceland(Eyjolfsdottir pers comm Knudsen and Vesterholt2008) the European Alps (Favre 1955 Kuumlhner 1975Kuumlhner and Lamoure 1986 Senn-Irlet 1993 Bon1985a 1998 Heilmann-Clausen et al 1998 Jamoni1995) the Pyrenees (Bon and Ballaraacute 1996) the Car-pathians (Ronikier 2008) Fennoscandia (Kuumlhner1975 Gulden et al 1985 Heilmann-Clausen et al1998 Gulden 2005 Knudsen and Vesterholt 2008)Svalbard (Gulden et al 1985) Siberia (Nezdoiminogo1997) and alpine areas in the Altai Mountains (Karaty-gin et al 1999)

Specimens examined FRANCE Savoie 24 Aug 1989 Bon89093 (LIP) NORWAY Finse 5 Aug 2010 CLC 2134(MONT) Svalbard Longyearbyen 20 Aug 2002 CLC 1896(MONT) SWEDEN Torne Lappmark 15 Aug 1999JV15148 (TURA) SWITZERLAND Grisons 20 Aug 1943ZA192c (Holotype G) USA COLORADO Pitkin CountySawatch Range Independence Pass 6 Aug 2000 CLC 1471(MONT) San Juan County San Juan Range Black BearPass 11 Aug 2001 CLC 1716 (MONT) 11 Aug 2001ZT9529 (MONT) San Juan County San Juan Range Cinna-mon Pass 27 July 2002 CLC 1801 (MONT) 15 Aug 2012EB138 (MONT) San Miguel County San Juan Range Imo-gene Pass 29 July 2002 CLC 1829 (MONT) MONTANACarbon County Beartooth Plateau Highline Trail 7 Aug1998 CLC 1221 (MONT) 8 Aug 1998 ZT6422 (MONT) 18Aug 2013 EB106-13 (MONT) WYOMING Park CountyBeartooth Plateau Frozen Lakes 21 Aug 1999 CLC 1403(MONT) Park County Beartooth Plateau Solifluction Ter-races 11 Aug 2012 EB125 (MONT)

Other specimens examined FINLAND Koillismaa 28 Aug2011 JV28432 (L hysginoides TURA)

428 MYCOLOGIA

Comments Lactarius nanus is morphologically andphylogenetically close to L hysginoides however thelatter has a more cinnamon-toned pileus smaller basi-diospores (6ndash8 6 5ndash7 mm av 68 6 57 mm) and isgenerally reported from boreal areas with Betula Piceaand Salix in Europe (Heilmann-Clausen et al 1998)although phylogenetic placement of environmentalsequences extend its range into boreal Alaska (FIG4) Its range likely overlaps that of L nanus in alpine-subalpine transition areas It is not reported from theRocky Mountains Lactarius mammosus is a more robustspecies with a coconut odor (Heilmann-Clausen et al1998) that associates with members of the Pinaceaeit has not been reported from the Rocky MountainsDifferences between L glyciosmus and L nanus are dis-cussed in the taxonomy section for L glyciosmus

Lactarius glyciosmus (Fr) Fr FIGS 6 7Short description Lactarius glyciosmus is recognized by

its small to medium size pale gray-brown to mauvepileus typically with a glaucous coating at least whenyoung cream pale pinkish or pale yellow-orangelamellae buff or pale salmon stipe watery unchan-ging latex odor of coconuts mild flavor and associa-tion with Betula

Pileus 15ndash50 mm diam broadly convex later becom-ing plane with or without a depressed center and smallpapilla smooth dry pale gray-brown to mauve (violet-brown) with whitish glaucous coating when immaturefading in age azonate to lightly zoned especially nearmargin margin incurved when young and remainingso or becoming straight when mature Lamellaeadnate to subdecurrent slightly crowded to crowdedcream to pale pinkish to pale yellow-orange Stipe10ndash40 6 3ndash12 mm equal to slightly clavate oftencurved central to eccentric smooth dry buff or palesalmon with faint glaucous coating at first as in pileusstuffed or hollow often white-mycelioid toward baseLatex scarce watery unchanging Context of pileusand stipe pale cream Odor of coconut Flavor mildBasidiospore deposit not observed Basidiospores 7ndash96 5ndash7 mm av 5 77 6 60 mm Q 5 117ndash142 Q 5

128 hyaline in 25 KOH broadly ellipsoid to ellip-soid ornamentation of amyloid scattered ridges andwarts forming an incomplete to nearly complete reticu-lum to 05 mm high Basidia 381ndash635 6 76ndash102 mmmostly four-spored some two-spored Pleuromacrocys-tidia 457ndash635 6 64ndash76 mm subclavate to lanceolatewith rounded to occasionally mucronate apex scarceto scattered Cheilomacrocystidia 33ndash66 6 51ndash89mm cylindrical to subclavate with rounded apex scat-tered to abundant Pileipellis a cutis with tufts ofascending hyphae

Distribution and habitat Lactarius glyciosmus was firstdescribed by Fries from a hemiboreal to boreal habitat

in the Femsjouml region of Sweden and the species lacks atype specimen Phylogenetic analyses including speci-mens from the type locality confirm this species inthe central and southern Rocky Mountain alpinezone and suggest it has a broad ecological and geogra-phical distribution confirmed from subalpine and arc-tic-alpine areas in Alaska western Canada IdahoWisconsin New Hampshire Svalbard and Fennoscan-dia In subalpine and alpine areas of the Rocky Moun-tains L glyciosmus associates with Betula glandulosaPhylogenetic placement (FIG 4) of ectomycorrhizalroot tip sequences confirm Betula nana L as a host inAlaska (GU998223 GU998695) and likely B occidenta-lis Hook in Idaho (JX198526 Bogar amp Kennedy2013) Elsewhere it is reported with B pubescens Ehrh(eg Elborne amp Knudsen 1990) and B papyrifera Mar-shall (eg Durall et al 2006)

Previous reports from arctic-alpine areas includeCanada (Ohenoja and Ohenoja 1993 2010) theRocky Mountain alpine zone (Cripps and Horak2008 Cripps and Barge 2013) Greenland (Lange1957 Knudsen and Borgen 1982 Lamoure et al1982 Elbourne and Knudsen 1990 Borgen 2006 Bor-gen et al 2006) Iceland (Eyjolfsdottir pers comm) theAlps (Bon 1998) Fennoscandia (Kallio and Kankainen1964 Kuumlhner 1975 Bon 1998) Svalbard (Ohenoja1971 Geml et al 2012) and Siberia (Karatygin et al1999) At least some reports might be L aff glyciosmusas defined here

Specimens examined FINLAND Fjell Ailigas 27 Aug 2012CLC 2874 (MONT) SWEDEN Femsjouml 13 Aug 1978 MMoser 19780191 (Type locality IB) 16 Aug 1978 M Moser19780234 (Type locality IB) 9 Aug 1981 M Moser19810148 (Type locality IB) USA COLORADO BoulderCounty Front Range Blue Lake 2 Aug 2001 CLC 1624(MONT) Boulder County Front Range Brainard Lake 13Aug 2000 20923 (DBG) Ouray County San Juan RangeIronton Park 13 Aug 2012 EB133 (MONT) MONTANACarbon County Beartooth Plateau Birch Site 29 July 1997CLC 1134 5 ZT6096 (MONT) 8 Aug 1998 CLC 1217(MONT) 10 Aug 1999 TWO 269 (MONT) 19 Aug 1999CLC 1380 (MONT) 13 Aug 2007 ZT12723 (MONT)

Other specimens examined ESTONIA Saare CountyLuumlmanda Commune 17 Sep 2012 TU118535 (L aff glycios-mus TU) Tartu County Votildennu Commune 12 Sept 2010TU106737 (L aff glyciosmus TU) 13 Sept 2010 TU106724(L aff glyciosmus TU)

Comments Lactarius glyciosmus is morphologically andphylogenetically similar to L mammosus however thelatter has a browner pileus stouter stature and moreelongate more reticulate basidiospores (Heilmann-Clausen et al 1998) Phylogenetic placement ofsequences isolated from ectomycorrhizal root tips ofPinus sylvestris L (AJ633116 Heinonsalo et al 2007)and Picea abies (L) Karst (FM992937 Kjoslashller andClemmensen 2009) (FIG 4) confirm that L mammosus

BARGE ET AL ROCKY MOUNTAIN ALPINE LACTARIUS 429

FIG 7 Comparison of micromorphological features of Rocky Mountain alpine specimens and Holotypes (where available)Basidiospores pleuromacrocystidia (left) and cheilomacrocystidia (right) of A Lactarius lanceolatus (CLC2319) B L lanceolatus(F 4239 HOLOTYPE) C L nanus (CLC1471) D L nanus (ZA 192c HOLOTYPE) E L pallidomarginatus Barge amp Cripps spnov (EB0041 HOLOTYPE) F L pseudouvidus (Va 71971 HOLOTYPE) G L salicis-reticulatae (CLC2776) H L salicis-reticulatae

430 MYCOLOGIA

associates with members of the Pinaceae however italso has been reported with Betula mainly in subalpineareas (Heilmann-Clausen et al 1998) Lactarius vietus(Fr) Fr is also similar but has a distinct pale zonenear the stipe apex latex that dries greenish graylonger pleuromacrocystidia and a mild to slightly fruityodor (Heilmann-Clausen et al 1998) It has not beenreported from alpine areas in the Rocky Mountainsalthough it has been reported from elsewhere (mostlynorthern areas) in North America with Betula (Heslerand Smith 1979) The similar-looking L nanus alsooccurs in alpine areas of the Rocky Mountains andhas larger basidiospores larger and differently shapedmacrocystidia lacks an odor of coconut and associateswith Salix Lactarius spinosulus is phylogenetically closeto L glyciosmus (as well as L nanus) (FIG 4) howevermorphologically they are very different Lactarius spino-sulus has a distinctly zoned coarsely squamulose pink-ish salmon- or brick-colored pileus with a finely hairymargin and fruity odor (Heilmann-Clausen et al1998) it has not been reported from North AmericaNo definitive morphological differences were detectedbetween L glyciosmus and L aff glyciosmus and the lat-ter is not reported from the Rocky Mountainalpine zone

Lactarius pallidomarginatus Barge amp CL Cripps spnov FIGS 6 7Mycobank MB812246

Typification USA COLORADO San Juan CountySan Juan Range Stony Pass scattered solitary onsoil under Salix planifolia 13 Aug 2011 EB0041 (holo-type MONT) GenBank KR090940 (ITS) andKR091010 (RPB2)

Etymology Referring to the pale pileus marginDiagnosis Lactarius pallidomarginatus is recognized by

its small to medium size blotchy light tan to lightbrown to gray-brown pileus which is typically lightertoward the margin white to pale yellow-cream lamel-lae pale cream stipe violet staining tissue mild odorand flavor and association with Salix Morphologicallyit is close to L pseudouvidus Kuumlhner but differs by hav-ing smaller basidiospores with an ornamentation ofthinner ridges as well as macrocystidia that becomemore lanceolate (as opposed to subfusiform) and atte-nuated Phylogenetically it is close to L aspideus (Fr)Fr but differs consistently in two nucleotide positionsin the rDNA ITS locus and at eight nucleotide posi-tions in the RPB2 locus morphologically L aspideushas larger yellow basidiomata

Pileus 20ndash50 mm diam convex to broadly convexbecoming broadly convex to plane with or without adepressed center smooth dry to slightly viscid azo-nate blotchy light tan to light brown to gray-browndeveloping violet stains lighter (to cream) toward mar-gin margin incurved to downturned when youngremaining downturned or becoming nearly straightwhen mature Lamellae adnate to subdecurrent subdi-stant to slightly crowded white to pale yellow-creamstaining violet where damaged Stipe 10ndash40 6 5ndash10mm equal to slightly clavate central smooth drywhite to cream staining violet where damaged hollowLatex scarce watery staining tissue violet Context ofpileus and stipe white to cream staining violet wheredamaged Odor mild Flavor mild Basidiosporedeposit not observed Basidiospores 8ndash10 6 65ndash8mm av 5 87 6 71 mm Q 5 114ndash136 Q5 123 hya-line in 25 KOH broadly ellipsoid to ellipsoid orna-mentation amyloid of thin ridges and a few isolatedwarts forming an incomplete to dense reticulum to05 mm high Basidia 508ndash711 6 102ndash127 mm four-spored Pleuromacrocystidia 813ndash1118 6 89ndash102mm cylindrical to lanceolate with acute to moniliformapex scarce to scattered Cheilomacrocystidia 483ndash1016 6 76ndash127 mm cylindrical to lanceolate withacute to moniliform apex scattered Pileipellis an(ixo-)cutis with some ascending hyphae embedded ina faint gelatinous layer

Distribution and habitat Rare in low alpine areas withSalix planifolia reported from Independence Pass inthe Sawatch Range and Stony Pass in the San JuanRange in Colorado An additional collection(ZT5229) from Union Peak in the Wind River Rangeof Wyoming with Salix glauca is likely conspecific how-ever this hypothesis has not been confirmed withmolecular data Due to confusion with L pseudouvidusit is possible that L pallidomarginatus is more wide-spread than confirmed here

Specimens examined USA COLORADO Pitkin CountySawatch Range Independence Pass 6 Aug 2000 CLC 1470(MONT) 9 Aug 2000 ZT9093 (MONT) San Juan CountySan Juan Range Stony Pass 13 Aug 2011 EB0041 (HolotypeMONT) WYOMING Sublette County Wind River RangeUnion Peak 22 Aug 1994 ZT5229 (MONT)

Other specimens examined AUSTRIA Tyrol 3 July 2007 UPeintner 20070035 (L aff pseudouvidus from near type local-ity IB) FINLAND Inarin Lappi 16 Aug 1995 JV10468 (Laff pseudouvidus TURA) Koillismaa JV28448F 30 Aug 2011(L aff pseudouvidus originally identified as L brunneoviola-ceus TURA) FRANCE Isegravere 13 Aug 1971 Va 7 1971 (Holo-type of L pseudouvidus G) ITALY Trentino 20 Aug 2004 U

r(K72-104 HOLOTYPE no cheilocystidia drawn) I L glyciosmus (TWO269) J L repraesentaneus (CLC1747) Bars 5 10 mm forbasidiospores and 20 mm for cystidia Drawings by E Barge

BARGE ET AL ROCKY MOUNTAIN ALPINE LACTARIUS 431

Peintner 20040156 (L aff pseudouvidus from near type local-ity originally identified as L robertianus IB) NORWAYFinse 10 Aug 2005 CLC 2133 (Lactarius aff brunneoviolaceusMONT) Stordal 26 Aug 2006 P Larsen 361395 (L aff brun-neoviolaceus originally identified as L pseudouvidus O) Sval-bard 18 Aug 2002 TWO809 (L aff pseudouvidus MONT)20 Aug 2002 CLC 1910 (Lactarius sp 5 MONT) Ulvik 11Aug 2011 E Soslashyland 73867 (L aff pseudouvidus O) SWE-DEN Latnjajaure 4 Aug 2011 EL101-11 (L aff pseudouvidusGB) Latnjavagge 17 Aug 2010 EL63-10 (L aff pseudouvi-dus GB)

Comments Morphological features of L pallidomar-ginatus fit the holotype description of L pseudouvidusand both are described as lighter near the pileus mar-gin (Kuumlhner 1975 Basso 1999) however basidios-pores of L pallidomarginatus are slightly smaller (8ndash10 6 65ndash8 mm as opposed to 75ndash12 6 65ndash8(85)mm) and have narrower ridges that form a less densereticulum and macrocystidia take on a more lanceo-late form with greater attenuation of the apex (FIG7) In phylogenetic analyses exsiccati identified as Lpseudouvidus from Europe (including specimensfrom near the type locality) appear as distinct fromRocky Mountain alpine material and may representa species complex Lactarius sp 3 (CLC1910) col-lected in Svalbard has darker more vinaceous pileithan L pallidomarginatus and may represent anotherundescribed species although only one collectionwas morphologically examined Members of the Laff brunneoviolaceus clade (FIG 5) that were exam-ined differ from L pallidomarginatus in having dar-ker more vinaceous pilei larger more elongatebasidiospores (to 13 mm) and green staining of exsic-cati in 25 KOH a feature not mentioned previouslyfor L brunneoviolaceus but which could be of taxo-nomic interest Based on taxa included in the phylo-genetic analyses (FIG 5) Lactarius aspideus appearsclosely related to L pallidomarginatus however theformer has more yellow coloration and typically lar-ger sporocarps (pileus 10ndash70 mm diam) and smallerbasidiospores (67ndash95 6 56ndash78 mm) traditionallyL aspideus has been placed with other yellow violet-staining taxa such as L flavidus Boud L salicis-reticu-latae and L salicis-herbaceae (Heilmann-Clausen et al1998) but it is separated here

Lactarius montanus another violet-staining speciesoriginally described from the Rocky Mountains dif-fers from L pallidomarginatus by its larger staturedarker grayish vinaceous to vinaceous-brown pileusand green staining flesh in 25 KOH it is typicallyfound in subalpine habitats with conifers but alsooccurs in the krummholz zone The violet-stainingLactarius pallescens Hesler amp AH Sm occurs in theRocky Mountains but its sporocarps are larger andlighter in color and it is reported from subalpineconifer forests (Hesler and Smith 1979) Other

gray-brown to brownish violet-staining species suchas L luridus L uvidus (Fr) Fr L violascens (JOtto) Fr L pallescens v palmerensis Hesler amp AHSm and L cordovaensis Hesler amp AH Sm are alsolarger and have a subalpine ecology none havebeen reported from the central or southern RockyMountains (Hesler and Smith 1979 Heilmann-Clau-sen et al 1998) Lactarius cistophilus Bon amp Trimbachtypically has larger basidiomata larger basidiospores(10ndash14 6 72ndash9 mm) and occurs under Cistus inthe Mediterranean region (Basso 1999) The smallpale gray-brown European alpine taxon L uvidus falpigines Jamoni amp Bon has microscopic featuresthat match those of L uvidus (Jamoni and Bon1991) but not L pallidomarginatus The small non-violet staining L nanus has basidiospores withthicker more jagged ridges and differently shapedmacrocystidia (often with more rounded apices)(FIG 7)

Lactarius repraesentaneus Britzelm FIGS 6 7Short description Lactarius repraesentaneus is recog-

nized by its medium to large size orange-brown to yel-low-brown hairy pileus with a heavily bearded margincream to pale-yellow lamellae stout cream to pale-yel-low often pitted stipe violet staining latex and tissuespicy-floral odor slightly acrid flavor and associationwith Betula Picea and possibly shrubby Salix speciessuch as S glauca

Pileus 60ndash100 mm diam convex to plano-convexusually with a depressed center and sometimes with apapilla smooth at center becoming increasingly hairyto bearded toward margin dry to viscid azonate sur-face orange-brown at center becoming pale yellow-brown to cream toward margin hairs pale yellow-brown to red-brown margin strongly incurvedremaining so or becoming nearly straight whenmature Lamellae adnate to decurrent crowdedcream to pale-yellow staining violet where damagedStipe 30ndash70 6 15ndash35 mm stout equal to clavate cen-tral dry to viscid cream to pale yellow surface oftenwith numerous dingy yellow golden yellow yellow-brown or light orange-brown pits hollow Latex scarceto abundant white becoming violet Context of pileusand stipe white staining violet where damaged Odorspicy-floral Flavor slightly acrid Basidiospore depositnot observed Basidiospores 8ndash105 6 6ndash85 mm av5 93 6 73 mm Q 5 113ndash143 Q5 127 hyaline in25 KOH broadly ellipsoid to ellipsoid ornamenta-tion amyloid of narrow to broad ridges and isolatedwarts forming an incomplete to dense reticulum to1 mm high Basidia 610ndash762 6 102ndash114 mm four-spored Pleuromacrocystidia 787ndash1448 6 89ndash127 mm subfusiform to lanceolate thin-walled withacute to moniliform apex scattered to abundant

432 MYCOLOGIA

Cheilomacrocystidia 559ndash1397 6 76ndash127 mm subfu-siform to lanceolate thin-walled with acute to monili-form apex scattered to abundant Pileipellis anixotrichoderm with well-developed gelatinous layer

Distribution and habitat Lactarius repraesentaneus firstwas described from Germany (Britzelmayr 1885) Phy-logenetic analyses including material from Germanyconfirm L repraesentaneus from alpine areas of theRocky Mountains and overall reinforce the traditionalconcept that this species has a broad ecological andgeographic range in arctic-alpine and subalpine habi-tats in North America northern central and southernEurope In alpine areas of the Rocky Mountains itassociates with Salix glauca however krummholz Piceaalso is typically (maybe always) present it is reportedwith Picea and Betula elsewhere

Previous reports from arctic-alpine areas mainlywith Betula include Alaska (Laursen and Ammirati1982 Geml et al 2009) the Rocky Mountain alpinezone (Cripps and Horak 2008 Cripps and Barge2013) Greenland (Knudsen and Borgen 1982 Watling1983 Elbourne and Knudsen 1990 Borgen 2006 Bor-gen et al 2006) Iceland (Eyjolfsdottir pers comm)Fennoscandia (Kallio and Kankainen 1964 Kuumlhner1975 Bon 1998) Siberia (Karatygin et al 1999) andthe Altai Mountains (Gorbunova 2010)

Specimens examined FINLAND Enontakiouml 16 Aug 2004JV21671 (TURA) Varsinais-Suomi 9 Aug 1998 JV13837F(TURA) SWEDEN Latnjavagge 12 Aug 2007 EL92-07(GB) USA COLORADO Chaffee County Sawatch RangeCottonwood Pass 4 Aug 2001 CLC 1643 (MONT) 16 Aug2011 EB0048 (MONT) Pitkin County Sawatch Range Inde-pendence Pass 14 Aug 2001 CLC 1747 (MONT) ZT9837(MONT) 15 Aug 2001 ZT9537 (MONT) MONTANA Car-bon County Beartooth Plateau Birch Site 12 Aug 2002CLC 1971 (MONT) 13 Aug 2007 CLC 2318 (MONT) 18Aug 2013 EB107-13 (MONT) WYOMING Park CountyBeartooth Plateau Frozen Lakes 21 Aug 1999 CLC1394 (MONT)

Other specimens examined SWEDEN Latnjavagge 14 Aug2010 EL57-10 (L dryadophilus GB) USA ALASKA 5 Aug2011 CLC 2729 (L dryadophilus MONT) Kenai PeninsulaCounty Palmer Creek Road 7 Aug 2011 CLC 2744 (L drya-dophilus MONT)

Comments The combination of a large pale yellow-brown to orange-brown pileus with a bearded marginand violet staining latex clearly separates L repraesenta-neus from other Lactarius species of the central andsouthern Rocky Mountain alpine zone Phylogeneti-cally close the arctic-alpine taxon L dryadophilus ismorphologically similar but has a cream to yellowsmoother pileus with hairs restricted to near the mar-gin basidiospore ornamentation consisting of sparsethinner ridges and ecological associations with Dryasand Salix It has not been reported from the RockyMountains

Lactarius salicis-reticulatae Kuumlhner FIGS 6 7Short description Lactarius salicis-reticulatae is recog-

nized by its small to medium cream pale yellow orpale ocher sporocarps with pale yellowish well-spacedlamellae often with a pinkish buff tint cream to pale yel-low stipe violet staining latex and tissue mild to slightlysweet odor mild flavor and association with dwarf (Sreticulata) and possibly also shrubby Salix species

Pileus 20ndash40 mm diam convex to broadly convexusually with a depressed center smooth dry to viscidazonate cream pale yellow or pale ocher sometimesdarker toward center staining violet where damagedmargin invcurved remaining so or becoming down-turned when mature Lamellae adnate to subdecur-rent fairly well-spaced cream pale yellow or paleorange-ocher often with a pinkish-buff tint stainingviolet where damaged Stipe 15ndash20 6 10ndash15 mmequal to clavate central dry to viscid cream to pale-yellow staining violet where damaged hollow Latexscarce watery-white becoming violet Context ofpileus and stipe white staining violet where damagedOdor mild to slightly sweet Flavor mild Basidiosporedeposit not observed Basidiospores (7ndash)85ndash115 6(7ndash)8ndash10 mm av 5 100 6 88 mm Q 5 (100ndash)113ndash143 Q 5 125 hyaline in 25 KOH sublgobose toellipsoid ornamentation amyloid of narrow ridgesand a few isolated warts forming an incomplete reticu-lum to 03 mm high Basidia 737ndash889 6 102ndash127 mm four-spored Pleuromacrocystidia 762ndash10166 76ndash114 mm subfusiform to fusiform thin-walledwith rounded to acute to moniliform apex scarceCheilomacrocystidia 686ndash9146 76ndash102 mm subfusi-form to fusiform thin-walled with acute to rounded tomoniliform apex scattered to abundant Pileipellis anixocutis

Distribution and habitat Lactarius salicis-reticulatae firstwas described from alpine tundra with Salix on MtLaringktatjaringkka Sweden (Kuumlhner 1975) Phylogenetic ana-lyses including material from the type locality confirmthis species from alpine areas on the Beartooth Plateauwith Salix reticulata and shrubby Salix spp and in arctic-alpine areas of Alaska Svalbard and Finland OverallL salicis-reticulatae appears largely confined to arctic-alpine habitats Colorado collections with S reticulataand S planifolia may represent a distinct taxonalthough support is low

Previous arctic-alpine reports include Greenland(Lamoure et al 1982 Heilmann-Clausen et al1998 Borgen et al 2006) the Rocky Mountainalpine zone (Cripps and Horak 2008 Cripps andBarge 2013) the Alps (Kuumlhner 1975 Kuumlhner andLamoure 1986 Jamoni 1995 2008 Bon 1998 Heil-mann-Clausen et al 1998) the Pyrenees (Ballaraacute1997) Fennoscandia (Kuumlhner 1975 Heilmann-Clau-sen et al 1998 Gulden 2005 Knudsen and

BARGE ET AL ROCKY MOUNTAIN ALPINE LACTARIUS 433

Vesterholt 2008) and Siberia (Nezdoiminogo 1997Karatygin et al 1999) previous reports of L aspi-deoides Burl from arctic-alpine areas in Alaska (Laur-sen and Ammirati 1982) Greenland (Knudsen andBorgen 1982) and the British Isles (Watling 1987)may refer to this species

Specimens examined FINLAND Near Utsjoki 28 Aug 2012CLC 2885 (MONT) SWEDEN Mt Laringktatjaringkka 12 Aug1972 K72-104 (Holotype G) 15 Aug 1999 JV15133 (Typelocality TURA) USA ALASKA Kenai Peninsula County Pal-mer Creek Road 7 Aug 2011 CLC 2745 (MONT) MON-TANA Carbon County Beartooth Plateau Birch Site 17Aug 2011 CLC 2776 (MONT) Carbon County BeartoothPlateau Highline Trail 8 Aug 1998 CLC 1211 (MONT)WYOMING Park County Beartooth Plateau GardnerLake 16 Aug 2014 EB0057-14 (MONT)

Other specimens examined FINLAND Koillismaa 31 Aug2005 JV23334 (L flavopalustris TURA) GREENLAND Sis-miut 19 Aug 2000 CLC 1536 (L salicis-herbaceae MONT)USA COLORADO Pitkin County Sawatch Range Indepen-dence Pass 13 Aug 2001 CLC 1741 (L aff salicis-reticulataeMONT) San Juan County San Juan Range CinnamonPass 10 Aug 2001 CLC 1710 (L aff salicis-reticulataeMONT) San Juan County San Juan Range Maggie Gulch11 Aug 2011 EB0036 (L aff salicis-reticulatae MONT)San Juan County San Juan Range Stony Pass 9 Aug 2001CLC 1689 (L aff salicis-reticulatae MONT) 13 Aug 2011EB0039 (L aff salicis-reticulatae MONT) MICHIGANEmmet County 10 Aug 1973 RL Shaffer 6957 (L aspi-deoides MICH)

Comments Some taxonomic confusion has beencaused by the fact that Kuumlhner (1975) originallynamed this species L aspideoides Kuumlhner but subse-quently renamed it because the name had been pre-viously applied L aspideoides Burl (1907) is generallylarger with a somewhat zonate pileus more crowdedlamellae that lack salmon-colored hues slightly smallerbasidiospores with broader ridges a bitter to slightlyacrid flavor and a subalpine ecology apparently ineastern and western North America (pers obs of RLShaffer 6957 Hesler and Smith 1979) Lactarius fla-vopalustris is also similar however it has a largermore brightly yellow pileus with faint zonationmore crowded lamellae a weakly scrobiculate stipe(sometimes) smaller basidiospores with thickerridges and a subalpine ecology (Kytoumlvuori 2009)Phylogenetic analyses (FIG 5) reveal three collec-tions of L flavopalustris (two from near the typelocality in Finland) nested in a well-supported cladewith L aspideoides from a subalpine forest in north-ern Michigan suggesting the possible conspecificityof these taxa Neither is reported from the RockyMountains Two other collections identified as L fla-vopalustris (from Estonia) were resolved as distinctfrom the L aspideoidesflavopalustris clade (FIG 5)their identity is unclear

Lactarius aspideus is differentiated from L salicis-reti-culate by its smaller basidiospores with denser reticula-tion more crowded creamier-colored lamellae andhabitat mainly in subalpine areas (Heilmann-Clausenet al 1998) although an environmental sequence iso-lated from an S polaris root tip from arctic Svalbardclusters with L aspideus in phylogenetic analyses (FIG5) Lactarius dryadophilus is often larger with a cream-colored pileus that has a tomentose to bearded margin(Heilmann-Clausen et al 1998) Lactarius flavidus is lar-ger with a sparsely zonate pileus more crowdedcream-colored lamellae darker violet-staining contextbasidiospores with more prominent ornamentationand a distally amyloid plage and subalpine ecology(Heilmann-Clausen et al 1998) Lactarius flavoaspideusKytoumlv has slightly smaller basidiospores on averagewith a denser reticulum and subalpine ecology withSalix (Kytoumlvuori 2009) it has been reported onlyfrom northern Europe Lactarius salicis-herbaceae asimilar arctic-alpine Salix associate has smaller (typi-cally) darker yellow to yellow-brown sporocarpspale cream to grayish buff lamellae and more denselyreticulate basidiospores (pers obs Heilmann-Clausenet al 1998) It is fairly widespread in arctic-alpineareas but is not reported from alpine areas in the cen-tral and southern Rocky Mountains Lactarius aff sali-cis-reticulatae from Colorado may be a distinct taxonand sporocarps can obtain a larger size (pileus to 90mm diam) but otherwise morphological featuresstrongly overlap

KEY TO ROCKY MOUNTAIN ALPINE LACTARIUS SPECIES

1 Latex white turning violet to dull lilac or flesh stain-ing violet to dull lilac 2

1 Latex watery to white unchanging and flesh notstaining violet to dull lilac 42 Fruiting body robust pileus 60ndash100 mm diam

yellow-brown to orange-brown hairy especiallynear margin lamellae cream to pale-yellow stipestout cream to pale-yellow often with numerousyellow-brown pits odor spicy-floral flavor mildlyacrid basidiospores 8ndash105 6 6ndash85 mm puta-tively associated with Betula and Picea (possiblyalso Salix) in Rocky Mountain alpine zone andsubalpine and arctic-alpine areas in North Amer-ica and Eurasia L repraesentaneus

2 Fruiting body smaller 20ndash50 mm diam pileus andmargin smooth 3

3 Pileus pale yellow to pale ocher lamellae cream paleyellow or pale orange-ocher stipe cream to pale yel-low odor mild to slightly sweet flavor mild basidios-pores 85ndash115 6 8ndash10 mm in Rocky Mountain alpinezone and other arctic-alpine areas in North Americaand Eurasia with Salix L salicis-reticulatae

3 Pileus blotchy light tan to light brown to gray-brownmargin lighter lamellae white to pale yellow-cream

434 MYCOLOGIA

stipe white to cream odor mild flavor mild basi-

diospores 8ndash10 6 65ndash8 mm in low alpine areas inColorado with Salix planifolia and possibly Wyom-

ing with Salix glauca L pallidomarginatus4 Pileus bright orange to orange-brown 10ndash45

mm diam margin smooth lamellae pale creamto pale orange stipe pale orange odor mildflavor mild in Rocky Mountain alpine zonearctic-alpine Alaska (possibly also subalpine)to Svalbard with Salix L lanceolatus

4 Pileus pinkish buff gray-brown brown ormauve 5

5 With odor of coconuts pileus 15ndash50 mm diam palepinkish buff to pale gray-brown with glaucous coat-ing lamellae cream to pale yellow-orange stipe buffor pale salmon flavor mild basidiospores 7ndash9 6 5ndash7mm in Rocky Mountain alpine zone and subalpinearctic-alpine areas in North America and Eurasiawith Betula L glyciosmus

5 Without odor of coconuts pileus 10ndash50 mm diamgray-brown to liver-brown with whitish glaucous coat-ing lamellae cream to light tan sometimes with pink-ish tinge discoloring to dingy tan where damagedStipe pale apricot to dingy cream-tan flavor mild toslightly acrid basidiospores 7ndash105 6 5ndash8 mm inRocky Mountain alpine zone and arctic-alpine areasin Eurasia and likely other arctic-alpine areas in NorthAmerica with Salix L nanus

DISCUSSION

The number of Lactarius species reported from theRocky Mountain alpine zone represents only a fractionof the total number of Lactarius species reported else-where from arctic-alpine areas however nearly halfof the core arctic-alpine species are now confirmedfor continental North America Core species oftencited as being restricted to arctic-alpine areas withdwarf and shrubby Salix andor Dryas species includeL brunneoviolaceus L dryadophilus L lanceolatus Lnanus L pseudouvidus L salicis-herbaceae and L salicis-reticulatae (Knudsen and Borgen 1982 Heilmann-Clau-sen et al 1998 Basso 1999 Knudsen and Vesterholt2008) Phylogenetic analyses and morphological exam-ination confirm three out of seven of these species forthe Rocky Mountain alpine zone (L lanceolatus Lnanus L salicis-reticulatae) and support the hypothesisthat these species are largely restricted to arctic-alpineareas with Salix One definitive new species L pallido-marginatus (with Salix) was revealed along with a possi-ble cryptic species L aff salicis-reticulatae (also withSalix) Many Lactarius species previously reportedfrom arctic-alpine areas are also commonly reportedfrom subalpine areas with Betulaceae andor Pinaceae

as well as with other plant families (Heilmann-Clausenet al 1998) From this set of species we report L gly-ciosmus which associates with Betula glandulosa in theRocky Mountain alpine zone and Betula spp acrossthe rest of its distribution and L repraesentaneus whichis commonly reported with Betula and Picea (Heslerand Smith 1979 Heilmann-Clausen et al 1998) andappears to associate with Salix glauca andor perhapskrummholz Picea in the Rocky Mountain alpine zone

Several taxa of interest were not molecularly distinctIn dataset A L lanceolatus did not form a monophyleticgroup but was paraphyletic with respect to Lactarius sp2 the L luculentus group and L aurantiacus In datasetB L nanus and L hysginoides were not clearly distinctIn dataset C L salicis-reticulatae did not form a clearmonophyletic group and L pseudouvidus L brunneovio-laceus and L luridus were not clearly distinguishedfrom each other especially at the RPB2 locus poormolecular resolution also has been encountered forthe latter three taxa by Das et al (2015)

Overall there was little genetic variation distinguish-ing closely related species in this study These findingsare similar to reports from other researchers who sug-gest that there is in general relatively little molecularvariation yet high morphological variation in genusLactarius (Verbeken and Nuytinck 2013) While manyof the species could be morphologically distinguishedall of the Rocky Mountain alpine taxa delineated inthis study are 97 or more similar at the ITS regionto one or more species For example L repraesentaneusand L dryadophilus are as much as 994 similar L sal-icis-reticulatae is approximately 98 similar to L salicis-herbaceae and L flavopalustris L pallidomarginatus isapproximately 97ndash98 similar to L aff brunneoviola-ceus L luridus L montanus and members of the Laff pseudouvidus group and approximately 99 similarto L aspideus L glyciosmus is approximately 98 simi-lar to L mammosus L nanus is approximately 98similar to L hysginoides and L lanceolatus is approxi-mately 97 similar to L substriatussubflammeus L aur-antiacus L luculentus v laetus and Lactarius sp 1 and 2and approximately 98 similar to L luculentus Thishigh degree of similarity could be problematic forenvironmental sequencing studies that use a strict97 ITS region similarity cutoff for recognizing spe-cies (eg Geml et al 2012 Timling et al 2012 2014)In addition Tedersoo et al (2014) suggested that theITS region in the sebacina lineage evolves moreslowly in high latitude areas which could be true forother genera The RPB2 locus proved better for differ-entiating some species and overall phylogenetic ana-lyses revealed several taxonomic issues that we werenot able to resolve A search for additional phylogen-etically informative loci for Lactarius should beinitiated along with more species-level phylogenetic

BARGE ET AL ROCKY MOUNTAIN ALPINE LACTARIUS 435

studies to provide a high quality molecular databasefor ecological studies Furthermore improvement ofmethods for sequencing older material such as typespecimens is critical

Most of the Rocky Mountain alpine Lactarius speciesdelineated in this study (except L pallidomarginatusand L aff salicis-reticulatae for which further studycould reveal broader ranges) appear to have broadintercontinental distributions in North America andEurasia The current distributions of these specieslikely were shaped by a number of biotic and abioticfactors that include host dispersal and distribution cli-mate glaciation and geography Because the genusLactarius is obligately ectomycorrhizal it is likely thatthe distribution of each fungal species was shaped lar-gely by the distribution of suitable host plants manyof which also have broad intercontinental distribu-tions Long-distance dispersal is hypothesized to haveplayed an important role in structuring present distri-bution patterns of arctic-alpine plants although manyappear clonal (Abbott and Brochmann 2003 Alsoset al 2007) and ectomycorrhizal fungi (Geml et al2012 Timling et al 2014) It also has been suggestedthat long-distance dispersal mechanisms have beenselected for in arctic flora due to repeated glaciationsand the need to track potential niches (Alsos et al2007) Pollen data from Blacktail Pond YellowstoneNational Park suggest that montane mid-elevationsin the northern greater Yellowstone ecosystem (Mon-tana Wyoming) were occupied by alpine tundra(Betula Salix many herbaceous taxa) similar to thatof the present Rocky Mountain alpine vegetation dur-ing the last glacial period (14 000ndash11 500 calibratedyears before present) (Krause and Whitlock 2013)Alpine vegetation also appears to have dominatedareas in the southern Rocky Mountains (WyomingColorado) 17 500ndash12 600 calibrated years before pre-sent (Minckley 2014) Both studies suggest the pre-sence of broad low-elevation glacial refugia for arctic-alpine life in the Rocky Mountains along the glacialfront and along with this study lend support to theidea that arctic-alpine Lactarius species (and manyother ectomycorrhizal fungi) may have occupied tun-dra areas south of the icesheets during cooler weatherduring the last glacial maximum and then dispersedupward in elevation and northward as the glaciersand ectomycorrhizal host plants retreated Broaderecological tolerances of taxa such as L glyciosmus andL repraesentaneus which are not restricted to arctic-alpine habitats likely let these species persist in tundraand non-tundra areas during the last glacial maximumas well as to track arctic-alpine vegetation as it movedupward in elevation and northward following theretreat of the icesheets For these species to haveachieved and maintained such broad distributions

gene flow must have been possible over long distancesduring the Pleistocene perhaps mediated by long-dis-tance dispersal (Geml et al 2012) range shifts due toclimate change and glacial expansion and retreat(Birks 2008) glacial and interglacial refugia (Billings1974) and the Bering land bridge (Shapiro et al2004 Geml et al 2006)

ACKNOWLEDGMENTS

We thank Dr Ellen Larsson Dr Pierre-Arthur Moreau JukkaVauras Dr Ursula Peintner Dr Egon Horak and herbaria GIB TU O GB TURA C LIP MICH DBG VPI and NY forloan of specimens Carol Johnson and Dr Matt Lavin areacknowledged for reviewing early stages of the manuscriptWe thank Don Bachman Marcy Barge and Charissa Bujakfor assistance with field work We also gratefully acknowledgesupport from the Montana Institute on Ecosystems graduateenhancement fund

LITERATURE CITED

Abbott RJ Brochmann C 2003 History and evolution of thearctic flora in the footsteps of Eric Hulteacuten Mol Ecol12299ndash313 doi101046j1365-294X200301731x

Alsos IG Eidesen PB Ehrich D Skrede I Westergaard KJacobsen GH Landvik JY Taberlet P Brochmann C2007 Frequent long-distance plant colonization in thechanging arctic Science 3161606ndash1609 doi101126science1139178

Ballaraacute J 1997 Nou estudi drsquoespegravecies fuacutengiques interessantsdels estatges alpiacute i subalpiacute dels Pirineus catalans RevCatalana Micol 201ndash24

Barge E 2015 Systematics of Lactarius in the Rocky Moun-tain alpine zone (masterrsquos thesis) Bozeman MontanaMontana State University 265 p

Basso MT 1999 Lactarius Pers Fungi Europaei 7 AlassioMykoflora (Italy) 845 p

Beker HJ Eberhardt U Vesterholt J 2010 Hebeloma hiemaleBres In arcticalpine habitats N Am Fungi 551ndash65

Bessette AE Harris DB Bessette AR 2009 Milk mushroomsof North America a field identification guide to thegenus Lactarius Syracuse New York Syracuse UniversityPress 297 p

Billings WD 1974 Adaptations and origins of alpine plantsArctic Alpine Res 6129ndash142 doi1023071550081

mdashmdashmdash 1988 Alpine vegetation In Barbour MG BillingsWD eds North American terrestrial vegetation NewYork Cambridge University Press p 392ndash420

Birks HH 2008 The late-quaternary history of arctic andalpine plants Plant Ecol Divers 1135ndash146 doi10108017550870802328652

Bogar LM Kennedy PG 2013 New wrinkles in an old para-digm neighborhood effects can modify the structureand specificity of Alnus-associated ectomycorrhizal fun-gal communities FEMS Microbiol Ecol 83767ndash777doi1011111574-694112032

Bon M 1985a Stage mycologie alpine Lanslebourg (Savoie)du 1 aug 3 sep 1984 Bull Feacuted Mycol Daupineacute-Savoie9619ndash25

436 MYCOLOGIA

FIG 6 A L lanceolatus (EB105-13) B L nanus (EB106-13) C L nanus (EB138) D L glyciosmus (ZT12723) E Lpallidomarginatus Barge amp Cripps sp nov (EB0041 HOLOTYPE) F L repraesentaneus (EB107-13) G L salicis-reticulatae(EB0057-14) H L aff salicis-reticulatae (EB0036) Photographs by E Barge (A-C E-H) E Horak (D) Bars 5 2 cm

BARGE ET AL ROCKY MOUNTAIN ALPINE LACTARIUS 427

acrid taste (pers obs of holotype AH Smith 83602Hesler and Smith 1979) Molecular phylogenetic ana-lyses (FIG 3) suggest that L subflammeus is conspecificwith L substriatus however sequencing of the holo-type of L substriatus is needed to confirm this observa-tion Lactarius luculentus L luculentus v laetus Lsubflammeus and L substriatus have been reportedonly from subalpine areas with conifers in westernNorth America (Hesler and Smith 1979)

Lactarius duplicatus AH Sm (syn L lapponicus Har-maja) a North American and European Betula associ-ate is also morphologically close to L lanceolatusHowever it can be distinguished by the productionof strongly yellowing latex We did not procure anymolecular data from material representing this taxonand no molecular data is publicly available on Gen-Bank or UNITE However L duplicatus should beincluded in future phylogenetic analyses investigatingthe L lanceolatusL aurantiacus group

Lactarius nanus J Favre FIGS 6 7Short description Lactarius nanus is recognized by its

typically small size gray-brown to liver brown pileustypically with a glaucous coating at least when youngcream to pinkish tan lamellae pale cream-tan to paleapricot stipe watery unchanging latex mild odormild to slightly acrid flavor and occurrence with Salixspecies in arctic-alpine areas

Pileus 10ndash50 mm diam shallowly convex to plano-convex becoming plane to infundibuliform with orwithout a depressed center and small papilla smoothslightly viscid to dry with whitish glaucous coatingwhen immature easily rubbing away or fading in ageoften in patches deep brown to gray-brown to liverbrown with lighter and darker areas present oftenlighter toward margin becoming lighter overall upondessication margin straight when immature becomingupturned and wavy to convoluted when matureLamellae adnate to subdecurrent subdistant to slightlycrowded cream when immature becoming dingycream to light tan in age sometimes with a faint pink-ish tinge discoloring to dingy tan where damagedStipe 5ndash30 6 3ndash15 mm equal to clavate centralsmooth dry at first covered with whitish glaucous coat-ing as in pileus pale apricot to dingy cream-tan hol-low Latex scarce watery unchanging Context ofpileus and stipe dingy cream to brownish Odor mildFlavor mild to slightly acrid Basidiospore deposit notobserved Basidiospores 7ndash105(ndash12) 6 5ndash8 mm av 584 6 66 mm Q 5 107ndash160 Q 5 127 hyaline in25 KOH subglobose to ellipsoid ornamentationof amyloid scattered ridges and warts forming anincomplete reticulum with some completely reticulateareas to 07 mm high Basidia 406ndash762 6 89ndash152 mm mostly four-spored some two-spored

Pleuromacrocystidia 533ndash965(ndash1143) 6 64ndash114mm lanceolate with acute to moniliform apexscattered to abundant Cheilomacrocystidia 260ndash9146 76ndash102 mm subulate to linear with undulatingmargin with acute to rounded apex scattered toabundant Pileipellis an (ixo-) cutis with some ascend-ing hyphae embedded in a very faint gelatinouslayer

Distribution and habitat Lactarius nanus originally wasdescribed by Jules Favre from the Swiss Alps with Salixherbacea L (Favre 1955) Phylogenetic analyses includ-ing a specimen from near the type locality confirmthis species in the Rocky Mountain alpine zone whereit is one of the most commonly encountered Lactariusspecies it associates with Salix arctica S reticulata andS planifolia Overall L nanus appears to be largelyrestricted to arctic-alpine areas with Salix

Previous arctic-alpine reports include Canada (Ohe-noja and Ohenoja 1993 2010) the Rocky Mountainalpine zone (Moser and McKnight 1987 Cripps andHorak 2008 Cripps and Barge 2013) Greenland(Knudsen and Borgen 1982 Lamoure et al 1982 Heil-mann-Clausen et al 1998 Borgen et al 2006) Iceland(Eyjolfsdottir pers comm Knudsen and Vesterholt2008) the European Alps (Favre 1955 Kuumlhner 1975Kuumlhner and Lamoure 1986 Senn-Irlet 1993 Bon1985a 1998 Heilmann-Clausen et al 1998 Jamoni1995) the Pyrenees (Bon and Ballaraacute 1996) the Car-pathians (Ronikier 2008) Fennoscandia (Kuumlhner1975 Gulden et al 1985 Heilmann-Clausen et al1998 Gulden 2005 Knudsen and Vesterholt 2008)Svalbard (Gulden et al 1985) Siberia (Nezdoiminogo1997) and alpine areas in the Altai Mountains (Karaty-gin et al 1999)

Specimens examined FRANCE Savoie 24 Aug 1989 Bon89093 (LIP) NORWAY Finse 5 Aug 2010 CLC 2134(MONT) Svalbard Longyearbyen 20 Aug 2002 CLC 1896(MONT) SWEDEN Torne Lappmark 15 Aug 1999JV15148 (TURA) SWITZERLAND Grisons 20 Aug 1943ZA192c (Holotype G) USA COLORADO Pitkin CountySawatch Range Independence Pass 6 Aug 2000 CLC 1471(MONT) San Juan County San Juan Range Black BearPass 11 Aug 2001 CLC 1716 (MONT) 11 Aug 2001ZT9529 (MONT) San Juan County San Juan Range Cinna-mon Pass 27 July 2002 CLC 1801 (MONT) 15 Aug 2012EB138 (MONT) San Miguel County San Juan Range Imo-gene Pass 29 July 2002 CLC 1829 (MONT) MONTANACarbon County Beartooth Plateau Highline Trail 7 Aug1998 CLC 1221 (MONT) 8 Aug 1998 ZT6422 (MONT) 18Aug 2013 EB106-13 (MONT) WYOMING Park CountyBeartooth Plateau Frozen Lakes 21 Aug 1999 CLC 1403(MONT) Park County Beartooth Plateau Solifluction Ter-races 11 Aug 2012 EB125 (MONT)

Other specimens examined FINLAND Koillismaa 28 Aug2011 JV28432 (L hysginoides TURA)

428 MYCOLOGIA

Comments Lactarius nanus is morphologically andphylogenetically close to L hysginoides however thelatter has a more cinnamon-toned pileus smaller basi-diospores (6ndash8 6 5ndash7 mm av 68 6 57 mm) and isgenerally reported from boreal areas with Betula Piceaand Salix in Europe (Heilmann-Clausen et al 1998)although phylogenetic placement of environmentalsequences extend its range into boreal Alaska (FIG4) Its range likely overlaps that of L nanus in alpine-subalpine transition areas It is not reported from theRocky Mountains Lactarius mammosus is a more robustspecies with a coconut odor (Heilmann-Clausen et al1998) that associates with members of the Pinaceaeit has not been reported from the Rocky MountainsDifferences between L glyciosmus and L nanus are dis-cussed in the taxonomy section for L glyciosmus

Lactarius glyciosmus (Fr) Fr FIGS 6 7Short description Lactarius glyciosmus is recognized by

its small to medium size pale gray-brown to mauvepileus typically with a glaucous coating at least whenyoung cream pale pinkish or pale yellow-orangelamellae buff or pale salmon stipe watery unchan-ging latex odor of coconuts mild flavor and associa-tion with Betula

Pileus 15ndash50 mm diam broadly convex later becom-ing plane with or without a depressed center and smallpapilla smooth dry pale gray-brown to mauve (violet-brown) with whitish glaucous coating when immaturefading in age azonate to lightly zoned especially nearmargin margin incurved when young and remainingso or becoming straight when mature Lamellaeadnate to subdecurrent slightly crowded to crowdedcream to pale pinkish to pale yellow-orange Stipe10ndash40 6 3ndash12 mm equal to slightly clavate oftencurved central to eccentric smooth dry buff or palesalmon with faint glaucous coating at first as in pileusstuffed or hollow often white-mycelioid toward baseLatex scarce watery unchanging Context of pileusand stipe pale cream Odor of coconut Flavor mildBasidiospore deposit not observed Basidiospores 7ndash96 5ndash7 mm av 5 77 6 60 mm Q 5 117ndash142 Q 5

128 hyaline in 25 KOH broadly ellipsoid to ellip-soid ornamentation of amyloid scattered ridges andwarts forming an incomplete to nearly complete reticu-lum to 05 mm high Basidia 381ndash635 6 76ndash102 mmmostly four-spored some two-spored Pleuromacrocys-tidia 457ndash635 6 64ndash76 mm subclavate to lanceolatewith rounded to occasionally mucronate apex scarceto scattered Cheilomacrocystidia 33ndash66 6 51ndash89mm cylindrical to subclavate with rounded apex scat-tered to abundant Pileipellis a cutis with tufts ofascending hyphae

Distribution and habitat Lactarius glyciosmus was firstdescribed by Fries from a hemiboreal to boreal habitat

in the Femsjouml region of Sweden and the species lacks atype specimen Phylogenetic analyses including speci-mens from the type locality confirm this species inthe central and southern Rocky Mountain alpinezone and suggest it has a broad ecological and geogra-phical distribution confirmed from subalpine and arc-tic-alpine areas in Alaska western Canada IdahoWisconsin New Hampshire Svalbard and Fennoscan-dia In subalpine and alpine areas of the Rocky Moun-tains L glyciosmus associates with Betula glandulosaPhylogenetic placement (FIG 4) of ectomycorrhizalroot tip sequences confirm Betula nana L as a host inAlaska (GU998223 GU998695) and likely B occidenta-lis Hook in Idaho (JX198526 Bogar amp Kennedy2013) Elsewhere it is reported with B pubescens Ehrh(eg Elborne amp Knudsen 1990) and B papyrifera Mar-shall (eg Durall et al 2006)

Previous reports from arctic-alpine areas includeCanada (Ohenoja and Ohenoja 1993 2010) theRocky Mountain alpine zone (Cripps and Horak2008 Cripps and Barge 2013) Greenland (Lange1957 Knudsen and Borgen 1982 Lamoure et al1982 Elbourne and Knudsen 1990 Borgen 2006 Bor-gen et al 2006) Iceland (Eyjolfsdottir pers comm) theAlps (Bon 1998) Fennoscandia (Kallio and Kankainen1964 Kuumlhner 1975 Bon 1998) Svalbard (Ohenoja1971 Geml et al 2012) and Siberia (Karatygin et al1999) At least some reports might be L aff glyciosmusas defined here

Specimens examined FINLAND Fjell Ailigas 27 Aug 2012CLC 2874 (MONT) SWEDEN Femsjouml 13 Aug 1978 MMoser 19780191 (Type locality IB) 16 Aug 1978 M Moser19780234 (Type locality IB) 9 Aug 1981 M Moser19810148 (Type locality IB) USA COLORADO BoulderCounty Front Range Blue Lake 2 Aug 2001 CLC 1624(MONT) Boulder County Front Range Brainard Lake 13Aug 2000 20923 (DBG) Ouray County San Juan RangeIronton Park 13 Aug 2012 EB133 (MONT) MONTANACarbon County Beartooth Plateau Birch Site 29 July 1997CLC 1134 5 ZT6096 (MONT) 8 Aug 1998 CLC 1217(MONT) 10 Aug 1999 TWO 269 (MONT) 19 Aug 1999CLC 1380 (MONT) 13 Aug 2007 ZT12723 (MONT)

Other specimens examined ESTONIA Saare CountyLuumlmanda Commune 17 Sep 2012 TU118535 (L aff glycios-mus TU) Tartu County Votildennu Commune 12 Sept 2010TU106737 (L aff glyciosmus TU) 13 Sept 2010 TU106724(L aff glyciosmus TU)

Comments Lactarius glyciosmus is morphologically andphylogenetically similar to L mammosus however thelatter has a browner pileus stouter stature and moreelongate more reticulate basidiospores (Heilmann-Clausen et al 1998) Phylogenetic placement ofsequences isolated from ectomycorrhizal root tips ofPinus sylvestris L (AJ633116 Heinonsalo et al 2007)and Picea abies (L) Karst (FM992937 Kjoslashller andClemmensen 2009) (FIG 4) confirm that L mammosus

BARGE ET AL ROCKY MOUNTAIN ALPINE LACTARIUS 429

FIG 7 Comparison of micromorphological features of Rocky Mountain alpine specimens and Holotypes (where available)Basidiospores pleuromacrocystidia (left) and cheilomacrocystidia (right) of A Lactarius lanceolatus (CLC2319) B L lanceolatus(F 4239 HOLOTYPE) C L nanus (CLC1471) D L nanus (ZA 192c HOLOTYPE) E L pallidomarginatus Barge amp Cripps spnov (EB0041 HOLOTYPE) F L pseudouvidus (Va 71971 HOLOTYPE) G L salicis-reticulatae (CLC2776) H L salicis-reticulatae

430 MYCOLOGIA

associates with members of the Pinaceae however italso has been reported with Betula mainly in subalpineareas (Heilmann-Clausen et al 1998) Lactarius vietus(Fr) Fr is also similar but has a distinct pale zonenear the stipe apex latex that dries greenish graylonger pleuromacrocystidia and a mild to slightly fruityodor (Heilmann-Clausen et al 1998) It has not beenreported from alpine areas in the Rocky Mountainsalthough it has been reported from elsewhere (mostlynorthern areas) in North America with Betula (Heslerand Smith 1979) The similar-looking L nanus alsooccurs in alpine areas of the Rocky Mountains andhas larger basidiospores larger and differently shapedmacrocystidia lacks an odor of coconut and associateswith Salix Lactarius spinosulus is phylogenetically closeto L glyciosmus (as well as L nanus) (FIG 4) howevermorphologically they are very different Lactarius spino-sulus has a distinctly zoned coarsely squamulose pink-ish salmon- or brick-colored pileus with a finely hairymargin and fruity odor (Heilmann-Clausen et al1998) it has not been reported from North AmericaNo definitive morphological differences were detectedbetween L glyciosmus and L aff glyciosmus and the lat-ter is not reported from the Rocky Mountainalpine zone

Lactarius pallidomarginatus Barge amp CL Cripps spnov FIGS 6 7Mycobank MB812246

Typification USA COLORADO San Juan CountySan Juan Range Stony Pass scattered solitary onsoil under Salix planifolia 13 Aug 2011 EB0041 (holo-type MONT) GenBank KR090940 (ITS) andKR091010 (RPB2)

Etymology Referring to the pale pileus marginDiagnosis Lactarius pallidomarginatus is recognized by

its small to medium size blotchy light tan to lightbrown to gray-brown pileus which is typically lightertoward the margin white to pale yellow-cream lamel-lae pale cream stipe violet staining tissue mild odorand flavor and association with Salix Morphologicallyit is close to L pseudouvidus Kuumlhner but differs by hav-ing smaller basidiospores with an ornamentation ofthinner ridges as well as macrocystidia that becomemore lanceolate (as opposed to subfusiform) and atte-nuated Phylogenetically it is close to L aspideus (Fr)Fr but differs consistently in two nucleotide positionsin the rDNA ITS locus and at eight nucleotide posi-tions in the RPB2 locus morphologically L aspideushas larger yellow basidiomata

Pileus 20ndash50 mm diam convex to broadly convexbecoming broadly convex to plane with or without adepressed center smooth dry to slightly viscid azo-nate blotchy light tan to light brown to gray-browndeveloping violet stains lighter (to cream) toward mar-gin margin incurved to downturned when youngremaining downturned or becoming nearly straightwhen mature Lamellae adnate to subdecurrent subdi-stant to slightly crowded white to pale yellow-creamstaining violet where damaged Stipe 10ndash40 6 5ndash10mm equal to slightly clavate central smooth drywhite to cream staining violet where damaged hollowLatex scarce watery staining tissue violet Context ofpileus and stipe white to cream staining violet wheredamaged Odor mild Flavor mild Basidiosporedeposit not observed Basidiospores 8ndash10 6 65ndash8mm av 5 87 6 71 mm Q 5 114ndash136 Q5 123 hya-line in 25 KOH broadly ellipsoid to ellipsoid orna-mentation amyloid of thin ridges and a few isolatedwarts forming an incomplete to dense reticulum to05 mm high Basidia 508ndash711 6 102ndash127 mm four-spored Pleuromacrocystidia 813ndash1118 6 89ndash102mm cylindrical to lanceolate with acute to moniliformapex scarce to scattered Cheilomacrocystidia 483ndash1016 6 76ndash127 mm cylindrical to lanceolate withacute to moniliform apex scattered Pileipellis an(ixo-)cutis with some ascending hyphae embedded ina faint gelatinous layer

Distribution and habitat Rare in low alpine areas withSalix planifolia reported from Independence Pass inthe Sawatch Range and Stony Pass in the San JuanRange in Colorado An additional collection(ZT5229) from Union Peak in the Wind River Rangeof Wyoming with Salix glauca is likely conspecific how-ever this hypothesis has not been confirmed withmolecular data Due to confusion with L pseudouvidusit is possible that L pallidomarginatus is more wide-spread than confirmed here

Specimens examined USA COLORADO Pitkin CountySawatch Range Independence Pass 6 Aug 2000 CLC 1470(MONT) 9 Aug 2000 ZT9093 (MONT) San Juan CountySan Juan Range Stony Pass 13 Aug 2011 EB0041 (HolotypeMONT) WYOMING Sublette County Wind River RangeUnion Peak 22 Aug 1994 ZT5229 (MONT)

Other specimens examined AUSTRIA Tyrol 3 July 2007 UPeintner 20070035 (L aff pseudouvidus from near type local-ity IB) FINLAND Inarin Lappi 16 Aug 1995 JV10468 (Laff pseudouvidus TURA) Koillismaa JV28448F 30 Aug 2011(L aff pseudouvidus originally identified as L brunneoviola-ceus TURA) FRANCE Isegravere 13 Aug 1971 Va 7 1971 (Holo-type of L pseudouvidus G) ITALY Trentino 20 Aug 2004 U

r(K72-104 HOLOTYPE no cheilocystidia drawn) I L glyciosmus (TWO269) J L repraesentaneus (CLC1747) Bars 5 10 mm forbasidiospores and 20 mm for cystidia Drawings by E Barge

BARGE ET AL ROCKY MOUNTAIN ALPINE LACTARIUS 431

Peintner 20040156 (L aff pseudouvidus from near type local-ity originally identified as L robertianus IB) NORWAYFinse 10 Aug 2005 CLC 2133 (Lactarius aff brunneoviolaceusMONT) Stordal 26 Aug 2006 P Larsen 361395 (L aff brun-neoviolaceus originally identified as L pseudouvidus O) Sval-bard 18 Aug 2002 TWO809 (L aff pseudouvidus MONT)20 Aug 2002 CLC 1910 (Lactarius sp 5 MONT) Ulvik 11Aug 2011 E Soslashyland 73867 (L aff pseudouvidus O) SWE-DEN Latnjajaure 4 Aug 2011 EL101-11 (L aff pseudouvidusGB) Latnjavagge 17 Aug 2010 EL63-10 (L aff pseudouvi-dus GB)

Comments Morphological features of L pallidomar-ginatus fit the holotype description of L pseudouvidusand both are described as lighter near the pileus mar-gin (Kuumlhner 1975 Basso 1999) however basidios-pores of L pallidomarginatus are slightly smaller (8ndash10 6 65ndash8 mm as opposed to 75ndash12 6 65ndash8(85)mm) and have narrower ridges that form a less densereticulum and macrocystidia take on a more lanceo-late form with greater attenuation of the apex (FIG7) In phylogenetic analyses exsiccati identified as Lpseudouvidus from Europe (including specimensfrom near the type locality) appear as distinct fromRocky Mountain alpine material and may representa species complex Lactarius sp 3 (CLC1910) col-lected in Svalbard has darker more vinaceous pileithan L pallidomarginatus and may represent anotherundescribed species although only one collectionwas morphologically examined Members of the Laff brunneoviolaceus clade (FIG 5) that were exam-ined differ from L pallidomarginatus in having dar-ker more vinaceous pilei larger more elongatebasidiospores (to 13 mm) and green staining of exsic-cati in 25 KOH a feature not mentioned previouslyfor L brunneoviolaceus but which could be of taxo-nomic interest Based on taxa included in the phylo-genetic analyses (FIG 5) Lactarius aspideus appearsclosely related to L pallidomarginatus however theformer has more yellow coloration and typically lar-ger sporocarps (pileus 10ndash70 mm diam) and smallerbasidiospores (67ndash95 6 56ndash78 mm) traditionallyL aspideus has been placed with other yellow violet-staining taxa such as L flavidus Boud L salicis-reticu-latae and L salicis-herbaceae (Heilmann-Clausen et al1998) but it is separated here

Lactarius montanus another violet-staining speciesoriginally described from the Rocky Mountains dif-fers from L pallidomarginatus by its larger staturedarker grayish vinaceous to vinaceous-brown pileusand green staining flesh in 25 KOH it is typicallyfound in subalpine habitats with conifers but alsooccurs in the krummholz zone The violet-stainingLactarius pallescens Hesler amp AH Sm occurs in theRocky Mountains but its sporocarps are larger andlighter in color and it is reported from subalpineconifer forests (Hesler and Smith 1979) Other

gray-brown to brownish violet-staining species suchas L luridus L uvidus (Fr) Fr L violascens (JOtto) Fr L pallescens v palmerensis Hesler amp AHSm and L cordovaensis Hesler amp AH Sm are alsolarger and have a subalpine ecology none havebeen reported from the central or southern RockyMountains (Hesler and Smith 1979 Heilmann-Clau-sen et al 1998) Lactarius cistophilus Bon amp Trimbachtypically has larger basidiomata larger basidiospores(10ndash14 6 72ndash9 mm) and occurs under Cistus inthe Mediterranean region (Basso 1999) The smallpale gray-brown European alpine taxon L uvidus falpigines Jamoni amp Bon has microscopic featuresthat match those of L uvidus (Jamoni and Bon1991) but not L pallidomarginatus The small non-violet staining L nanus has basidiospores withthicker more jagged ridges and differently shapedmacrocystidia (often with more rounded apices)(FIG 7)

Lactarius repraesentaneus Britzelm FIGS 6 7Short description Lactarius repraesentaneus is recog-

nized by its medium to large size orange-brown to yel-low-brown hairy pileus with a heavily bearded margincream to pale-yellow lamellae stout cream to pale-yel-low often pitted stipe violet staining latex and tissuespicy-floral odor slightly acrid flavor and associationwith Betula Picea and possibly shrubby Salix speciessuch as S glauca

Pileus 60ndash100 mm diam convex to plano-convexusually with a depressed center and sometimes with apapilla smooth at center becoming increasingly hairyto bearded toward margin dry to viscid azonate sur-face orange-brown at center becoming pale yellow-brown to cream toward margin hairs pale yellow-brown to red-brown margin strongly incurvedremaining so or becoming nearly straight whenmature Lamellae adnate to decurrent crowdedcream to pale-yellow staining violet where damagedStipe 30ndash70 6 15ndash35 mm stout equal to clavate cen-tral dry to viscid cream to pale yellow surface oftenwith numerous dingy yellow golden yellow yellow-brown or light orange-brown pits hollow Latex scarceto abundant white becoming violet Context of pileusand stipe white staining violet where damaged Odorspicy-floral Flavor slightly acrid Basidiospore depositnot observed Basidiospores 8ndash105 6 6ndash85 mm av5 93 6 73 mm Q 5 113ndash143 Q5 127 hyaline in25 KOH broadly ellipsoid to ellipsoid ornamenta-tion amyloid of narrow to broad ridges and isolatedwarts forming an incomplete to dense reticulum to1 mm high Basidia 610ndash762 6 102ndash114 mm four-spored Pleuromacrocystidia 787ndash1448 6 89ndash127 mm subfusiform to lanceolate thin-walled withacute to moniliform apex scattered to abundant

432 MYCOLOGIA

Cheilomacrocystidia 559ndash1397 6 76ndash127 mm subfu-siform to lanceolate thin-walled with acute to monili-form apex scattered to abundant Pileipellis anixotrichoderm with well-developed gelatinous layer

Distribution and habitat Lactarius repraesentaneus firstwas described from Germany (Britzelmayr 1885) Phy-logenetic analyses including material from Germanyconfirm L repraesentaneus from alpine areas of theRocky Mountains and overall reinforce the traditionalconcept that this species has a broad ecological andgeographic range in arctic-alpine and subalpine habi-tats in North America northern central and southernEurope In alpine areas of the Rocky Mountains itassociates with Salix glauca however krummholz Piceaalso is typically (maybe always) present it is reportedwith Picea and Betula elsewhere

Previous reports from arctic-alpine areas mainlywith Betula include Alaska (Laursen and Ammirati1982 Geml et al 2009) the Rocky Mountain alpinezone (Cripps and Horak 2008 Cripps and Barge2013) Greenland (Knudsen and Borgen 1982 Watling1983 Elbourne and Knudsen 1990 Borgen 2006 Bor-gen et al 2006) Iceland (Eyjolfsdottir pers comm)Fennoscandia (Kallio and Kankainen 1964 Kuumlhner1975 Bon 1998) Siberia (Karatygin et al 1999) andthe Altai Mountains (Gorbunova 2010)

Specimens examined FINLAND Enontakiouml 16 Aug 2004JV21671 (TURA) Varsinais-Suomi 9 Aug 1998 JV13837F(TURA) SWEDEN Latnjavagge 12 Aug 2007 EL92-07(GB) USA COLORADO Chaffee County Sawatch RangeCottonwood Pass 4 Aug 2001 CLC 1643 (MONT) 16 Aug2011 EB0048 (MONT) Pitkin County Sawatch Range Inde-pendence Pass 14 Aug 2001 CLC 1747 (MONT) ZT9837(MONT) 15 Aug 2001 ZT9537 (MONT) MONTANA Car-bon County Beartooth Plateau Birch Site 12 Aug 2002CLC 1971 (MONT) 13 Aug 2007 CLC 2318 (MONT) 18Aug 2013 EB107-13 (MONT) WYOMING Park CountyBeartooth Plateau Frozen Lakes 21 Aug 1999 CLC1394 (MONT)

Other specimens examined SWEDEN Latnjavagge 14 Aug2010 EL57-10 (L dryadophilus GB) USA ALASKA 5 Aug2011 CLC 2729 (L dryadophilus MONT) Kenai PeninsulaCounty Palmer Creek Road 7 Aug 2011 CLC 2744 (L drya-dophilus MONT)

Comments The combination of a large pale yellow-brown to orange-brown pileus with a bearded marginand violet staining latex clearly separates L repraesenta-neus from other Lactarius species of the central andsouthern Rocky Mountain alpine zone Phylogeneti-cally close the arctic-alpine taxon L dryadophilus ismorphologically similar but has a cream to yellowsmoother pileus with hairs restricted to near the mar-gin basidiospore ornamentation consisting of sparsethinner ridges and ecological associations with Dryasand Salix It has not been reported from the RockyMountains

Lactarius salicis-reticulatae Kuumlhner FIGS 6 7Short description Lactarius salicis-reticulatae is recog-

nized by its small to medium cream pale yellow orpale ocher sporocarps with pale yellowish well-spacedlamellae often with a pinkish buff tint cream to pale yel-low stipe violet staining latex and tissue mild to slightlysweet odor mild flavor and association with dwarf (Sreticulata) and possibly also shrubby Salix species

Pileus 20ndash40 mm diam convex to broadly convexusually with a depressed center smooth dry to viscidazonate cream pale yellow or pale ocher sometimesdarker toward center staining violet where damagedmargin invcurved remaining so or becoming down-turned when mature Lamellae adnate to subdecur-rent fairly well-spaced cream pale yellow or paleorange-ocher often with a pinkish-buff tint stainingviolet where damaged Stipe 15ndash20 6 10ndash15 mmequal to clavate central dry to viscid cream to pale-yellow staining violet where damaged hollow Latexscarce watery-white becoming violet Context ofpileus and stipe white staining violet where damagedOdor mild to slightly sweet Flavor mild Basidiosporedeposit not observed Basidiospores (7ndash)85ndash115 6(7ndash)8ndash10 mm av 5 100 6 88 mm Q 5 (100ndash)113ndash143 Q 5 125 hyaline in 25 KOH sublgobose toellipsoid ornamentation amyloid of narrow ridgesand a few isolated warts forming an incomplete reticu-lum to 03 mm high Basidia 737ndash889 6 102ndash127 mm four-spored Pleuromacrocystidia 762ndash10166 76ndash114 mm subfusiform to fusiform thin-walledwith rounded to acute to moniliform apex scarceCheilomacrocystidia 686ndash9146 76ndash102 mm subfusi-form to fusiform thin-walled with acute to rounded tomoniliform apex scattered to abundant Pileipellis anixocutis

Distribution and habitat Lactarius salicis-reticulatae firstwas described from alpine tundra with Salix on MtLaringktatjaringkka Sweden (Kuumlhner 1975) Phylogenetic ana-lyses including material from the type locality confirmthis species from alpine areas on the Beartooth Plateauwith Salix reticulata and shrubby Salix spp and in arctic-alpine areas of Alaska Svalbard and Finland OverallL salicis-reticulatae appears largely confined to arctic-alpine habitats Colorado collections with S reticulataand S planifolia may represent a distinct taxonalthough support is low

Previous arctic-alpine reports include Greenland(Lamoure et al 1982 Heilmann-Clausen et al1998 Borgen et al 2006) the Rocky Mountainalpine zone (Cripps and Horak 2008 Cripps andBarge 2013) the Alps (Kuumlhner 1975 Kuumlhner andLamoure 1986 Jamoni 1995 2008 Bon 1998 Heil-mann-Clausen et al 1998) the Pyrenees (Ballaraacute1997) Fennoscandia (Kuumlhner 1975 Heilmann-Clau-sen et al 1998 Gulden 2005 Knudsen and

BARGE ET AL ROCKY MOUNTAIN ALPINE LACTARIUS 433

Vesterholt 2008) and Siberia (Nezdoiminogo 1997Karatygin et al 1999) previous reports of L aspi-deoides Burl from arctic-alpine areas in Alaska (Laur-sen and Ammirati 1982) Greenland (Knudsen andBorgen 1982) and the British Isles (Watling 1987)may refer to this species

Specimens examined FINLAND Near Utsjoki 28 Aug 2012CLC 2885 (MONT) SWEDEN Mt Laringktatjaringkka 12 Aug1972 K72-104 (Holotype G) 15 Aug 1999 JV15133 (Typelocality TURA) USA ALASKA Kenai Peninsula County Pal-mer Creek Road 7 Aug 2011 CLC 2745 (MONT) MON-TANA Carbon County Beartooth Plateau Birch Site 17Aug 2011 CLC 2776 (MONT) Carbon County BeartoothPlateau Highline Trail 8 Aug 1998 CLC 1211 (MONT)WYOMING Park County Beartooth Plateau GardnerLake 16 Aug 2014 EB0057-14 (MONT)

Other specimens examined FINLAND Koillismaa 31 Aug2005 JV23334 (L flavopalustris TURA) GREENLAND Sis-miut 19 Aug 2000 CLC 1536 (L salicis-herbaceae MONT)USA COLORADO Pitkin County Sawatch Range Indepen-dence Pass 13 Aug 2001 CLC 1741 (L aff salicis-reticulataeMONT) San Juan County San Juan Range CinnamonPass 10 Aug 2001 CLC 1710 (L aff salicis-reticulataeMONT) San Juan County San Juan Range Maggie Gulch11 Aug 2011 EB0036 (L aff salicis-reticulatae MONT)San Juan County San Juan Range Stony Pass 9 Aug 2001CLC 1689 (L aff salicis-reticulatae MONT) 13 Aug 2011EB0039 (L aff salicis-reticulatae MONT) MICHIGANEmmet County 10 Aug 1973 RL Shaffer 6957 (L aspi-deoides MICH)

Comments Some taxonomic confusion has beencaused by the fact that Kuumlhner (1975) originallynamed this species L aspideoides Kuumlhner but subse-quently renamed it because the name had been pre-viously applied L aspideoides Burl (1907) is generallylarger with a somewhat zonate pileus more crowdedlamellae that lack salmon-colored hues slightly smallerbasidiospores with broader ridges a bitter to slightlyacrid flavor and a subalpine ecology apparently ineastern and western North America (pers obs of RLShaffer 6957 Hesler and Smith 1979) Lactarius fla-vopalustris is also similar however it has a largermore brightly yellow pileus with faint zonationmore crowded lamellae a weakly scrobiculate stipe(sometimes) smaller basidiospores with thickerridges and a subalpine ecology (Kytoumlvuori 2009)Phylogenetic analyses (FIG 5) reveal three collec-tions of L flavopalustris (two from near the typelocality in Finland) nested in a well-supported cladewith L aspideoides from a subalpine forest in north-ern Michigan suggesting the possible conspecificityof these taxa Neither is reported from the RockyMountains Two other collections identified as L fla-vopalustris (from Estonia) were resolved as distinctfrom the L aspideoidesflavopalustris clade (FIG 5)their identity is unclear

Lactarius aspideus is differentiated from L salicis-reti-culate by its smaller basidiospores with denser reticula-tion more crowded creamier-colored lamellae andhabitat mainly in subalpine areas (Heilmann-Clausenet al 1998) although an environmental sequence iso-lated from an S polaris root tip from arctic Svalbardclusters with L aspideus in phylogenetic analyses (FIG5) Lactarius dryadophilus is often larger with a cream-colored pileus that has a tomentose to bearded margin(Heilmann-Clausen et al 1998) Lactarius flavidus is lar-ger with a sparsely zonate pileus more crowdedcream-colored lamellae darker violet-staining contextbasidiospores with more prominent ornamentationand a distally amyloid plage and subalpine ecology(Heilmann-Clausen et al 1998) Lactarius flavoaspideusKytoumlv has slightly smaller basidiospores on averagewith a denser reticulum and subalpine ecology withSalix (Kytoumlvuori 2009) it has been reported onlyfrom northern Europe Lactarius salicis-herbaceae asimilar arctic-alpine Salix associate has smaller (typi-cally) darker yellow to yellow-brown sporocarpspale cream to grayish buff lamellae and more denselyreticulate basidiospores (pers obs Heilmann-Clausenet al 1998) It is fairly widespread in arctic-alpineareas but is not reported from alpine areas in the cen-tral and southern Rocky Mountains Lactarius aff sali-cis-reticulatae from Colorado may be a distinct taxonand sporocarps can obtain a larger size (pileus to 90mm diam) but otherwise morphological featuresstrongly overlap

KEY TO ROCKY MOUNTAIN ALPINE LACTARIUS SPECIES

1 Latex white turning violet to dull lilac or flesh stain-ing violet to dull lilac 2

1 Latex watery to white unchanging and flesh notstaining violet to dull lilac 42 Fruiting body robust pileus 60ndash100 mm diam

yellow-brown to orange-brown hairy especiallynear margin lamellae cream to pale-yellow stipestout cream to pale-yellow often with numerousyellow-brown pits odor spicy-floral flavor mildlyacrid basidiospores 8ndash105 6 6ndash85 mm puta-tively associated with Betula and Picea (possiblyalso Salix) in Rocky Mountain alpine zone andsubalpine and arctic-alpine areas in North Amer-ica and Eurasia L repraesentaneus

2 Fruiting body smaller 20ndash50 mm diam pileus andmargin smooth 3

3 Pileus pale yellow to pale ocher lamellae cream paleyellow or pale orange-ocher stipe cream to pale yel-low odor mild to slightly sweet flavor mild basidios-pores 85ndash115 6 8ndash10 mm in Rocky Mountain alpinezone and other arctic-alpine areas in North Americaand Eurasia with Salix L salicis-reticulatae

3 Pileus blotchy light tan to light brown to gray-brownmargin lighter lamellae white to pale yellow-cream

434 MYCOLOGIA

stipe white to cream odor mild flavor mild basi-

diospores 8ndash10 6 65ndash8 mm in low alpine areas inColorado with Salix planifolia and possibly Wyom-

ing with Salix glauca L pallidomarginatus4 Pileus bright orange to orange-brown 10ndash45

mm diam margin smooth lamellae pale creamto pale orange stipe pale orange odor mildflavor mild in Rocky Mountain alpine zonearctic-alpine Alaska (possibly also subalpine)to Svalbard with Salix L lanceolatus

4 Pileus pinkish buff gray-brown brown ormauve 5

5 With odor of coconuts pileus 15ndash50 mm diam palepinkish buff to pale gray-brown with glaucous coat-ing lamellae cream to pale yellow-orange stipe buffor pale salmon flavor mild basidiospores 7ndash9 6 5ndash7mm in Rocky Mountain alpine zone and subalpinearctic-alpine areas in North America and Eurasiawith Betula L glyciosmus

5 Without odor of coconuts pileus 10ndash50 mm diamgray-brown to liver-brown with whitish glaucous coat-ing lamellae cream to light tan sometimes with pink-ish tinge discoloring to dingy tan where damagedStipe pale apricot to dingy cream-tan flavor mild toslightly acrid basidiospores 7ndash105 6 5ndash8 mm inRocky Mountain alpine zone and arctic-alpine areasin Eurasia and likely other arctic-alpine areas in NorthAmerica with Salix L nanus

DISCUSSION

The number of Lactarius species reported from theRocky Mountain alpine zone represents only a fractionof the total number of Lactarius species reported else-where from arctic-alpine areas however nearly halfof the core arctic-alpine species are now confirmedfor continental North America Core species oftencited as being restricted to arctic-alpine areas withdwarf and shrubby Salix andor Dryas species includeL brunneoviolaceus L dryadophilus L lanceolatus Lnanus L pseudouvidus L salicis-herbaceae and L salicis-reticulatae (Knudsen and Borgen 1982 Heilmann-Clau-sen et al 1998 Basso 1999 Knudsen and Vesterholt2008) Phylogenetic analyses and morphological exam-ination confirm three out of seven of these species forthe Rocky Mountain alpine zone (L lanceolatus Lnanus L salicis-reticulatae) and support the hypothesisthat these species are largely restricted to arctic-alpineareas with Salix One definitive new species L pallido-marginatus (with Salix) was revealed along with a possi-ble cryptic species L aff salicis-reticulatae (also withSalix) Many Lactarius species previously reportedfrom arctic-alpine areas are also commonly reportedfrom subalpine areas with Betulaceae andor Pinaceae

as well as with other plant families (Heilmann-Clausenet al 1998) From this set of species we report L gly-ciosmus which associates with Betula glandulosa in theRocky Mountain alpine zone and Betula spp acrossthe rest of its distribution and L repraesentaneus whichis commonly reported with Betula and Picea (Heslerand Smith 1979 Heilmann-Clausen et al 1998) andappears to associate with Salix glauca andor perhapskrummholz Picea in the Rocky Mountain alpine zone

Several taxa of interest were not molecularly distinctIn dataset A L lanceolatus did not form a monophyleticgroup but was paraphyletic with respect to Lactarius sp2 the L luculentus group and L aurantiacus In datasetB L nanus and L hysginoides were not clearly distinctIn dataset C L salicis-reticulatae did not form a clearmonophyletic group and L pseudouvidus L brunneovio-laceus and L luridus were not clearly distinguishedfrom each other especially at the RPB2 locus poormolecular resolution also has been encountered forthe latter three taxa by Das et al (2015)

Overall there was little genetic variation distinguish-ing closely related species in this study These findingsare similar to reports from other researchers who sug-gest that there is in general relatively little molecularvariation yet high morphological variation in genusLactarius (Verbeken and Nuytinck 2013) While manyof the species could be morphologically distinguishedall of the Rocky Mountain alpine taxa delineated inthis study are 97 or more similar at the ITS regionto one or more species For example L repraesentaneusand L dryadophilus are as much as 994 similar L sal-icis-reticulatae is approximately 98 similar to L salicis-herbaceae and L flavopalustris L pallidomarginatus isapproximately 97ndash98 similar to L aff brunneoviola-ceus L luridus L montanus and members of the Laff pseudouvidus group and approximately 99 similarto L aspideus L glyciosmus is approximately 98 simi-lar to L mammosus L nanus is approximately 98similar to L hysginoides and L lanceolatus is approxi-mately 97 similar to L substriatussubflammeus L aur-antiacus L luculentus v laetus and Lactarius sp 1 and 2and approximately 98 similar to L luculentus Thishigh degree of similarity could be problematic forenvironmental sequencing studies that use a strict97 ITS region similarity cutoff for recognizing spe-cies (eg Geml et al 2012 Timling et al 2012 2014)In addition Tedersoo et al (2014) suggested that theITS region in the sebacina lineage evolves moreslowly in high latitude areas which could be true forother genera The RPB2 locus proved better for differ-entiating some species and overall phylogenetic ana-lyses revealed several taxonomic issues that we werenot able to resolve A search for additional phylogen-etically informative loci for Lactarius should beinitiated along with more species-level phylogenetic

BARGE ET AL ROCKY MOUNTAIN ALPINE LACTARIUS 435

studies to provide a high quality molecular databasefor ecological studies Furthermore improvement ofmethods for sequencing older material such as typespecimens is critical

Most of the Rocky Mountain alpine Lactarius speciesdelineated in this study (except L pallidomarginatusand L aff salicis-reticulatae for which further studycould reveal broader ranges) appear to have broadintercontinental distributions in North America andEurasia The current distributions of these specieslikely were shaped by a number of biotic and abioticfactors that include host dispersal and distribution cli-mate glaciation and geography Because the genusLactarius is obligately ectomycorrhizal it is likely thatthe distribution of each fungal species was shaped lar-gely by the distribution of suitable host plants manyof which also have broad intercontinental distribu-tions Long-distance dispersal is hypothesized to haveplayed an important role in structuring present distri-bution patterns of arctic-alpine plants although manyappear clonal (Abbott and Brochmann 2003 Alsoset al 2007) and ectomycorrhizal fungi (Geml et al2012 Timling et al 2014) It also has been suggestedthat long-distance dispersal mechanisms have beenselected for in arctic flora due to repeated glaciationsand the need to track potential niches (Alsos et al2007) Pollen data from Blacktail Pond YellowstoneNational Park suggest that montane mid-elevationsin the northern greater Yellowstone ecosystem (Mon-tana Wyoming) were occupied by alpine tundra(Betula Salix many herbaceous taxa) similar to thatof the present Rocky Mountain alpine vegetation dur-ing the last glacial period (14 000ndash11 500 calibratedyears before present) (Krause and Whitlock 2013)Alpine vegetation also appears to have dominatedareas in the southern Rocky Mountains (WyomingColorado) 17 500ndash12 600 calibrated years before pre-sent (Minckley 2014) Both studies suggest the pre-sence of broad low-elevation glacial refugia for arctic-alpine life in the Rocky Mountains along the glacialfront and along with this study lend support to theidea that arctic-alpine Lactarius species (and manyother ectomycorrhizal fungi) may have occupied tun-dra areas south of the icesheets during cooler weatherduring the last glacial maximum and then dispersedupward in elevation and northward as the glaciersand ectomycorrhizal host plants retreated Broaderecological tolerances of taxa such as L glyciosmus andL repraesentaneus which are not restricted to arctic-alpine habitats likely let these species persist in tundraand non-tundra areas during the last glacial maximumas well as to track arctic-alpine vegetation as it movedupward in elevation and northward following theretreat of the icesheets For these species to haveachieved and maintained such broad distributions

gene flow must have been possible over long distancesduring the Pleistocene perhaps mediated by long-dis-tance dispersal (Geml et al 2012) range shifts due toclimate change and glacial expansion and retreat(Birks 2008) glacial and interglacial refugia (Billings1974) and the Bering land bridge (Shapiro et al2004 Geml et al 2006)

ACKNOWLEDGMENTS

We thank Dr Ellen Larsson Dr Pierre-Arthur Moreau JukkaVauras Dr Ursula Peintner Dr Egon Horak and herbaria GIB TU O GB TURA C LIP MICH DBG VPI and NY forloan of specimens Carol Johnson and Dr Matt Lavin areacknowledged for reviewing early stages of the manuscriptWe thank Don Bachman Marcy Barge and Charissa Bujakfor assistance with field work We also gratefully acknowledgesupport from the Montana Institute on Ecosystems graduateenhancement fund

LITERATURE CITED

Abbott RJ Brochmann C 2003 History and evolution of thearctic flora in the footsteps of Eric Hulteacuten Mol Ecol12299ndash313 doi101046j1365-294X200301731x

Alsos IG Eidesen PB Ehrich D Skrede I Westergaard KJacobsen GH Landvik JY Taberlet P Brochmann C2007 Frequent long-distance plant colonization in thechanging arctic Science 3161606ndash1609 doi101126science1139178

Ballaraacute J 1997 Nou estudi drsquoespegravecies fuacutengiques interessantsdels estatges alpiacute i subalpiacute dels Pirineus catalans RevCatalana Micol 201ndash24

Barge E 2015 Systematics of Lactarius in the Rocky Moun-tain alpine zone (masterrsquos thesis) Bozeman MontanaMontana State University 265 p

Basso MT 1999 Lactarius Pers Fungi Europaei 7 AlassioMykoflora (Italy) 845 p

Beker HJ Eberhardt U Vesterholt J 2010 Hebeloma hiemaleBres In arcticalpine habitats N Am Fungi 551ndash65

Bessette AE Harris DB Bessette AR 2009 Milk mushroomsof North America a field identification guide to thegenus Lactarius Syracuse New York Syracuse UniversityPress 297 p

Billings WD 1974 Adaptations and origins of alpine plantsArctic Alpine Res 6129ndash142 doi1023071550081

mdashmdashmdash 1988 Alpine vegetation In Barbour MG BillingsWD eds North American terrestrial vegetation NewYork Cambridge University Press p 392ndash420

Birks HH 2008 The late-quaternary history of arctic andalpine plants Plant Ecol Divers 1135ndash146 doi10108017550870802328652

Bogar LM Kennedy PG 2013 New wrinkles in an old para-digm neighborhood effects can modify the structureand specificity of Alnus-associated ectomycorrhizal fun-gal communities FEMS Microbiol Ecol 83767ndash777doi1011111574-694112032

Bon M 1985a Stage mycologie alpine Lanslebourg (Savoie)du 1 aug 3 sep 1984 Bull Feacuted Mycol Daupineacute-Savoie9619ndash25

436 MYCOLOGIA

acrid taste (pers obs of holotype AH Smith 83602Hesler and Smith 1979) Molecular phylogenetic ana-lyses (FIG 3) suggest that L subflammeus is conspecificwith L substriatus however sequencing of the holo-type of L substriatus is needed to confirm this observa-tion Lactarius luculentus L luculentus v laetus Lsubflammeus and L substriatus have been reportedonly from subalpine areas with conifers in westernNorth America (Hesler and Smith 1979)

Lactarius duplicatus AH Sm (syn L lapponicus Har-maja) a North American and European Betula associ-ate is also morphologically close to L lanceolatusHowever it can be distinguished by the productionof strongly yellowing latex We did not procure anymolecular data from material representing this taxonand no molecular data is publicly available on Gen-Bank or UNITE However L duplicatus should beincluded in future phylogenetic analyses investigatingthe L lanceolatusL aurantiacus group

Lactarius nanus J Favre FIGS 6 7Short description Lactarius nanus is recognized by its

typically small size gray-brown to liver brown pileustypically with a glaucous coating at least when youngcream to pinkish tan lamellae pale cream-tan to paleapricot stipe watery unchanging latex mild odormild to slightly acrid flavor and occurrence with Salixspecies in arctic-alpine areas

Pileus 10ndash50 mm diam shallowly convex to plano-convex becoming plane to infundibuliform with orwithout a depressed center and small papilla smoothslightly viscid to dry with whitish glaucous coatingwhen immature easily rubbing away or fading in ageoften in patches deep brown to gray-brown to liverbrown with lighter and darker areas present oftenlighter toward margin becoming lighter overall upondessication margin straight when immature becomingupturned and wavy to convoluted when matureLamellae adnate to subdecurrent subdistant to slightlycrowded cream when immature becoming dingycream to light tan in age sometimes with a faint pink-ish tinge discoloring to dingy tan where damagedStipe 5ndash30 6 3ndash15 mm equal to clavate centralsmooth dry at first covered with whitish glaucous coat-ing as in pileus pale apricot to dingy cream-tan hol-low Latex scarce watery unchanging Context ofpileus and stipe dingy cream to brownish Odor mildFlavor mild to slightly acrid Basidiospore deposit notobserved Basidiospores 7ndash105(ndash12) 6 5ndash8 mm av 584 6 66 mm Q 5 107ndash160 Q 5 127 hyaline in25 KOH subglobose to ellipsoid ornamentationof amyloid scattered ridges and warts forming anincomplete reticulum with some completely reticulateareas to 07 mm high Basidia 406ndash762 6 89ndash152 mm mostly four-spored some two-spored

Pleuromacrocystidia 533ndash965(ndash1143) 6 64ndash114mm lanceolate with acute to moniliform apexscattered to abundant Cheilomacrocystidia 260ndash9146 76ndash102 mm subulate to linear with undulatingmargin with acute to rounded apex scattered toabundant Pileipellis an (ixo-) cutis with some ascend-ing hyphae embedded in a very faint gelatinouslayer

Distribution and habitat Lactarius nanus originally wasdescribed by Jules Favre from the Swiss Alps with Salixherbacea L (Favre 1955) Phylogenetic analyses includ-ing a specimen from near the type locality confirmthis species in the Rocky Mountain alpine zone whereit is one of the most commonly encountered Lactariusspecies it associates with Salix arctica S reticulata andS planifolia Overall L nanus appears to be largelyrestricted to arctic-alpine areas with Salix

Previous arctic-alpine reports include Canada (Ohe-noja and Ohenoja 1993 2010) the Rocky Mountainalpine zone (Moser and McKnight 1987 Cripps andHorak 2008 Cripps and Barge 2013) Greenland(Knudsen and Borgen 1982 Lamoure et al 1982 Heil-mann-Clausen et al 1998 Borgen et al 2006) Iceland(Eyjolfsdottir pers comm Knudsen and Vesterholt2008) the European Alps (Favre 1955 Kuumlhner 1975Kuumlhner and Lamoure 1986 Senn-Irlet 1993 Bon1985a 1998 Heilmann-Clausen et al 1998 Jamoni1995) the Pyrenees (Bon and Ballaraacute 1996) the Car-pathians (Ronikier 2008) Fennoscandia (Kuumlhner1975 Gulden et al 1985 Heilmann-Clausen et al1998 Gulden 2005 Knudsen and Vesterholt 2008)Svalbard (Gulden et al 1985) Siberia (Nezdoiminogo1997) and alpine areas in the Altai Mountains (Karaty-gin et al 1999)

Specimens examined FRANCE Savoie 24 Aug 1989 Bon89093 (LIP) NORWAY Finse 5 Aug 2010 CLC 2134(MONT) Svalbard Longyearbyen 20 Aug 2002 CLC 1896(MONT) SWEDEN Torne Lappmark 15 Aug 1999JV15148 (TURA) SWITZERLAND Grisons 20 Aug 1943ZA192c (Holotype G) USA COLORADO Pitkin CountySawatch Range Independence Pass 6 Aug 2000 CLC 1471(MONT) San Juan County San Juan Range Black BearPass 11 Aug 2001 CLC 1716 (MONT) 11 Aug 2001ZT9529 (MONT) San Juan County San Juan Range Cinna-mon Pass 27 July 2002 CLC 1801 (MONT) 15 Aug 2012EB138 (MONT) San Miguel County San Juan Range Imo-gene Pass 29 July 2002 CLC 1829 (MONT) MONTANACarbon County Beartooth Plateau Highline Trail 7 Aug1998 CLC 1221 (MONT) 8 Aug 1998 ZT6422 (MONT) 18Aug 2013 EB106-13 (MONT) WYOMING Park CountyBeartooth Plateau Frozen Lakes 21 Aug 1999 CLC 1403(MONT) Park County Beartooth Plateau Solifluction Ter-races 11 Aug 2012 EB125 (MONT)

Other specimens examined FINLAND Koillismaa 28 Aug2011 JV28432 (L hysginoides TURA)

428 MYCOLOGIA

Comments Lactarius nanus is morphologically andphylogenetically close to L hysginoides however thelatter has a more cinnamon-toned pileus smaller basi-diospores (6ndash8 6 5ndash7 mm av 68 6 57 mm) and isgenerally reported from boreal areas with Betula Piceaand Salix in Europe (Heilmann-Clausen et al 1998)although phylogenetic placement of environmentalsequences extend its range into boreal Alaska (FIG4) Its range likely overlaps that of L nanus in alpine-subalpine transition areas It is not reported from theRocky Mountains Lactarius mammosus is a more robustspecies with a coconut odor (Heilmann-Clausen et al1998) that associates with members of the Pinaceaeit has not been reported from the Rocky MountainsDifferences between L glyciosmus and L nanus are dis-cussed in the taxonomy section for L glyciosmus

Lactarius glyciosmus (Fr) Fr FIGS 6 7Short description Lactarius glyciosmus is recognized by

its small to medium size pale gray-brown to mauvepileus typically with a glaucous coating at least whenyoung cream pale pinkish or pale yellow-orangelamellae buff or pale salmon stipe watery unchan-ging latex odor of coconuts mild flavor and associa-tion with Betula

Pileus 15ndash50 mm diam broadly convex later becom-ing plane with or without a depressed center and smallpapilla smooth dry pale gray-brown to mauve (violet-brown) with whitish glaucous coating when immaturefading in age azonate to lightly zoned especially nearmargin margin incurved when young and remainingso or becoming straight when mature Lamellaeadnate to subdecurrent slightly crowded to crowdedcream to pale pinkish to pale yellow-orange Stipe10ndash40 6 3ndash12 mm equal to slightly clavate oftencurved central to eccentric smooth dry buff or palesalmon with faint glaucous coating at first as in pileusstuffed or hollow often white-mycelioid toward baseLatex scarce watery unchanging Context of pileusand stipe pale cream Odor of coconut Flavor mildBasidiospore deposit not observed Basidiospores 7ndash96 5ndash7 mm av 5 77 6 60 mm Q 5 117ndash142 Q 5

128 hyaline in 25 KOH broadly ellipsoid to ellip-soid ornamentation of amyloid scattered ridges andwarts forming an incomplete to nearly complete reticu-lum to 05 mm high Basidia 381ndash635 6 76ndash102 mmmostly four-spored some two-spored Pleuromacrocys-tidia 457ndash635 6 64ndash76 mm subclavate to lanceolatewith rounded to occasionally mucronate apex scarceto scattered Cheilomacrocystidia 33ndash66 6 51ndash89mm cylindrical to subclavate with rounded apex scat-tered to abundant Pileipellis a cutis with tufts ofascending hyphae

Distribution and habitat Lactarius glyciosmus was firstdescribed by Fries from a hemiboreal to boreal habitat

in the Femsjouml region of Sweden and the species lacks atype specimen Phylogenetic analyses including speci-mens from the type locality confirm this species inthe central and southern Rocky Mountain alpinezone and suggest it has a broad ecological and geogra-phical distribution confirmed from subalpine and arc-tic-alpine areas in Alaska western Canada IdahoWisconsin New Hampshire Svalbard and Fennoscan-dia In subalpine and alpine areas of the Rocky Moun-tains L glyciosmus associates with Betula glandulosaPhylogenetic placement (FIG 4) of ectomycorrhizalroot tip sequences confirm Betula nana L as a host inAlaska (GU998223 GU998695) and likely B occidenta-lis Hook in Idaho (JX198526 Bogar amp Kennedy2013) Elsewhere it is reported with B pubescens Ehrh(eg Elborne amp Knudsen 1990) and B papyrifera Mar-shall (eg Durall et al 2006)

Previous reports from arctic-alpine areas includeCanada (Ohenoja and Ohenoja 1993 2010) theRocky Mountain alpine zone (Cripps and Horak2008 Cripps and Barge 2013) Greenland (Lange1957 Knudsen and Borgen 1982 Lamoure et al1982 Elbourne and Knudsen 1990 Borgen 2006 Bor-gen et al 2006) Iceland (Eyjolfsdottir pers comm) theAlps (Bon 1998) Fennoscandia (Kallio and Kankainen1964 Kuumlhner 1975 Bon 1998) Svalbard (Ohenoja1971 Geml et al 2012) and Siberia (Karatygin et al1999) At least some reports might be L aff glyciosmusas defined here

Specimens examined FINLAND Fjell Ailigas 27 Aug 2012CLC 2874 (MONT) SWEDEN Femsjouml 13 Aug 1978 MMoser 19780191 (Type locality IB) 16 Aug 1978 M Moser19780234 (Type locality IB) 9 Aug 1981 M Moser19810148 (Type locality IB) USA COLORADO BoulderCounty Front Range Blue Lake 2 Aug 2001 CLC 1624(MONT) Boulder County Front Range Brainard Lake 13Aug 2000 20923 (DBG) Ouray County San Juan RangeIronton Park 13 Aug 2012 EB133 (MONT) MONTANACarbon County Beartooth Plateau Birch Site 29 July 1997CLC 1134 5 ZT6096 (MONT) 8 Aug 1998 CLC 1217(MONT) 10 Aug 1999 TWO 269 (MONT) 19 Aug 1999CLC 1380 (MONT) 13 Aug 2007 ZT12723 (MONT)

Other specimens examined ESTONIA Saare CountyLuumlmanda Commune 17 Sep 2012 TU118535 (L aff glycios-mus TU) Tartu County Votildennu Commune 12 Sept 2010TU106737 (L aff glyciosmus TU) 13 Sept 2010 TU106724(L aff glyciosmus TU)

Comments Lactarius glyciosmus is morphologically andphylogenetically similar to L mammosus however thelatter has a browner pileus stouter stature and moreelongate more reticulate basidiospores (Heilmann-Clausen et al 1998) Phylogenetic placement ofsequences isolated from ectomycorrhizal root tips ofPinus sylvestris L (AJ633116 Heinonsalo et al 2007)and Picea abies (L) Karst (FM992937 Kjoslashller andClemmensen 2009) (FIG 4) confirm that L mammosus

BARGE ET AL ROCKY MOUNTAIN ALPINE LACTARIUS 429

FIG 7 Comparison of micromorphological features of Rocky Mountain alpine specimens and Holotypes (where available)Basidiospores pleuromacrocystidia (left) and cheilomacrocystidia (right) of A Lactarius lanceolatus (CLC2319) B L lanceolatus(F 4239 HOLOTYPE) C L nanus (CLC1471) D L nanus (ZA 192c HOLOTYPE) E L pallidomarginatus Barge amp Cripps spnov (EB0041 HOLOTYPE) F L pseudouvidus (Va 71971 HOLOTYPE) G L salicis-reticulatae (CLC2776) H L salicis-reticulatae

430 MYCOLOGIA

associates with members of the Pinaceae however italso has been reported with Betula mainly in subalpineareas (Heilmann-Clausen et al 1998) Lactarius vietus(Fr) Fr is also similar but has a distinct pale zonenear the stipe apex latex that dries greenish graylonger pleuromacrocystidia and a mild to slightly fruityodor (Heilmann-Clausen et al 1998) It has not beenreported from alpine areas in the Rocky Mountainsalthough it has been reported from elsewhere (mostlynorthern areas) in North America with Betula (Heslerand Smith 1979) The similar-looking L nanus alsooccurs in alpine areas of the Rocky Mountains andhas larger basidiospores larger and differently shapedmacrocystidia lacks an odor of coconut and associateswith Salix Lactarius spinosulus is phylogenetically closeto L glyciosmus (as well as L nanus) (FIG 4) howevermorphologically they are very different Lactarius spino-sulus has a distinctly zoned coarsely squamulose pink-ish salmon- or brick-colored pileus with a finely hairymargin and fruity odor (Heilmann-Clausen et al1998) it has not been reported from North AmericaNo definitive morphological differences were detectedbetween L glyciosmus and L aff glyciosmus and the lat-ter is not reported from the Rocky Mountainalpine zone

Lactarius pallidomarginatus Barge amp CL Cripps spnov FIGS 6 7Mycobank MB812246

Typification USA COLORADO San Juan CountySan Juan Range Stony Pass scattered solitary onsoil under Salix planifolia 13 Aug 2011 EB0041 (holo-type MONT) GenBank KR090940 (ITS) andKR091010 (RPB2)

Etymology Referring to the pale pileus marginDiagnosis Lactarius pallidomarginatus is recognized by

its small to medium size blotchy light tan to lightbrown to gray-brown pileus which is typically lightertoward the margin white to pale yellow-cream lamel-lae pale cream stipe violet staining tissue mild odorand flavor and association with Salix Morphologicallyit is close to L pseudouvidus Kuumlhner but differs by hav-ing smaller basidiospores with an ornamentation ofthinner ridges as well as macrocystidia that becomemore lanceolate (as opposed to subfusiform) and atte-nuated Phylogenetically it is close to L aspideus (Fr)Fr but differs consistently in two nucleotide positionsin the rDNA ITS locus and at eight nucleotide posi-tions in the RPB2 locus morphologically L aspideushas larger yellow basidiomata

Pileus 20ndash50 mm diam convex to broadly convexbecoming broadly convex to plane with or without adepressed center smooth dry to slightly viscid azo-nate blotchy light tan to light brown to gray-browndeveloping violet stains lighter (to cream) toward mar-gin margin incurved to downturned when youngremaining downturned or becoming nearly straightwhen mature Lamellae adnate to subdecurrent subdi-stant to slightly crowded white to pale yellow-creamstaining violet where damaged Stipe 10ndash40 6 5ndash10mm equal to slightly clavate central smooth drywhite to cream staining violet where damaged hollowLatex scarce watery staining tissue violet Context ofpileus and stipe white to cream staining violet wheredamaged Odor mild Flavor mild Basidiosporedeposit not observed Basidiospores 8ndash10 6 65ndash8mm av 5 87 6 71 mm Q 5 114ndash136 Q5 123 hya-line in 25 KOH broadly ellipsoid to ellipsoid orna-mentation amyloid of thin ridges and a few isolatedwarts forming an incomplete to dense reticulum to05 mm high Basidia 508ndash711 6 102ndash127 mm four-spored Pleuromacrocystidia 813ndash1118 6 89ndash102mm cylindrical to lanceolate with acute to moniliformapex scarce to scattered Cheilomacrocystidia 483ndash1016 6 76ndash127 mm cylindrical to lanceolate withacute to moniliform apex scattered Pileipellis an(ixo-)cutis with some ascending hyphae embedded ina faint gelatinous layer

Distribution and habitat Rare in low alpine areas withSalix planifolia reported from Independence Pass inthe Sawatch Range and Stony Pass in the San JuanRange in Colorado An additional collection(ZT5229) from Union Peak in the Wind River Rangeof Wyoming with Salix glauca is likely conspecific how-ever this hypothesis has not been confirmed withmolecular data Due to confusion with L pseudouvidusit is possible that L pallidomarginatus is more wide-spread than confirmed here

Specimens examined USA COLORADO Pitkin CountySawatch Range Independence Pass 6 Aug 2000 CLC 1470(MONT) 9 Aug 2000 ZT9093 (MONT) San Juan CountySan Juan Range Stony Pass 13 Aug 2011 EB0041 (HolotypeMONT) WYOMING Sublette County Wind River RangeUnion Peak 22 Aug 1994 ZT5229 (MONT)

Other specimens examined AUSTRIA Tyrol 3 July 2007 UPeintner 20070035 (L aff pseudouvidus from near type local-ity IB) FINLAND Inarin Lappi 16 Aug 1995 JV10468 (Laff pseudouvidus TURA) Koillismaa JV28448F 30 Aug 2011(L aff pseudouvidus originally identified as L brunneoviola-ceus TURA) FRANCE Isegravere 13 Aug 1971 Va 7 1971 (Holo-type of L pseudouvidus G) ITALY Trentino 20 Aug 2004 U

r(K72-104 HOLOTYPE no cheilocystidia drawn) I L glyciosmus (TWO269) J L repraesentaneus (CLC1747) Bars 5 10 mm forbasidiospores and 20 mm for cystidia Drawings by E Barge

BARGE ET AL ROCKY MOUNTAIN ALPINE LACTARIUS 431

Peintner 20040156 (L aff pseudouvidus from near type local-ity originally identified as L robertianus IB) NORWAYFinse 10 Aug 2005 CLC 2133 (Lactarius aff brunneoviolaceusMONT) Stordal 26 Aug 2006 P Larsen 361395 (L aff brun-neoviolaceus originally identified as L pseudouvidus O) Sval-bard 18 Aug 2002 TWO809 (L aff pseudouvidus MONT)20 Aug 2002 CLC 1910 (Lactarius sp 5 MONT) Ulvik 11Aug 2011 E Soslashyland 73867 (L aff pseudouvidus O) SWE-DEN Latnjajaure 4 Aug 2011 EL101-11 (L aff pseudouvidusGB) Latnjavagge 17 Aug 2010 EL63-10 (L aff pseudouvi-dus GB)

Comments Morphological features of L pallidomar-ginatus fit the holotype description of L pseudouvidusand both are described as lighter near the pileus mar-gin (Kuumlhner 1975 Basso 1999) however basidios-pores of L pallidomarginatus are slightly smaller (8ndash10 6 65ndash8 mm as opposed to 75ndash12 6 65ndash8(85)mm) and have narrower ridges that form a less densereticulum and macrocystidia take on a more lanceo-late form with greater attenuation of the apex (FIG7) In phylogenetic analyses exsiccati identified as Lpseudouvidus from Europe (including specimensfrom near the type locality) appear as distinct fromRocky Mountain alpine material and may representa species complex Lactarius sp 3 (CLC1910) col-lected in Svalbard has darker more vinaceous pileithan L pallidomarginatus and may represent anotherundescribed species although only one collectionwas morphologically examined Members of the Laff brunneoviolaceus clade (FIG 5) that were exam-ined differ from L pallidomarginatus in having dar-ker more vinaceous pilei larger more elongatebasidiospores (to 13 mm) and green staining of exsic-cati in 25 KOH a feature not mentioned previouslyfor L brunneoviolaceus but which could be of taxo-nomic interest Based on taxa included in the phylo-genetic analyses (FIG 5) Lactarius aspideus appearsclosely related to L pallidomarginatus however theformer has more yellow coloration and typically lar-ger sporocarps (pileus 10ndash70 mm diam) and smallerbasidiospores (67ndash95 6 56ndash78 mm) traditionallyL aspideus has been placed with other yellow violet-staining taxa such as L flavidus Boud L salicis-reticu-latae and L salicis-herbaceae (Heilmann-Clausen et al1998) but it is separated here

Lactarius montanus another violet-staining speciesoriginally described from the Rocky Mountains dif-fers from L pallidomarginatus by its larger staturedarker grayish vinaceous to vinaceous-brown pileusand green staining flesh in 25 KOH it is typicallyfound in subalpine habitats with conifers but alsooccurs in the krummholz zone The violet-stainingLactarius pallescens Hesler amp AH Sm occurs in theRocky Mountains but its sporocarps are larger andlighter in color and it is reported from subalpineconifer forests (Hesler and Smith 1979) Other

gray-brown to brownish violet-staining species suchas L luridus L uvidus (Fr) Fr L violascens (JOtto) Fr L pallescens v palmerensis Hesler amp AHSm and L cordovaensis Hesler amp AH Sm are alsolarger and have a subalpine ecology none havebeen reported from the central or southern RockyMountains (Hesler and Smith 1979 Heilmann-Clau-sen et al 1998) Lactarius cistophilus Bon amp Trimbachtypically has larger basidiomata larger basidiospores(10ndash14 6 72ndash9 mm) and occurs under Cistus inthe Mediterranean region (Basso 1999) The smallpale gray-brown European alpine taxon L uvidus falpigines Jamoni amp Bon has microscopic featuresthat match those of L uvidus (Jamoni and Bon1991) but not L pallidomarginatus The small non-violet staining L nanus has basidiospores withthicker more jagged ridges and differently shapedmacrocystidia (often with more rounded apices)(FIG 7)

Lactarius repraesentaneus Britzelm FIGS 6 7Short description Lactarius repraesentaneus is recog-

nized by its medium to large size orange-brown to yel-low-brown hairy pileus with a heavily bearded margincream to pale-yellow lamellae stout cream to pale-yel-low often pitted stipe violet staining latex and tissuespicy-floral odor slightly acrid flavor and associationwith Betula Picea and possibly shrubby Salix speciessuch as S glauca

Pileus 60ndash100 mm diam convex to plano-convexusually with a depressed center and sometimes with apapilla smooth at center becoming increasingly hairyto bearded toward margin dry to viscid azonate sur-face orange-brown at center becoming pale yellow-brown to cream toward margin hairs pale yellow-brown to red-brown margin strongly incurvedremaining so or becoming nearly straight whenmature Lamellae adnate to decurrent crowdedcream to pale-yellow staining violet where damagedStipe 30ndash70 6 15ndash35 mm stout equal to clavate cen-tral dry to viscid cream to pale yellow surface oftenwith numerous dingy yellow golden yellow yellow-brown or light orange-brown pits hollow Latex scarceto abundant white becoming violet Context of pileusand stipe white staining violet where damaged Odorspicy-floral Flavor slightly acrid Basidiospore depositnot observed Basidiospores 8ndash105 6 6ndash85 mm av5 93 6 73 mm Q 5 113ndash143 Q5 127 hyaline in25 KOH broadly ellipsoid to ellipsoid ornamenta-tion amyloid of narrow to broad ridges and isolatedwarts forming an incomplete to dense reticulum to1 mm high Basidia 610ndash762 6 102ndash114 mm four-spored Pleuromacrocystidia 787ndash1448 6 89ndash127 mm subfusiform to lanceolate thin-walled withacute to moniliform apex scattered to abundant

432 MYCOLOGIA

Cheilomacrocystidia 559ndash1397 6 76ndash127 mm subfu-siform to lanceolate thin-walled with acute to monili-form apex scattered to abundant Pileipellis anixotrichoderm with well-developed gelatinous layer

Distribution and habitat Lactarius repraesentaneus firstwas described from Germany (Britzelmayr 1885) Phy-logenetic analyses including material from Germanyconfirm L repraesentaneus from alpine areas of theRocky Mountains and overall reinforce the traditionalconcept that this species has a broad ecological andgeographic range in arctic-alpine and subalpine habi-tats in North America northern central and southernEurope In alpine areas of the Rocky Mountains itassociates with Salix glauca however krummholz Piceaalso is typically (maybe always) present it is reportedwith Picea and Betula elsewhere

Previous reports from arctic-alpine areas mainlywith Betula include Alaska (Laursen and Ammirati1982 Geml et al 2009) the Rocky Mountain alpinezone (Cripps and Horak 2008 Cripps and Barge2013) Greenland (Knudsen and Borgen 1982 Watling1983 Elbourne and Knudsen 1990 Borgen 2006 Bor-gen et al 2006) Iceland (Eyjolfsdottir pers comm)Fennoscandia (Kallio and Kankainen 1964 Kuumlhner1975 Bon 1998) Siberia (Karatygin et al 1999) andthe Altai Mountains (Gorbunova 2010)

Specimens examined FINLAND Enontakiouml 16 Aug 2004JV21671 (TURA) Varsinais-Suomi 9 Aug 1998 JV13837F(TURA) SWEDEN Latnjavagge 12 Aug 2007 EL92-07(GB) USA COLORADO Chaffee County Sawatch RangeCottonwood Pass 4 Aug 2001 CLC 1643 (MONT) 16 Aug2011 EB0048 (MONT) Pitkin County Sawatch Range Inde-pendence Pass 14 Aug 2001 CLC 1747 (MONT) ZT9837(MONT) 15 Aug 2001 ZT9537 (MONT) MONTANA Car-bon County Beartooth Plateau Birch Site 12 Aug 2002CLC 1971 (MONT) 13 Aug 2007 CLC 2318 (MONT) 18Aug 2013 EB107-13 (MONT) WYOMING Park CountyBeartooth Plateau Frozen Lakes 21 Aug 1999 CLC1394 (MONT)

Other specimens examined SWEDEN Latnjavagge 14 Aug2010 EL57-10 (L dryadophilus GB) USA ALASKA 5 Aug2011 CLC 2729 (L dryadophilus MONT) Kenai PeninsulaCounty Palmer Creek Road 7 Aug 2011 CLC 2744 (L drya-dophilus MONT)

Comments The combination of a large pale yellow-brown to orange-brown pileus with a bearded marginand violet staining latex clearly separates L repraesenta-neus from other Lactarius species of the central andsouthern Rocky Mountain alpine zone Phylogeneti-cally close the arctic-alpine taxon L dryadophilus ismorphologically similar but has a cream to yellowsmoother pileus with hairs restricted to near the mar-gin basidiospore ornamentation consisting of sparsethinner ridges and ecological associations with Dryasand Salix It has not been reported from the RockyMountains

Lactarius salicis-reticulatae Kuumlhner FIGS 6 7Short description Lactarius salicis-reticulatae is recog-

nized by its small to medium cream pale yellow orpale ocher sporocarps with pale yellowish well-spacedlamellae often with a pinkish buff tint cream to pale yel-low stipe violet staining latex and tissue mild to slightlysweet odor mild flavor and association with dwarf (Sreticulata) and possibly also shrubby Salix species

Pileus 20ndash40 mm diam convex to broadly convexusually with a depressed center smooth dry to viscidazonate cream pale yellow or pale ocher sometimesdarker toward center staining violet where damagedmargin invcurved remaining so or becoming down-turned when mature Lamellae adnate to subdecur-rent fairly well-spaced cream pale yellow or paleorange-ocher often with a pinkish-buff tint stainingviolet where damaged Stipe 15ndash20 6 10ndash15 mmequal to clavate central dry to viscid cream to pale-yellow staining violet where damaged hollow Latexscarce watery-white becoming violet Context ofpileus and stipe white staining violet where damagedOdor mild to slightly sweet Flavor mild Basidiosporedeposit not observed Basidiospores (7ndash)85ndash115 6(7ndash)8ndash10 mm av 5 100 6 88 mm Q 5 (100ndash)113ndash143 Q 5 125 hyaline in 25 KOH sublgobose toellipsoid ornamentation amyloid of narrow ridgesand a few isolated warts forming an incomplete reticu-lum to 03 mm high Basidia 737ndash889 6 102ndash127 mm four-spored Pleuromacrocystidia 762ndash10166 76ndash114 mm subfusiform to fusiform thin-walledwith rounded to acute to moniliform apex scarceCheilomacrocystidia 686ndash9146 76ndash102 mm subfusi-form to fusiform thin-walled with acute to rounded tomoniliform apex scattered to abundant Pileipellis anixocutis

Distribution and habitat Lactarius salicis-reticulatae firstwas described from alpine tundra with Salix on MtLaringktatjaringkka Sweden (Kuumlhner 1975) Phylogenetic ana-lyses including material from the type locality confirmthis species from alpine areas on the Beartooth Plateauwith Salix reticulata and shrubby Salix spp and in arctic-alpine areas of Alaska Svalbard and Finland OverallL salicis-reticulatae appears largely confined to arctic-alpine habitats Colorado collections with S reticulataand S planifolia may represent a distinct taxonalthough support is low

Previous arctic-alpine reports include Greenland(Lamoure et al 1982 Heilmann-Clausen et al1998 Borgen et al 2006) the Rocky Mountainalpine zone (Cripps and Horak 2008 Cripps andBarge 2013) the Alps (Kuumlhner 1975 Kuumlhner andLamoure 1986 Jamoni 1995 2008 Bon 1998 Heil-mann-Clausen et al 1998) the Pyrenees (Ballaraacute1997) Fennoscandia (Kuumlhner 1975 Heilmann-Clau-sen et al 1998 Gulden 2005 Knudsen and

BARGE ET AL ROCKY MOUNTAIN ALPINE LACTARIUS 433

Vesterholt 2008) and Siberia (Nezdoiminogo 1997Karatygin et al 1999) previous reports of L aspi-deoides Burl from arctic-alpine areas in Alaska (Laur-sen and Ammirati 1982) Greenland (Knudsen andBorgen 1982) and the British Isles (Watling 1987)may refer to this species

Specimens examined FINLAND Near Utsjoki 28 Aug 2012CLC 2885 (MONT) SWEDEN Mt Laringktatjaringkka 12 Aug1972 K72-104 (Holotype G) 15 Aug 1999 JV15133 (Typelocality TURA) USA ALASKA Kenai Peninsula County Pal-mer Creek Road 7 Aug 2011 CLC 2745 (MONT) MON-TANA Carbon County Beartooth Plateau Birch Site 17Aug 2011 CLC 2776 (MONT) Carbon County BeartoothPlateau Highline Trail 8 Aug 1998 CLC 1211 (MONT)WYOMING Park County Beartooth Plateau GardnerLake 16 Aug 2014 EB0057-14 (MONT)

Other specimens examined FINLAND Koillismaa 31 Aug2005 JV23334 (L flavopalustris TURA) GREENLAND Sis-miut 19 Aug 2000 CLC 1536 (L salicis-herbaceae MONT)USA COLORADO Pitkin County Sawatch Range Indepen-dence Pass 13 Aug 2001 CLC 1741 (L aff salicis-reticulataeMONT) San Juan County San Juan Range CinnamonPass 10 Aug 2001 CLC 1710 (L aff salicis-reticulataeMONT) San Juan County San Juan Range Maggie Gulch11 Aug 2011 EB0036 (L aff salicis-reticulatae MONT)San Juan County San Juan Range Stony Pass 9 Aug 2001CLC 1689 (L aff salicis-reticulatae MONT) 13 Aug 2011EB0039 (L aff salicis-reticulatae MONT) MICHIGANEmmet County 10 Aug 1973 RL Shaffer 6957 (L aspi-deoides MICH)

Comments Some taxonomic confusion has beencaused by the fact that Kuumlhner (1975) originallynamed this species L aspideoides Kuumlhner but subse-quently renamed it because the name had been pre-viously applied L aspideoides Burl (1907) is generallylarger with a somewhat zonate pileus more crowdedlamellae that lack salmon-colored hues slightly smallerbasidiospores with broader ridges a bitter to slightlyacrid flavor and a subalpine ecology apparently ineastern and western North America (pers obs of RLShaffer 6957 Hesler and Smith 1979) Lactarius fla-vopalustris is also similar however it has a largermore brightly yellow pileus with faint zonationmore crowded lamellae a weakly scrobiculate stipe(sometimes) smaller basidiospores with thickerridges and a subalpine ecology (Kytoumlvuori 2009)Phylogenetic analyses (FIG 5) reveal three collec-tions of L flavopalustris (two from near the typelocality in Finland) nested in a well-supported cladewith L aspideoides from a subalpine forest in north-ern Michigan suggesting the possible conspecificityof these taxa Neither is reported from the RockyMountains Two other collections identified as L fla-vopalustris (from Estonia) were resolved as distinctfrom the L aspideoidesflavopalustris clade (FIG 5)their identity is unclear

Lactarius aspideus is differentiated from L salicis-reti-culate by its smaller basidiospores with denser reticula-tion more crowded creamier-colored lamellae andhabitat mainly in subalpine areas (Heilmann-Clausenet al 1998) although an environmental sequence iso-lated from an S polaris root tip from arctic Svalbardclusters with L aspideus in phylogenetic analyses (FIG5) Lactarius dryadophilus is often larger with a cream-colored pileus that has a tomentose to bearded margin(Heilmann-Clausen et al 1998) Lactarius flavidus is lar-ger with a sparsely zonate pileus more crowdedcream-colored lamellae darker violet-staining contextbasidiospores with more prominent ornamentationand a distally amyloid plage and subalpine ecology(Heilmann-Clausen et al 1998) Lactarius flavoaspideusKytoumlv has slightly smaller basidiospores on averagewith a denser reticulum and subalpine ecology withSalix (Kytoumlvuori 2009) it has been reported onlyfrom northern Europe Lactarius salicis-herbaceae asimilar arctic-alpine Salix associate has smaller (typi-cally) darker yellow to yellow-brown sporocarpspale cream to grayish buff lamellae and more denselyreticulate basidiospores (pers obs Heilmann-Clausenet al 1998) It is fairly widespread in arctic-alpineareas but is not reported from alpine areas in the cen-tral and southern Rocky Mountains Lactarius aff sali-cis-reticulatae from Colorado may be a distinct taxonand sporocarps can obtain a larger size (pileus to 90mm diam) but otherwise morphological featuresstrongly overlap

KEY TO ROCKY MOUNTAIN ALPINE LACTARIUS SPECIES

1 Latex white turning violet to dull lilac or flesh stain-ing violet to dull lilac 2

1 Latex watery to white unchanging and flesh notstaining violet to dull lilac 42 Fruiting body robust pileus 60ndash100 mm diam

yellow-brown to orange-brown hairy especiallynear margin lamellae cream to pale-yellow stipestout cream to pale-yellow often with numerousyellow-brown pits odor spicy-floral flavor mildlyacrid basidiospores 8ndash105 6 6ndash85 mm puta-tively associated with Betula and Picea (possiblyalso Salix) in Rocky Mountain alpine zone andsubalpine and arctic-alpine areas in North Amer-ica and Eurasia L repraesentaneus

2 Fruiting body smaller 20ndash50 mm diam pileus andmargin smooth 3

3 Pileus pale yellow to pale ocher lamellae cream paleyellow or pale orange-ocher stipe cream to pale yel-low odor mild to slightly sweet flavor mild basidios-pores 85ndash115 6 8ndash10 mm in Rocky Mountain alpinezone and other arctic-alpine areas in North Americaand Eurasia with Salix L salicis-reticulatae

3 Pileus blotchy light tan to light brown to gray-brownmargin lighter lamellae white to pale yellow-cream

434 MYCOLOGIA

stipe white to cream odor mild flavor mild basi-

diospores 8ndash10 6 65ndash8 mm in low alpine areas inColorado with Salix planifolia and possibly Wyom-

ing with Salix glauca L pallidomarginatus4 Pileus bright orange to orange-brown 10ndash45

mm diam margin smooth lamellae pale creamto pale orange stipe pale orange odor mildflavor mild in Rocky Mountain alpine zonearctic-alpine Alaska (possibly also subalpine)to Svalbard with Salix L lanceolatus

4 Pileus pinkish buff gray-brown brown ormauve 5

5 With odor of coconuts pileus 15ndash50 mm diam palepinkish buff to pale gray-brown with glaucous coat-ing lamellae cream to pale yellow-orange stipe buffor pale salmon flavor mild basidiospores 7ndash9 6 5ndash7mm in Rocky Mountain alpine zone and subalpinearctic-alpine areas in North America and Eurasiawith Betula L glyciosmus

5 Without odor of coconuts pileus 10ndash50 mm diamgray-brown to liver-brown with whitish glaucous coat-ing lamellae cream to light tan sometimes with pink-ish tinge discoloring to dingy tan where damagedStipe pale apricot to dingy cream-tan flavor mild toslightly acrid basidiospores 7ndash105 6 5ndash8 mm inRocky Mountain alpine zone and arctic-alpine areasin Eurasia and likely other arctic-alpine areas in NorthAmerica with Salix L nanus

DISCUSSION

The number of Lactarius species reported from theRocky Mountain alpine zone represents only a fractionof the total number of Lactarius species reported else-where from arctic-alpine areas however nearly halfof the core arctic-alpine species are now confirmedfor continental North America Core species oftencited as being restricted to arctic-alpine areas withdwarf and shrubby Salix andor Dryas species includeL brunneoviolaceus L dryadophilus L lanceolatus Lnanus L pseudouvidus L salicis-herbaceae and L salicis-reticulatae (Knudsen and Borgen 1982 Heilmann-Clau-sen et al 1998 Basso 1999 Knudsen and Vesterholt2008) Phylogenetic analyses and morphological exam-ination confirm three out of seven of these species forthe Rocky Mountain alpine zone (L lanceolatus Lnanus L salicis-reticulatae) and support the hypothesisthat these species are largely restricted to arctic-alpineareas with Salix One definitive new species L pallido-marginatus (with Salix) was revealed along with a possi-ble cryptic species L aff salicis-reticulatae (also withSalix) Many Lactarius species previously reportedfrom arctic-alpine areas are also commonly reportedfrom subalpine areas with Betulaceae andor Pinaceae

as well as with other plant families (Heilmann-Clausenet al 1998) From this set of species we report L gly-ciosmus which associates with Betula glandulosa in theRocky Mountain alpine zone and Betula spp acrossthe rest of its distribution and L repraesentaneus whichis commonly reported with Betula and Picea (Heslerand Smith 1979 Heilmann-Clausen et al 1998) andappears to associate with Salix glauca andor perhapskrummholz Picea in the Rocky Mountain alpine zone

Several taxa of interest were not molecularly distinctIn dataset A L lanceolatus did not form a monophyleticgroup but was paraphyletic with respect to Lactarius sp2 the L luculentus group and L aurantiacus In datasetB L nanus and L hysginoides were not clearly distinctIn dataset C L salicis-reticulatae did not form a clearmonophyletic group and L pseudouvidus L brunneovio-laceus and L luridus were not clearly distinguishedfrom each other especially at the RPB2 locus poormolecular resolution also has been encountered forthe latter three taxa by Das et al (2015)

Overall there was little genetic variation distinguish-ing closely related species in this study These findingsare similar to reports from other researchers who sug-gest that there is in general relatively little molecularvariation yet high morphological variation in genusLactarius (Verbeken and Nuytinck 2013) While manyof the species could be morphologically distinguishedall of the Rocky Mountain alpine taxa delineated inthis study are 97 or more similar at the ITS regionto one or more species For example L repraesentaneusand L dryadophilus are as much as 994 similar L sal-icis-reticulatae is approximately 98 similar to L salicis-herbaceae and L flavopalustris L pallidomarginatus isapproximately 97ndash98 similar to L aff brunneoviola-ceus L luridus L montanus and members of the Laff pseudouvidus group and approximately 99 similarto L aspideus L glyciosmus is approximately 98 simi-lar to L mammosus L nanus is approximately 98similar to L hysginoides and L lanceolatus is approxi-mately 97 similar to L substriatussubflammeus L aur-antiacus L luculentus v laetus and Lactarius sp 1 and 2and approximately 98 similar to L luculentus Thishigh degree of similarity could be problematic forenvironmental sequencing studies that use a strict97 ITS region similarity cutoff for recognizing spe-cies (eg Geml et al 2012 Timling et al 2012 2014)In addition Tedersoo et al (2014) suggested that theITS region in the sebacina lineage evolves moreslowly in high latitude areas which could be true forother genera The RPB2 locus proved better for differ-entiating some species and overall phylogenetic ana-lyses revealed several taxonomic issues that we werenot able to resolve A search for additional phylogen-etically informative loci for Lactarius should beinitiated along with more species-level phylogenetic

BARGE ET AL ROCKY MOUNTAIN ALPINE LACTARIUS 435

studies to provide a high quality molecular databasefor ecological studies Furthermore improvement ofmethods for sequencing older material such as typespecimens is critical

Most of the Rocky Mountain alpine Lactarius speciesdelineated in this study (except L pallidomarginatusand L aff salicis-reticulatae for which further studycould reveal broader ranges) appear to have broadintercontinental distributions in North America andEurasia The current distributions of these specieslikely were shaped by a number of biotic and abioticfactors that include host dispersal and distribution cli-mate glaciation and geography Because the genusLactarius is obligately ectomycorrhizal it is likely thatthe distribution of each fungal species was shaped lar-gely by the distribution of suitable host plants manyof which also have broad intercontinental distribu-tions Long-distance dispersal is hypothesized to haveplayed an important role in structuring present distri-bution patterns of arctic-alpine plants although manyappear clonal (Abbott and Brochmann 2003 Alsoset al 2007) and ectomycorrhizal fungi (Geml et al2012 Timling et al 2014) It also has been suggestedthat long-distance dispersal mechanisms have beenselected for in arctic flora due to repeated glaciationsand the need to track potential niches (Alsos et al2007) Pollen data from Blacktail Pond YellowstoneNational Park suggest that montane mid-elevationsin the northern greater Yellowstone ecosystem (Mon-tana Wyoming) were occupied by alpine tundra(Betula Salix many herbaceous taxa) similar to thatof the present Rocky Mountain alpine vegetation dur-ing the last glacial period (14 000ndash11 500 calibratedyears before present) (Krause and Whitlock 2013)Alpine vegetation also appears to have dominatedareas in the southern Rocky Mountains (WyomingColorado) 17 500ndash12 600 calibrated years before pre-sent (Minckley 2014) Both studies suggest the pre-sence of broad low-elevation glacial refugia for arctic-alpine life in the Rocky Mountains along the glacialfront and along with this study lend support to theidea that arctic-alpine Lactarius species (and manyother ectomycorrhizal fungi) may have occupied tun-dra areas south of the icesheets during cooler weatherduring the last glacial maximum and then dispersedupward in elevation and northward as the glaciersand ectomycorrhizal host plants retreated Broaderecological tolerances of taxa such as L glyciosmus andL repraesentaneus which are not restricted to arctic-alpine habitats likely let these species persist in tundraand non-tundra areas during the last glacial maximumas well as to track arctic-alpine vegetation as it movedupward in elevation and northward following theretreat of the icesheets For these species to haveachieved and maintained such broad distributions

gene flow must have been possible over long distancesduring the Pleistocene perhaps mediated by long-dis-tance dispersal (Geml et al 2012) range shifts due toclimate change and glacial expansion and retreat(Birks 2008) glacial and interglacial refugia (Billings1974) and the Bering land bridge (Shapiro et al2004 Geml et al 2006)

ACKNOWLEDGMENTS

We thank Dr Ellen Larsson Dr Pierre-Arthur Moreau JukkaVauras Dr Ursula Peintner Dr Egon Horak and herbaria GIB TU O GB TURA C LIP MICH DBG VPI and NY forloan of specimens Carol Johnson and Dr Matt Lavin areacknowledged for reviewing early stages of the manuscriptWe thank Don Bachman Marcy Barge and Charissa Bujakfor assistance with field work We also gratefully acknowledgesupport from the Montana Institute on Ecosystems graduateenhancement fund

LITERATURE CITED

Abbott RJ Brochmann C 2003 History and evolution of thearctic flora in the footsteps of Eric Hulteacuten Mol Ecol12299ndash313 doi101046j1365-294X200301731x

Alsos IG Eidesen PB Ehrich D Skrede I Westergaard KJacobsen GH Landvik JY Taberlet P Brochmann C2007 Frequent long-distance plant colonization in thechanging arctic Science 3161606ndash1609 doi101126science1139178

Ballaraacute J 1997 Nou estudi drsquoespegravecies fuacutengiques interessantsdels estatges alpiacute i subalpiacute dels Pirineus catalans RevCatalana Micol 201ndash24

Barge E 2015 Systematics of Lactarius in the Rocky Moun-tain alpine zone (masterrsquos thesis) Bozeman MontanaMontana State University 265 p

Basso MT 1999 Lactarius Pers Fungi Europaei 7 AlassioMykoflora (Italy) 845 p

Beker HJ Eberhardt U Vesterholt J 2010 Hebeloma hiemaleBres In arcticalpine habitats N Am Fungi 551ndash65

Bessette AE Harris DB Bessette AR 2009 Milk mushroomsof North America a field identification guide to thegenus Lactarius Syracuse New York Syracuse UniversityPress 297 p

Billings WD 1974 Adaptations and origins of alpine plantsArctic Alpine Res 6129ndash142 doi1023071550081

mdashmdashmdash 1988 Alpine vegetation In Barbour MG BillingsWD eds North American terrestrial vegetation NewYork Cambridge University Press p 392ndash420

Birks HH 2008 The late-quaternary history of arctic andalpine plants Plant Ecol Divers 1135ndash146 doi10108017550870802328652

Bogar LM Kennedy PG 2013 New wrinkles in an old para-digm neighborhood effects can modify the structureand specificity of Alnus-associated ectomycorrhizal fun-gal communities FEMS Microbiol Ecol 83767ndash777doi1011111574-694112032

Bon M 1985a Stage mycologie alpine Lanslebourg (Savoie)du 1 aug 3 sep 1984 Bull Feacuted Mycol Daupineacute-Savoie9619ndash25

436 MYCOLOGIA

Comments Lactarius nanus is morphologically andphylogenetically close to L hysginoides however thelatter has a more cinnamon-toned pileus smaller basi-diospores (6ndash8 6 5ndash7 mm av 68 6 57 mm) and isgenerally reported from boreal areas with Betula Piceaand Salix in Europe (Heilmann-Clausen et al 1998)although phylogenetic placement of environmentalsequences extend its range into boreal Alaska (FIG4) Its range likely overlaps that of L nanus in alpine-subalpine transition areas It is not reported from theRocky Mountains Lactarius mammosus is a more robustspecies with a coconut odor (Heilmann-Clausen et al1998) that associates with members of the Pinaceaeit has not been reported from the Rocky MountainsDifferences between L glyciosmus and L nanus are dis-cussed in the taxonomy section for L glyciosmus

Lactarius glyciosmus (Fr) Fr FIGS 6 7Short description Lactarius glyciosmus is recognized by

its small to medium size pale gray-brown to mauvepileus typically with a glaucous coating at least whenyoung cream pale pinkish or pale yellow-orangelamellae buff or pale salmon stipe watery unchan-ging latex odor of coconuts mild flavor and associa-tion with Betula

Pileus 15ndash50 mm diam broadly convex later becom-ing plane with or without a depressed center and smallpapilla smooth dry pale gray-brown to mauve (violet-brown) with whitish glaucous coating when immaturefading in age azonate to lightly zoned especially nearmargin margin incurved when young and remainingso or becoming straight when mature Lamellaeadnate to subdecurrent slightly crowded to crowdedcream to pale pinkish to pale yellow-orange Stipe10ndash40 6 3ndash12 mm equal to slightly clavate oftencurved central to eccentric smooth dry buff or palesalmon with faint glaucous coating at first as in pileusstuffed or hollow often white-mycelioid toward baseLatex scarce watery unchanging Context of pileusand stipe pale cream Odor of coconut Flavor mildBasidiospore deposit not observed Basidiospores 7ndash96 5ndash7 mm av 5 77 6 60 mm Q 5 117ndash142 Q 5

128 hyaline in 25 KOH broadly ellipsoid to ellip-soid ornamentation of amyloid scattered ridges andwarts forming an incomplete to nearly complete reticu-lum to 05 mm high Basidia 381ndash635 6 76ndash102 mmmostly four-spored some two-spored Pleuromacrocys-tidia 457ndash635 6 64ndash76 mm subclavate to lanceolatewith rounded to occasionally mucronate apex scarceto scattered Cheilomacrocystidia 33ndash66 6 51ndash89mm cylindrical to subclavate with rounded apex scat-tered to abundant Pileipellis a cutis with tufts ofascending hyphae

Distribution and habitat Lactarius glyciosmus was firstdescribed by Fries from a hemiboreal to boreal habitat

in the Femsjouml region of Sweden and the species lacks atype specimen Phylogenetic analyses including speci-mens from the type locality confirm this species inthe central and southern Rocky Mountain alpinezone and suggest it has a broad ecological and geogra-phical distribution confirmed from subalpine and arc-tic-alpine areas in Alaska western Canada IdahoWisconsin New Hampshire Svalbard and Fennoscan-dia In subalpine and alpine areas of the Rocky Moun-tains L glyciosmus associates with Betula glandulosaPhylogenetic placement (FIG 4) of ectomycorrhizalroot tip sequences confirm Betula nana L as a host inAlaska (GU998223 GU998695) and likely B occidenta-lis Hook in Idaho (JX198526 Bogar amp Kennedy2013) Elsewhere it is reported with B pubescens Ehrh(eg Elborne amp Knudsen 1990) and B papyrifera Mar-shall (eg Durall et al 2006)

Previous reports from arctic-alpine areas includeCanada (Ohenoja and Ohenoja 1993 2010) theRocky Mountain alpine zone (Cripps and Horak2008 Cripps and Barge 2013) Greenland (Lange1957 Knudsen and Borgen 1982 Lamoure et al1982 Elbourne and Knudsen 1990 Borgen 2006 Bor-gen et al 2006) Iceland (Eyjolfsdottir pers comm) theAlps (Bon 1998) Fennoscandia (Kallio and Kankainen1964 Kuumlhner 1975 Bon 1998) Svalbard (Ohenoja1971 Geml et al 2012) and Siberia (Karatygin et al1999) At least some reports might be L aff glyciosmusas defined here

Specimens examined FINLAND Fjell Ailigas 27 Aug 2012CLC 2874 (MONT) SWEDEN Femsjouml 13 Aug 1978 MMoser 19780191 (Type locality IB) 16 Aug 1978 M Moser19780234 (Type locality IB) 9 Aug 1981 M Moser19810148 (Type locality IB) USA COLORADO BoulderCounty Front Range Blue Lake 2 Aug 2001 CLC 1624(MONT) Boulder County Front Range Brainard Lake 13Aug 2000 20923 (DBG) Ouray County San Juan RangeIronton Park 13 Aug 2012 EB133 (MONT) MONTANACarbon County Beartooth Plateau Birch Site 29 July 1997CLC 1134 5 ZT6096 (MONT) 8 Aug 1998 CLC 1217(MONT) 10 Aug 1999 TWO 269 (MONT) 19 Aug 1999CLC 1380 (MONT) 13 Aug 2007 ZT12723 (MONT)

Other specimens examined ESTONIA Saare CountyLuumlmanda Commune 17 Sep 2012 TU118535 (L aff glycios-mus TU) Tartu County Votildennu Commune 12 Sept 2010TU106737 (L aff glyciosmus TU) 13 Sept 2010 TU106724(L aff glyciosmus TU)

Comments Lactarius glyciosmus is morphologically andphylogenetically similar to L mammosus however thelatter has a browner pileus stouter stature and moreelongate more reticulate basidiospores (Heilmann-Clausen et al 1998) Phylogenetic placement ofsequences isolated from ectomycorrhizal root tips ofPinus sylvestris L (AJ633116 Heinonsalo et al 2007)and Picea abies (L) Karst (FM992937 Kjoslashller andClemmensen 2009) (FIG 4) confirm that L mammosus

BARGE ET AL ROCKY MOUNTAIN ALPINE LACTARIUS 429

FIG 7 Comparison of micromorphological features of Rocky Mountain alpine specimens and Holotypes (where available)Basidiospores pleuromacrocystidia (left) and cheilomacrocystidia (right) of A Lactarius lanceolatus (CLC2319) B L lanceolatus(F 4239 HOLOTYPE) C L nanus (CLC1471) D L nanus (ZA 192c HOLOTYPE) E L pallidomarginatus Barge amp Cripps spnov (EB0041 HOLOTYPE) F L pseudouvidus (Va 71971 HOLOTYPE) G L salicis-reticulatae (CLC2776) H L salicis-reticulatae

430 MYCOLOGIA

associates with members of the Pinaceae however italso has been reported with Betula mainly in subalpineareas (Heilmann-Clausen et al 1998) Lactarius vietus(Fr) Fr is also similar but has a distinct pale zonenear the stipe apex latex that dries greenish graylonger pleuromacrocystidia and a mild to slightly fruityodor (Heilmann-Clausen et al 1998) It has not beenreported from alpine areas in the Rocky Mountainsalthough it has been reported from elsewhere (mostlynorthern areas) in North America with Betula (Heslerand Smith 1979) The similar-looking L nanus alsooccurs in alpine areas of the Rocky Mountains andhas larger basidiospores larger and differently shapedmacrocystidia lacks an odor of coconut and associateswith Salix Lactarius spinosulus is phylogenetically closeto L glyciosmus (as well as L nanus) (FIG 4) howevermorphologically they are very different Lactarius spino-sulus has a distinctly zoned coarsely squamulose pink-ish salmon- or brick-colored pileus with a finely hairymargin and fruity odor (Heilmann-Clausen et al1998) it has not been reported from North AmericaNo definitive morphological differences were detectedbetween L glyciosmus and L aff glyciosmus and the lat-ter is not reported from the Rocky Mountainalpine zone

Lactarius pallidomarginatus Barge amp CL Cripps spnov FIGS 6 7Mycobank MB812246

Typification USA COLORADO San Juan CountySan Juan Range Stony Pass scattered solitary onsoil under Salix planifolia 13 Aug 2011 EB0041 (holo-type MONT) GenBank KR090940 (ITS) andKR091010 (RPB2)

Etymology Referring to the pale pileus marginDiagnosis Lactarius pallidomarginatus is recognized by

its small to medium size blotchy light tan to lightbrown to gray-brown pileus which is typically lightertoward the margin white to pale yellow-cream lamel-lae pale cream stipe violet staining tissue mild odorand flavor and association with Salix Morphologicallyit is close to L pseudouvidus Kuumlhner but differs by hav-ing smaller basidiospores with an ornamentation ofthinner ridges as well as macrocystidia that becomemore lanceolate (as opposed to subfusiform) and atte-nuated Phylogenetically it is close to L aspideus (Fr)Fr but differs consistently in two nucleotide positionsin the rDNA ITS locus and at eight nucleotide posi-tions in the RPB2 locus morphologically L aspideushas larger yellow basidiomata

Pileus 20ndash50 mm diam convex to broadly convexbecoming broadly convex to plane with or without adepressed center smooth dry to slightly viscid azo-nate blotchy light tan to light brown to gray-browndeveloping violet stains lighter (to cream) toward mar-gin margin incurved to downturned when youngremaining downturned or becoming nearly straightwhen mature Lamellae adnate to subdecurrent subdi-stant to slightly crowded white to pale yellow-creamstaining violet where damaged Stipe 10ndash40 6 5ndash10mm equal to slightly clavate central smooth drywhite to cream staining violet where damaged hollowLatex scarce watery staining tissue violet Context ofpileus and stipe white to cream staining violet wheredamaged Odor mild Flavor mild Basidiosporedeposit not observed Basidiospores 8ndash10 6 65ndash8mm av 5 87 6 71 mm Q 5 114ndash136 Q5 123 hya-line in 25 KOH broadly ellipsoid to ellipsoid orna-mentation amyloid of thin ridges and a few isolatedwarts forming an incomplete to dense reticulum to05 mm high Basidia 508ndash711 6 102ndash127 mm four-spored Pleuromacrocystidia 813ndash1118 6 89ndash102mm cylindrical to lanceolate with acute to moniliformapex scarce to scattered Cheilomacrocystidia 483ndash1016 6 76ndash127 mm cylindrical to lanceolate withacute to moniliform apex scattered Pileipellis an(ixo-)cutis with some ascending hyphae embedded ina faint gelatinous layer

Distribution and habitat Rare in low alpine areas withSalix planifolia reported from Independence Pass inthe Sawatch Range and Stony Pass in the San JuanRange in Colorado An additional collection(ZT5229) from Union Peak in the Wind River Rangeof Wyoming with Salix glauca is likely conspecific how-ever this hypothesis has not been confirmed withmolecular data Due to confusion with L pseudouvidusit is possible that L pallidomarginatus is more wide-spread than confirmed here

Specimens examined USA COLORADO Pitkin CountySawatch Range Independence Pass 6 Aug 2000 CLC 1470(MONT) 9 Aug 2000 ZT9093 (MONT) San Juan CountySan Juan Range Stony Pass 13 Aug 2011 EB0041 (HolotypeMONT) WYOMING Sublette County Wind River RangeUnion Peak 22 Aug 1994 ZT5229 (MONT)

Other specimens examined AUSTRIA Tyrol 3 July 2007 UPeintner 20070035 (L aff pseudouvidus from near type local-ity IB) FINLAND Inarin Lappi 16 Aug 1995 JV10468 (Laff pseudouvidus TURA) Koillismaa JV28448F 30 Aug 2011(L aff pseudouvidus originally identified as L brunneoviola-ceus TURA) FRANCE Isegravere 13 Aug 1971 Va 7 1971 (Holo-type of L pseudouvidus G) ITALY Trentino 20 Aug 2004 U

r(K72-104 HOLOTYPE no cheilocystidia drawn) I L glyciosmus (TWO269) J L repraesentaneus (CLC1747) Bars 5 10 mm forbasidiospores and 20 mm for cystidia Drawings by E Barge

BARGE ET AL ROCKY MOUNTAIN ALPINE LACTARIUS 431

Peintner 20040156 (L aff pseudouvidus from near type local-ity originally identified as L robertianus IB) NORWAYFinse 10 Aug 2005 CLC 2133 (Lactarius aff brunneoviolaceusMONT) Stordal 26 Aug 2006 P Larsen 361395 (L aff brun-neoviolaceus originally identified as L pseudouvidus O) Sval-bard 18 Aug 2002 TWO809 (L aff pseudouvidus MONT)20 Aug 2002 CLC 1910 (Lactarius sp 5 MONT) Ulvik 11Aug 2011 E Soslashyland 73867 (L aff pseudouvidus O) SWE-DEN Latnjajaure 4 Aug 2011 EL101-11 (L aff pseudouvidusGB) Latnjavagge 17 Aug 2010 EL63-10 (L aff pseudouvi-dus GB)

Comments Morphological features of L pallidomar-ginatus fit the holotype description of L pseudouvidusand both are described as lighter near the pileus mar-gin (Kuumlhner 1975 Basso 1999) however basidios-pores of L pallidomarginatus are slightly smaller (8ndash10 6 65ndash8 mm as opposed to 75ndash12 6 65ndash8(85)mm) and have narrower ridges that form a less densereticulum and macrocystidia take on a more lanceo-late form with greater attenuation of the apex (FIG7) In phylogenetic analyses exsiccati identified as Lpseudouvidus from Europe (including specimensfrom near the type locality) appear as distinct fromRocky Mountain alpine material and may representa species complex Lactarius sp 3 (CLC1910) col-lected in Svalbard has darker more vinaceous pileithan L pallidomarginatus and may represent anotherundescribed species although only one collectionwas morphologically examined Members of the Laff brunneoviolaceus clade (FIG 5) that were exam-ined differ from L pallidomarginatus in having dar-ker more vinaceous pilei larger more elongatebasidiospores (to 13 mm) and green staining of exsic-cati in 25 KOH a feature not mentioned previouslyfor L brunneoviolaceus but which could be of taxo-nomic interest Based on taxa included in the phylo-genetic analyses (FIG 5) Lactarius aspideus appearsclosely related to L pallidomarginatus however theformer has more yellow coloration and typically lar-ger sporocarps (pileus 10ndash70 mm diam) and smallerbasidiospores (67ndash95 6 56ndash78 mm) traditionallyL aspideus has been placed with other yellow violet-staining taxa such as L flavidus Boud L salicis-reticu-latae and L salicis-herbaceae (Heilmann-Clausen et al1998) but it is separated here

Lactarius montanus another violet-staining speciesoriginally described from the Rocky Mountains dif-fers from L pallidomarginatus by its larger staturedarker grayish vinaceous to vinaceous-brown pileusand green staining flesh in 25 KOH it is typicallyfound in subalpine habitats with conifers but alsooccurs in the krummholz zone The violet-stainingLactarius pallescens Hesler amp AH Sm occurs in theRocky Mountains but its sporocarps are larger andlighter in color and it is reported from subalpineconifer forests (Hesler and Smith 1979) Other

gray-brown to brownish violet-staining species suchas L luridus L uvidus (Fr) Fr L violascens (JOtto) Fr L pallescens v palmerensis Hesler amp AHSm and L cordovaensis Hesler amp AH Sm are alsolarger and have a subalpine ecology none havebeen reported from the central or southern RockyMountains (Hesler and Smith 1979 Heilmann-Clau-sen et al 1998) Lactarius cistophilus Bon amp Trimbachtypically has larger basidiomata larger basidiospores(10ndash14 6 72ndash9 mm) and occurs under Cistus inthe Mediterranean region (Basso 1999) The smallpale gray-brown European alpine taxon L uvidus falpigines Jamoni amp Bon has microscopic featuresthat match those of L uvidus (Jamoni and Bon1991) but not L pallidomarginatus The small non-violet staining L nanus has basidiospores withthicker more jagged ridges and differently shapedmacrocystidia (often with more rounded apices)(FIG 7)

Lactarius repraesentaneus Britzelm FIGS 6 7Short description Lactarius repraesentaneus is recog-

nized by its medium to large size orange-brown to yel-low-brown hairy pileus with a heavily bearded margincream to pale-yellow lamellae stout cream to pale-yel-low often pitted stipe violet staining latex and tissuespicy-floral odor slightly acrid flavor and associationwith Betula Picea and possibly shrubby Salix speciessuch as S glauca

Pileus 60ndash100 mm diam convex to plano-convexusually with a depressed center and sometimes with apapilla smooth at center becoming increasingly hairyto bearded toward margin dry to viscid azonate sur-face orange-brown at center becoming pale yellow-brown to cream toward margin hairs pale yellow-brown to red-brown margin strongly incurvedremaining so or becoming nearly straight whenmature Lamellae adnate to decurrent crowdedcream to pale-yellow staining violet where damagedStipe 30ndash70 6 15ndash35 mm stout equal to clavate cen-tral dry to viscid cream to pale yellow surface oftenwith numerous dingy yellow golden yellow yellow-brown or light orange-brown pits hollow Latex scarceto abundant white becoming violet Context of pileusand stipe white staining violet where damaged Odorspicy-floral Flavor slightly acrid Basidiospore depositnot observed Basidiospores 8ndash105 6 6ndash85 mm av5 93 6 73 mm Q 5 113ndash143 Q5 127 hyaline in25 KOH broadly ellipsoid to ellipsoid ornamenta-tion amyloid of narrow to broad ridges and isolatedwarts forming an incomplete to dense reticulum to1 mm high Basidia 610ndash762 6 102ndash114 mm four-spored Pleuromacrocystidia 787ndash1448 6 89ndash127 mm subfusiform to lanceolate thin-walled withacute to moniliform apex scattered to abundant

432 MYCOLOGIA

Cheilomacrocystidia 559ndash1397 6 76ndash127 mm subfu-siform to lanceolate thin-walled with acute to monili-form apex scattered to abundant Pileipellis anixotrichoderm with well-developed gelatinous layer

Distribution and habitat Lactarius repraesentaneus firstwas described from Germany (Britzelmayr 1885) Phy-logenetic analyses including material from Germanyconfirm L repraesentaneus from alpine areas of theRocky Mountains and overall reinforce the traditionalconcept that this species has a broad ecological andgeographic range in arctic-alpine and subalpine habi-tats in North America northern central and southernEurope In alpine areas of the Rocky Mountains itassociates with Salix glauca however krummholz Piceaalso is typically (maybe always) present it is reportedwith Picea and Betula elsewhere

Previous reports from arctic-alpine areas mainlywith Betula include Alaska (Laursen and Ammirati1982 Geml et al 2009) the Rocky Mountain alpinezone (Cripps and Horak 2008 Cripps and Barge2013) Greenland (Knudsen and Borgen 1982 Watling1983 Elbourne and Knudsen 1990 Borgen 2006 Bor-gen et al 2006) Iceland (Eyjolfsdottir pers comm)Fennoscandia (Kallio and Kankainen 1964 Kuumlhner1975 Bon 1998) Siberia (Karatygin et al 1999) andthe Altai Mountains (Gorbunova 2010)

Specimens examined FINLAND Enontakiouml 16 Aug 2004JV21671 (TURA) Varsinais-Suomi 9 Aug 1998 JV13837F(TURA) SWEDEN Latnjavagge 12 Aug 2007 EL92-07(GB) USA COLORADO Chaffee County Sawatch RangeCottonwood Pass 4 Aug 2001 CLC 1643 (MONT) 16 Aug2011 EB0048 (MONT) Pitkin County Sawatch Range Inde-pendence Pass 14 Aug 2001 CLC 1747 (MONT) ZT9837(MONT) 15 Aug 2001 ZT9537 (MONT) MONTANA Car-bon County Beartooth Plateau Birch Site 12 Aug 2002CLC 1971 (MONT) 13 Aug 2007 CLC 2318 (MONT) 18Aug 2013 EB107-13 (MONT) WYOMING Park CountyBeartooth Plateau Frozen Lakes 21 Aug 1999 CLC1394 (MONT)

Other specimens examined SWEDEN Latnjavagge 14 Aug2010 EL57-10 (L dryadophilus GB) USA ALASKA 5 Aug2011 CLC 2729 (L dryadophilus MONT) Kenai PeninsulaCounty Palmer Creek Road 7 Aug 2011 CLC 2744 (L drya-dophilus MONT)

Comments The combination of a large pale yellow-brown to orange-brown pileus with a bearded marginand violet staining latex clearly separates L repraesenta-neus from other Lactarius species of the central andsouthern Rocky Mountain alpine zone Phylogeneti-cally close the arctic-alpine taxon L dryadophilus ismorphologically similar but has a cream to yellowsmoother pileus with hairs restricted to near the mar-gin basidiospore ornamentation consisting of sparsethinner ridges and ecological associations with Dryasand Salix It has not been reported from the RockyMountains

Lactarius salicis-reticulatae Kuumlhner FIGS 6 7Short description Lactarius salicis-reticulatae is recog-

nized by its small to medium cream pale yellow orpale ocher sporocarps with pale yellowish well-spacedlamellae often with a pinkish buff tint cream to pale yel-low stipe violet staining latex and tissue mild to slightlysweet odor mild flavor and association with dwarf (Sreticulata) and possibly also shrubby Salix species

Pileus 20ndash40 mm diam convex to broadly convexusually with a depressed center smooth dry to viscidazonate cream pale yellow or pale ocher sometimesdarker toward center staining violet where damagedmargin invcurved remaining so or becoming down-turned when mature Lamellae adnate to subdecur-rent fairly well-spaced cream pale yellow or paleorange-ocher often with a pinkish-buff tint stainingviolet where damaged Stipe 15ndash20 6 10ndash15 mmequal to clavate central dry to viscid cream to pale-yellow staining violet where damaged hollow Latexscarce watery-white becoming violet Context ofpileus and stipe white staining violet where damagedOdor mild to slightly sweet Flavor mild Basidiosporedeposit not observed Basidiospores (7ndash)85ndash115 6(7ndash)8ndash10 mm av 5 100 6 88 mm Q 5 (100ndash)113ndash143 Q 5 125 hyaline in 25 KOH sublgobose toellipsoid ornamentation amyloid of narrow ridgesand a few isolated warts forming an incomplete reticu-lum to 03 mm high Basidia 737ndash889 6 102ndash127 mm four-spored Pleuromacrocystidia 762ndash10166 76ndash114 mm subfusiform to fusiform thin-walledwith rounded to acute to moniliform apex scarceCheilomacrocystidia 686ndash9146 76ndash102 mm subfusi-form to fusiform thin-walled with acute to rounded tomoniliform apex scattered to abundant Pileipellis anixocutis

Distribution and habitat Lactarius salicis-reticulatae firstwas described from alpine tundra with Salix on MtLaringktatjaringkka Sweden (Kuumlhner 1975) Phylogenetic ana-lyses including material from the type locality confirmthis species from alpine areas on the Beartooth Plateauwith Salix reticulata and shrubby Salix spp and in arctic-alpine areas of Alaska Svalbard and Finland OverallL salicis-reticulatae appears largely confined to arctic-alpine habitats Colorado collections with S reticulataand S planifolia may represent a distinct taxonalthough support is low

Previous arctic-alpine reports include Greenland(Lamoure et al 1982 Heilmann-Clausen et al1998 Borgen et al 2006) the Rocky Mountainalpine zone (Cripps and Horak 2008 Cripps andBarge 2013) the Alps (Kuumlhner 1975 Kuumlhner andLamoure 1986 Jamoni 1995 2008 Bon 1998 Heil-mann-Clausen et al 1998) the Pyrenees (Ballaraacute1997) Fennoscandia (Kuumlhner 1975 Heilmann-Clau-sen et al 1998 Gulden 2005 Knudsen and

BARGE ET AL ROCKY MOUNTAIN ALPINE LACTARIUS 433

Vesterholt 2008) and Siberia (Nezdoiminogo 1997Karatygin et al 1999) previous reports of L aspi-deoides Burl from arctic-alpine areas in Alaska (Laur-sen and Ammirati 1982) Greenland (Knudsen andBorgen 1982) and the British Isles (Watling 1987)may refer to this species

Specimens examined FINLAND Near Utsjoki 28 Aug 2012CLC 2885 (MONT) SWEDEN Mt Laringktatjaringkka 12 Aug1972 K72-104 (Holotype G) 15 Aug 1999 JV15133 (Typelocality TURA) USA ALASKA Kenai Peninsula County Pal-mer Creek Road 7 Aug 2011 CLC 2745 (MONT) MON-TANA Carbon County Beartooth Plateau Birch Site 17Aug 2011 CLC 2776 (MONT) Carbon County BeartoothPlateau Highline Trail 8 Aug 1998 CLC 1211 (MONT)WYOMING Park County Beartooth Plateau GardnerLake 16 Aug 2014 EB0057-14 (MONT)

Other specimens examined FINLAND Koillismaa 31 Aug2005 JV23334 (L flavopalustris TURA) GREENLAND Sis-miut 19 Aug 2000 CLC 1536 (L salicis-herbaceae MONT)USA COLORADO Pitkin County Sawatch Range Indepen-dence Pass 13 Aug 2001 CLC 1741 (L aff salicis-reticulataeMONT) San Juan County San Juan Range CinnamonPass 10 Aug 2001 CLC 1710 (L aff salicis-reticulataeMONT) San Juan County San Juan Range Maggie Gulch11 Aug 2011 EB0036 (L aff salicis-reticulatae MONT)San Juan County San Juan Range Stony Pass 9 Aug 2001CLC 1689 (L aff salicis-reticulatae MONT) 13 Aug 2011EB0039 (L aff salicis-reticulatae MONT) MICHIGANEmmet County 10 Aug 1973 RL Shaffer 6957 (L aspi-deoides MICH)

Comments Some taxonomic confusion has beencaused by the fact that Kuumlhner (1975) originallynamed this species L aspideoides Kuumlhner but subse-quently renamed it because the name had been pre-viously applied L aspideoides Burl (1907) is generallylarger with a somewhat zonate pileus more crowdedlamellae that lack salmon-colored hues slightly smallerbasidiospores with broader ridges a bitter to slightlyacrid flavor and a subalpine ecology apparently ineastern and western North America (pers obs of RLShaffer 6957 Hesler and Smith 1979) Lactarius fla-vopalustris is also similar however it has a largermore brightly yellow pileus with faint zonationmore crowded lamellae a weakly scrobiculate stipe(sometimes) smaller basidiospores with thickerridges and a subalpine ecology (Kytoumlvuori 2009)Phylogenetic analyses (FIG 5) reveal three collec-tions of L flavopalustris (two from near the typelocality in Finland) nested in a well-supported cladewith L aspideoides from a subalpine forest in north-ern Michigan suggesting the possible conspecificityof these taxa Neither is reported from the RockyMountains Two other collections identified as L fla-vopalustris (from Estonia) were resolved as distinctfrom the L aspideoidesflavopalustris clade (FIG 5)their identity is unclear

Lactarius aspideus is differentiated from L salicis-reti-culate by its smaller basidiospores with denser reticula-tion more crowded creamier-colored lamellae andhabitat mainly in subalpine areas (Heilmann-Clausenet al 1998) although an environmental sequence iso-lated from an S polaris root tip from arctic Svalbardclusters with L aspideus in phylogenetic analyses (FIG5) Lactarius dryadophilus is often larger with a cream-colored pileus that has a tomentose to bearded margin(Heilmann-Clausen et al 1998) Lactarius flavidus is lar-ger with a sparsely zonate pileus more crowdedcream-colored lamellae darker violet-staining contextbasidiospores with more prominent ornamentationand a distally amyloid plage and subalpine ecology(Heilmann-Clausen et al 1998) Lactarius flavoaspideusKytoumlv has slightly smaller basidiospores on averagewith a denser reticulum and subalpine ecology withSalix (Kytoumlvuori 2009) it has been reported onlyfrom northern Europe Lactarius salicis-herbaceae asimilar arctic-alpine Salix associate has smaller (typi-cally) darker yellow to yellow-brown sporocarpspale cream to grayish buff lamellae and more denselyreticulate basidiospores (pers obs Heilmann-Clausenet al 1998) It is fairly widespread in arctic-alpineareas but is not reported from alpine areas in the cen-tral and southern Rocky Mountains Lactarius aff sali-cis-reticulatae from Colorado may be a distinct taxonand sporocarps can obtain a larger size (pileus to 90mm diam) but otherwise morphological featuresstrongly overlap

KEY TO ROCKY MOUNTAIN ALPINE LACTARIUS SPECIES

1 Latex white turning violet to dull lilac or flesh stain-ing violet to dull lilac 2

1 Latex watery to white unchanging and flesh notstaining violet to dull lilac 42 Fruiting body robust pileus 60ndash100 mm diam

yellow-brown to orange-brown hairy especiallynear margin lamellae cream to pale-yellow stipestout cream to pale-yellow often with numerousyellow-brown pits odor spicy-floral flavor mildlyacrid basidiospores 8ndash105 6 6ndash85 mm puta-tively associated with Betula and Picea (possiblyalso Salix) in Rocky Mountain alpine zone andsubalpine and arctic-alpine areas in North Amer-ica and Eurasia L repraesentaneus

2 Fruiting body smaller 20ndash50 mm diam pileus andmargin smooth 3

3 Pileus pale yellow to pale ocher lamellae cream paleyellow or pale orange-ocher stipe cream to pale yel-low odor mild to slightly sweet flavor mild basidios-pores 85ndash115 6 8ndash10 mm in Rocky Mountain alpinezone and other arctic-alpine areas in North Americaand Eurasia with Salix L salicis-reticulatae

3 Pileus blotchy light tan to light brown to gray-brownmargin lighter lamellae white to pale yellow-cream

434 MYCOLOGIA

stipe white to cream odor mild flavor mild basi-

diospores 8ndash10 6 65ndash8 mm in low alpine areas inColorado with Salix planifolia and possibly Wyom-

ing with Salix glauca L pallidomarginatus4 Pileus bright orange to orange-brown 10ndash45

mm diam margin smooth lamellae pale creamto pale orange stipe pale orange odor mildflavor mild in Rocky Mountain alpine zonearctic-alpine Alaska (possibly also subalpine)to Svalbard with Salix L lanceolatus

4 Pileus pinkish buff gray-brown brown ormauve 5

5 With odor of coconuts pileus 15ndash50 mm diam palepinkish buff to pale gray-brown with glaucous coat-ing lamellae cream to pale yellow-orange stipe buffor pale salmon flavor mild basidiospores 7ndash9 6 5ndash7mm in Rocky Mountain alpine zone and subalpinearctic-alpine areas in North America and Eurasiawith Betula L glyciosmus

5 Without odor of coconuts pileus 10ndash50 mm diamgray-brown to liver-brown with whitish glaucous coat-ing lamellae cream to light tan sometimes with pink-ish tinge discoloring to dingy tan where damagedStipe pale apricot to dingy cream-tan flavor mild toslightly acrid basidiospores 7ndash105 6 5ndash8 mm inRocky Mountain alpine zone and arctic-alpine areasin Eurasia and likely other arctic-alpine areas in NorthAmerica with Salix L nanus

DISCUSSION

The number of Lactarius species reported from theRocky Mountain alpine zone represents only a fractionof the total number of Lactarius species reported else-where from arctic-alpine areas however nearly halfof the core arctic-alpine species are now confirmedfor continental North America Core species oftencited as being restricted to arctic-alpine areas withdwarf and shrubby Salix andor Dryas species includeL brunneoviolaceus L dryadophilus L lanceolatus Lnanus L pseudouvidus L salicis-herbaceae and L salicis-reticulatae (Knudsen and Borgen 1982 Heilmann-Clau-sen et al 1998 Basso 1999 Knudsen and Vesterholt2008) Phylogenetic analyses and morphological exam-ination confirm three out of seven of these species forthe Rocky Mountain alpine zone (L lanceolatus Lnanus L salicis-reticulatae) and support the hypothesisthat these species are largely restricted to arctic-alpineareas with Salix One definitive new species L pallido-marginatus (with Salix) was revealed along with a possi-ble cryptic species L aff salicis-reticulatae (also withSalix) Many Lactarius species previously reportedfrom arctic-alpine areas are also commonly reportedfrom subalpine areas with Betulaceae andor Pinaceae

as well as with other plant families (Heilmann-Clausenet al 1998) From this set of species we report L gly-ciosmus which associates with Betula glandulosa in theRocky Mountain alpine zone and Betula spp acrossthe rest of its distribution and L repraesentaneus whichis commonly reported with Betula and Picea (Heslerand Smith 1979 Heilmann-Clausen et al 1998) andappears to associate with Salix glauca andor perhapskrummholz Picea in the Rocky Mountain alpine zone

Several taxa of interest were not molecularly distinctIn dataset A L lanceolatus did not form a monophyleticgroup but was paraphyletic with respect to Lactarius sp2 the L luculentus group and L aurantiacus In datasetB L nanus and L hysginoides were not clearly distinctIn dataset C L salicis-reticulatae did not form a clearmonophyletic group and L pseudouvidus L brunneovio-laceus and L luridus were not clearly distinguishedfrom each other especially at the RPB2 locus poormolecular resolution also has been encountered forthe latter three taxa by Das et al (2015)

Overall there was little genetic variation distinguish-ing closely related species in this study These findingsare similar to reports from other researchers who sug-gest that there is in general relatively little molecularvariation yet high morphological variation in genusLactarius (Verbeken and Nuytinck 2013) While manyof the species could be morphologically distinguishedall of the Rocky Mountain alpine taxa delineated inthis study are 97 or more similar at the ITS regionto one or more species For example L repraesentaneusand L dryadophilus are as much as 994 similar L sal-icis-reticulatae is approximately 98 similar to L salicis-herbaceae and L flavopalustris L pallidomarginatus isapproximately 97ndash98 similar to L aff brunneoviola-ceus L luridus L montanus and members of the Laff pseudouvidus group and approximately 99 similarto L aspideus L glyciosmus is approximately 98 simi-lar to L mammosus L nanus is approximately 98similar to L hysginoides and L lanceolatus is approxi-mately 97 similar to L substriatussubflammeus L aur-antiacus L luculentus v laetus and Lactarius sp 1 and 2and approximately 98 similar to L luculentus Thishigh degree of similarity could be problematic forenvironmental sequencing studies that use a strict97 ITS region similarity cutoff for recognizing spe-cies (eg Geml et al 2012 Timling et al 2012 2014)In addition Tedersoo et al (2014) suggested that theITS region in the sebacina lineage evolves moreslowly in high latitude areas which could be true forother genera The RPB2 locus proved better for differ-entiating some species and overall phylogenetic ana-lyses revealed several taxonomic issues that we werenot able to resolve A search for additional phylogen-etically informative loci for Lactarius should beinitiated along with more species-level phylogenetic

BARGE ET AL ROCKY MOUNTAIN ALPINE LACTARIUS 435

studies to provide a high quality molecular databasefor ecological studies Furthermore improvement ofmethods for sequencing older material such as typespecimens is critical

Most of the Rocky Mountain alpine Lactarius speciesdelineated in this study (except L pallidomarginatusand L aff salicis-reticulatae for which further studycould reveal broader ranges) appear to have broadintercontinental distributions in North America andEurasia The current distributions of these specieslikely were shaped by a number of biotic and abioticfactors that include host dispersal and distribution cli-mate glaciation and geography Because the genusLactarius is obligately ectomycorrhizal it is likely thatthe distribution of each fungal species was shaped lar-gely by the distribution of suitable host plants manyof which also have broad intercontinental distribu-tions Long-distance dispersal is hypothesized to haveplayed an important role in structuring present distri-bution patterns of arctic-alpine plants although manyappear clonal (Abbott and Brochmann 2003 Alsoset al 2007) and ectomycorrhizal fungi (Geml et al2012 Timling et al 2014) It also has been suggestedthat long-distance dispersal mechanisms have beenselected for in arctic flora due to repeated glaciationsand the need to track potential niches (Alsos et al2007) Pollen data from Blacktail Pond YellowstoneNational Park suggest that montane mid-elevationsin the northern greater Yellowstone ecosystem (Mon-tana Wyoming) were occupied by alpine tundra(Betula Salix many herbaceous taxa) similar to thatof the present Rocky Mountain alpine vegetation dur-ing the last glacial period (14 000ndash11 500 calibratedyears before present) (Krause and Whitlock 2013)Alpine vegetation also appears to have dominatedareas in the southern Rocky Mountains (WyomingColorado) 17 500ndash12 600 calibrated years before pre-sent (Minckley 2014) Both studies suggest the pre-sence of broad low-elevation glacial refugia for arctic-alpine life in the Rocky Mountains along the glacialfront and along with this study lend support to theidea that arctic-alpine Lactarius species (and manyother ectomycorrhizal fungi) may have occupied tun-dra areas south of the icesheets during cooler weatherduring the last glacial maximum and then dispersedupward in elevation and northward as the glaciersand ectomycorrhizal host plants retreated Broaderecological tolerances of taxa such as L glyciosmus andL repraesentaneus which are not restricted to arctic-alpine habitats likely let these species persist in tundraand non-tundra areas during the last glacial maximumas well as to track arctic-alpine vegetation as it movedupward in elevation and northward following theretreat of the icesheets For these species to haveachieved and maintained such broad distributions

gene flow must have been possible over long distancesduring the Pleistocene perhaps mediated by long-dis-tance dispersal (Geml et al 2012) range shifts due toclimate change and glacial expansion and retreat(Birks 2008) glacial and interglacial refugia (Billings1974) and the Bering land bridge (Shapiro et al2004 Geml et al 2006)

ACKNOWLEDGMENTS

We thank Dr Ellen Larsson Dr Pierre-Arthur Moreau JukkaVauras Dr Ursula Peintner Dr Egon Horak and herbaria GIB TU O GB TURA C LIP MICH DBG VPI and NY forloan of specimens Carol Johnson and Dr Matt Lavin areacknowledged for reviewing early stages of the manuscriptWe thank Don Bachman Marcy Barge and Charissa Bujakfor assistance with field work We also gratefully acknowledgesupport from the Montana Institute on Ecosystems graduateenhancement fund

LITERATURE CITED

Abbott RJ Brochmann C 2003 History and evolution of thearctic flora in the footsteps of Eric Hulteacuten Mol Ecol12299ndash313 doi101046j1365-294X200301731x

Alsos IG Eidesen PB Ehrich D Skrede I Westergaard KJacobsen GH Landvik JY Taberlet P Brochmann C2007 Frequent long-distance plant colonization in thechanging arctic Science 3161606ndash1609 doi101126science1139178

Ballaraacute J 1997 Nou estudi drsquoespegravecies fuacutengiques interessantsdels estatges alpiacute i subalpiacute dels Pirineus catalans RevCatalana Micol 201ndash24

Barge E 2015 Systematics of Lactarius in the Rocky Moun-tain alpine zone (masterrsquos thesis) Bozeman MontanaMontana State University 265 p

Basso MT 1999 Lactarius Pers Fungi Europaei 7 AlassioMykoflora (Italy) 845 p

Beker HJ Eberhardt U Vesterholt J 2010 Hebeloma hiemaleBres In arcticalpine habitats N Am Fungi 551ndash65

Bessette AE Harris DB Bessette AR 2009 Milk mushroomsof North America a field identification guide to thegenus Lactarius Syracuse New York Syracuse UniversityPress 297 p

Billings WD 1974 Adaptations and origins of alpine plantsArctic Alpine Res 6129ndash142 doi1023071550081

mdashmdashmdash 1988 Alpine vegetation In Barbour MG BillingsWD eds North American terrestrial vegetation NewYork Cambridge University Press p 392ndash420

Birks HH 2008 The late-quaternary history of arctic andalpine plants Plant Ecol Divers 1135ndash146 doi10108017550870802328652

Bogar LM Kennedy PG 2013 New wrinkles in an old para-digm neighborhood effects can modify the structureand specificity of Alnus-associated ectomycorrhizal fun-gal communities FEMS Microbiol Ecol 83767ndash777doi1011111574-694112032

Bon M 1985a Stage mycologie alpine Lanslebourg (Savoie)du 1 aug 3 sep 1984 Bull Feacuted Mycol Daupineacute-Savoie9619ndash25

436 MYCOLOGIA

FIG 7 Comparison of micromorphological features of Rocky Mountain alpine specimens and Holotypes (where available)Basidiospores pleuromacrocystidia (left) and cheilomacrocystidia (right) of A Lactarius lanceolatus (CLC2319) B L lanceolatus(F 4239 HOLOTYPE) C L nanus (CLC1471) D L nanus (ZA 192c HOLOTYPE) E L pallidomarginatus Barge amp Cripps spnov (EB0041 HOLOTYPE) F L pseudouvidus (Va 71971 HOLOTYPE) G L salicis-reticulatae (CLC2776) H L salicis-reticulatae

430 MYCOLOGIA

associates with members of the Pinaceae however italso has been reported with Betula mainly in subalpineareas (Heilmann-Clausen et al 1998) Lactarius vietus(Fr) Fr is also similar but has a distinct pale zonenear the stipe apex latex that dries greenish graylonger pleuromacrocystidia and a mild to slightly fruityodor (Heilmann-Clausen et al 1998) It has not beenreported from alpine areas in the Rocky Mountainsalthough it has been reported from elsewhere (mostlynorthern areas) in North America with Betula (Heslerand Smith 1979) The similar-looking L nanus alsooccurs in alpine areas of the Rocky Mountains andhas larger basidiospores larger and differently shapedmacrocystidia lacks an odor of coconut and associateswith Salix Lactarius spinosulus is phylogenetically closeto L glyciosmus (as well as L nanus) (FIG 4) howevermorphologically they are very different Lactarius spino-sulus has a distinctly zoned coarsely squamulose pink-ish salmon- or brick-colored pileus with a finely hairymargin and fruity odor (Heilmann-Clausen et al1998) it has not been reported from North AmericaNo definitive morphological differences were detectedbetween L glyciosmus and L aff glyciosmus and the lat-ter is not reported from the Rocky Mountainalpine zone

Lactarius pallidomarginatus Barge amp CL Cripps spnov FIGS 6 7Mycobank MB812246

Typification USA COLORADO San Juan CountySan Juan Range Stony Pass scattered solitary onsoil under Salix planifolia 13 Aug 2011 EB0041 (holo-type MONT) GenBank KR090940 (ITS) andKR091010 (RPB2)

Etymology Referring to the pale pileus marginDiagnosis Lactarius pallidomarginatus is recognized by

its small to medium size blotchy light tan to lightbrown to gray-brown pileus which is typically lightertoward the margin white to pale yellow-cream lamel-lae pale cream stipe violet staining tissue mild odorand flavor and association with Salix Morphologicallyit is close to L pseudouvidus Kuumlhner but differs by hav-ing smaller basidiospores with an ornamentation ofthinner ridges as well as macrocystidia that becomemore lanceolate (as opposed to subfusiform) and atte-nuated Phylogenetically it is close to L aspideus (Fr)Fr but differs consistently in two nucleotide positionsin the rDNA ITS locus and at eight nucleotide posi-tions in the RPB2 locus morphologically L aspideushas larger yellow basidiomata

Pileus 20ndash50 mm diam convex to broadly convexbecoming broadly convex to plane with or without adepressed center smooth dry to slightly viscid azo-nate blotchy light tan to light brown to gray-browndeveloping violet stains lighter (to cream) toward mar-gin margin incurved to downturned when youngremaining downturned or becoming nearly straightwhen mature Lamellae adnate to subdecurrent subdi-stant to slightly crowded white to pale yellow-creamstaining violet where damaged Stipe 10ndash40 6 5ndash10mm equal to slightly clavate central smooth drywhite to cream staining violet where damaged hollowLatex scarce watery staining tissue violet Context ofpileus and stipe white to cream staining violet wheredamaged Odor mild Flavor mild Basidiosporedeposit not observed Basidiospores 8ndash10 6 65ndash8mm av 5 87 6 71 mm Q 5 114ndash136 Q5 123 hya-line in 25 KOH broadly ellipsoid to ellipsoid orna-mentation amyloid of thin ridges and a few isolatedwarts forming an incomplete to dense reticulum to05 mm high Basidia 508ndash711 6 102ndash127 mm four-spored Pleuromacrocystidia 813ndash1118 6 89ndash102mm cylindrical to lanceolate with acute to moniliformapex scarce to scattered Cheilomacrocystidia 483ndash1016 6 76ndash127 mm cylindrical to lanceolate withacute to moniliform apex scattered Pileipellis an(ixo-)cutis with some ascending hyphae embedded ina faint gelatinous layer

Distribution and habitat Rare in low alpine areas withSalix planifolia reported from Independence Pass inthe Sawatch Range and Stony Pass in the San JuanRange in Colorado An additional collection(ZT5229) from Union Peak in the Wind River Rangeof Wyoming with Salix glauca is likely conspecific how-ever this hypothesis has not been confirmed withmolecular data Due to confusion with L pseudouvidusit is possible that L pallidomarginatus is more wide-spread than confirmed here

Specimens examined USA COLORADO Pitkin CountySawatch Range Independence Pass 6 Aug 2000 CLC 1470(MONT) 9 Aug 2000 ZT9093 (MONT) San Juan CountySan Juan Range Stony Pass 13 Aug 2011 EB0041 (HolotypeMONT) WYOMING Sublette County Wind River RangeUnion Peak 22 Aug 1994 ZT5229 (MONT)

Other specimens examined AUSTRIA Tyrol 3 July 2007 UPeintner 20070035 (L aff pseudouvidus from near type local-ity IB) FINLAND Inarin Lappi 16 Aug 1995 JV10468 (Laff pseudouvidus TURA) Koillismaa JV28448F 30 Aug 2011(L aff pseudouvidus originally identified as L brunneoviola-ceus TURA) FRANCE Isegravere 13 Aug 1971 Va 7 1971 (Holo-type of L pseudouvidus G) ITALY Trentino 20 Aug 2004 U

r(K72-104 HOLOTYPE no cheilocystidia drawn) I L glyciosmus (TWO269) J L repraesentaneus (CLC1747) Bars 5 10 mm forbasidiospores and 20 mm for cystidia Drawings by E Barge

BARGE ET AL ROCKY MOUNTAIN ALPINE LACTARIUS 431

Peintner 20040156 (L aff pseudouvidus from near type local-ity originally identified as L robertianus IB) NORWAYFinse 10 Aug 2005 CLC 2133 (Lactarius aff brunneoviolaceusMONT) Stordal 26 Aug 2006 P Larsen 361395 (L aff brun-neoviolaceus originally identified as L pseudouvidus O) Sval-bard 18 Aug 2002 TWO809 (L aff pseudouvidus MONT)20 Aug 2002 CLC 1910 (Lactarius sp 5 MONT) Ulvik 11Aug 2011 E Soslashyland 73867 (L aff pseudouvidus O) SWE-DEN Latnjajaure 4 Aug 2011 EL101-11 (L aff pseudouvidusGB) Latnjavagge 17 Aug 2010 EL63-10 (L aff pseudouvi-dus GB)

Comments Morphological features of L pallidomar-ginatus fit the holotype description of L pseudouvidusand both are described as lighter near the pileus mar-gin (Kuumlhner 1975 Basso 1999) however basidios-pores of L pallidomarginatus are slightly smaller (8ndash10 6 65ndash8 mm as opposed to 75ndash12 6 65ndash8(85)mm) and have narrower ridges that form a less densereticulum and macrocystidia take on a more lanceo-late form with greater attenuation of the apex (FIG7) In phylogenetic analyses exsiccati identified as Lpseudouvidus from Europe (including specimensfrom near the type locality) appear as distinct fromRocky Mountain alpine material and may representa species complex Lactarius sp 3 (CLC1910) col-lected in Svalbard has darker more vinaceous pileithan L pallidomarginatus and may represent anotherundescribed species although only one collectionwas morphologically examined Members of the Laff brunneoviolaceus clade (FIG 5) that were exam-ined differ from L pallidomarginatus in having dar-ker more vinaceous pilei larger more elongatebasidiospores (to 13 mm) and green staining of exsic-cati in 25 KOH a feature not mentioned previouslyfor L brunneoviolaceus but which could be of taxo-nomic interest Based on taxa included in the phylo-genetic analyses (FIG 5) Lactarius aspideus appearsclosely related to L pallidomarginatus however theformer has more yellow coloration and typically lar-ger sporocarps (pileus 10ndash70 mm diam) and smallerbasidiospores (67ndash95 6 56ndash78 mm) traditionallyL aspideus has been placed with other yellow violet-staining taxa such as L flavidus Boud L salicis-reticu-latae and L salicis-herbaceae (Heilmann-Clausen et al1998) but it is separated here

Lactarius montanus another violet-staining speciesoriginally described from the Rocky Mountains dif-fers from L pallidomarginatus by its larger staturedarker grayish vinaceous to vinaceous-brown pileusand green staining flesh in 25 KOH it is typicallyfound in subalpine habitats with conifers but alsooccurs in the krummholz zone The violet-stainingLactarius pallescens Hesler amp AH Sm occurs in theRocky Mountains but its sporocarps are larger andlighter in color and it is reported from subalpineconifer forests (Hesler and Smith 1979) Other

gray-brown to brownish violet-staining species suchas L luridus L uvidus (Fr) Fr L violascens (JOtto) Fr L pallescens v palmerensis Hesler amp AHSm and L cordovaensis Hesler amp AH Sm are alsolarger and have a subalpine ecology none havebeen reported from the central or southern RockyMountains (Hesler and Smith 1979 Heilmann-Clau-sen et al 1998) Lactarius cistophilus Bon amp Trimbachtypically has larger basidiomata larger basidiospores(10ndash14 6 72ndash9 mm) and occurs under Cistus inthe Mediterranean region (Basso 1999) The smallpale gray-brown European alpine taxon L uvidus falpigines Jamoni amp Bon has microscopic featuresthat match those of L uvidus (Jamoni and Bon1991) but not L pallidomarginatus The small non-violet staining L nanus has basidiospores withthicker more jagged ridges and differently shapedmacrocystidia (often with more rounded apices)(FIG 7)

Lactarius repraesentaneus Britzelm FIGS 6 7Short description Lactarius repraesentaneus is recog-

nized by its medium to large size orange-brown to yel-low-brown hairy pileus with a heavily bearded margincream to pale-yellow lamellae stout cream to pale-yel-low often pitted stipe violet staining latex and tissuespicy-floral odor slightly acrid flavor and associationwith Betula Picea and possibly shrubby Salix speciessuch as S glauca

Pileus 60ndash100 mm diam convex to plano-convexusually with a depressed center and sometimes with apapilla smooth at center becoming increasingly hairyto bearded toward margin dry to viscid azonate sur-face orange-brown at center becoming pale yellow-brown to cream toward margin hairs pale yellow-brown to red-brown margin strongly incurvedremaining so or becoming nearly straight whenmature Lamellae adnate to decurrent crowdedcream to pale-yellow staining violet where damagedStipe 30ndash70 6 15ndash35 mm stout equal to clavate cen-tral dry to viscid cream to pale yellow surface oftenwith numerous dingy yellow golden yellow yellow-brown or light orange-brown pits hollow Latex scarceto abundant white becoming violet Context of pileusand stipe white staining violet where damaged Odorspicy-floral Flavor slightly acrid Basidiospore depositnot observed Basidiospores 8ndash105 6 6ndash85 mm av5 93 6 73 mm Q 5 113ndash143 Q5 127 hyaline in25 KOH broadly ellipsoid to ellipsoid ornamenta-tion amyloid of narrow to broad ridges and isolatedwarts forming an incomplete to dense reticulum to1 mm high Basidia 610ndash762 6 102ndash114 mm four-spored Pleuromacrocystidia 787ndash1448 6 89ndash127 mm subfusiform to lanceolate thin-walled withacute to moniliform apex scattered to abundant

432 MYCOLOGIA

Cheilomacrocystidia 559ndash1397 6 76ndash127 mm subfu-siform to lanceolate thin-walled with acute to monili-form apex scattered to abundant Pileipellis anixotrichoderm with well-developed gelatinous layer

Distribution and habitat Lactarius repraesentaneus firstwas described from Germany (Britzelmayr 1885) Phy-logenetic analyses including material from Germanyconfirm L repraesentaneus from alpine areas of theRocky Mountains and overall reinforce the traditionalconcept that this species has a broad ecological andgeographic range in arctic-alpine and subalpine habi-tats in North America northern central and southernEurope In alpine areas of the Rocky Mountains itassociates with Salix glauca however krummholz Piceaalso is typically (maybe always) present it is reportedwith Picea and Betula elsewhere

Previous reports from arctic-alpine areas mainlywith Betula include Alaska (Laursen and Ammirati1982 Geml et al 2009) the Rocky Mountain alpinezone (Cripps and Horak 2008 Cripps and Barge2013) Greenland (Knudsen and Borgen 1982 Watling1983 Elbourne and Knudsen 1990 Borgen 2006 Bor-gen et al 2006) Iceland (Eyjolfsdottir pers comm)Fennoscandia (Kallio and Kankainen 1964 Kuumlhner1975 Bon 1998) Siberia (Karatygin et al 1999) andthe Altai Mountains (Gorbunova 2010)

Specimens examined FINLAND Enontakiouml 16 Aug 2004JV21671 (TURA) Varsinais-Suomi 9 Aug 1998 JV13837F(TURA) SWEDEN Latnjavagge 12 Aug 2007 EL92-07(GB) USA COLORADO Chaffee County Sawatch RangeCottonwood Pass 4 Aug 2001 CLC 1643 (MONT) 16 Aug2011 EB0048 (MONT) Pitkin County Sawatch Range Inde-pendence Pass 14 Aug 2001 CLC 1747 (MONT) ZT9837(MONT) 15 Aug 2001 ZT9537 (MONT) MONTANA Car-bon County Beartooth Plateau Birch Site 12 Aug 2002CLC 1971 (MONT) 13 Aug 2007 CLC 2318 (MONT) 18Aug 2013 EB107-13 (MONT) WYOMING Park CountyBeartooth Plateau Frozen Lakes 21 Aug 1999 CLC1394 (MONT)

Other specimens examined SWEDEN Latnjavagge 14 Aug2010 EL57-10 (L dryadophilus GB) USA ALASKA 5 Aug2011 CLC 2729 (L dryadophilus MONT) Kenai PeninsulaCounty Palmer Creek Road 7 Aug 2011 CLC 2744 (L drya-dophilus MONT)

Comments The combination of a large pale yellow-brown to orange-brown pileus with a bearded marginand violet staining latex clearly separates L repraesenta-neus from other Lactarius species of the central andsouthern Rocky Mountain alpine zone Phylogeneti-cally close the arctic-alpine taxon L dryadophilus ismorphologically similar but has a cream to yellowsmoother pileus with hairs restricted to near the mar-gin basidiospore ornamentation consisting of sparsethinner ridges and ecological associations with Dryasand Salix It has not been reported from the RockyMountains

Lactarius salicis-reticulatae Kuumlhner FIGS 6 7Short description Lactarius salicis-reticulatae is recog-

nized by its small to medium cream pale yellow orpale ocher sporocarps with pale yellowish well-spacedlamellae often with a pinkish buff tint cream to pale yel-low stipe violet staining latex and tissue mild to slightlysweet odor mild flavor and association with dwarf (Sreticulata) and possibly also shrubby Salix species

Pileus 20ndash40 mm diam convex to broadly convexusually with a depressed center smooth dry to viscidazonate cream pale yellow or pale ocher sometimesdarker toward center staining violet where damagedmargin invcurved remaining so or becoming down-turned when mature Lamellae adnate to subdecur-rent fairly well-spaced cream pale yellow or paleorange-ocher often with a pinkish-buff tint stainingviolet where damaged Stipe 15ndash20 6 10ndash15 mmequal to clavate central dry to viscid cream to pale-yellow staining violet where damaged hollow Latexscarce watery-white becoming violet Context ofpileus and stipe white staining violet where damagedOdor mild to slightly sweet Flavor mild Basidiosporedeposit not observed Basidiospores (7ndash)85ndash115 6(7ndash)8ndash10 mm av 5 100 6 88 mm Q 5 (100ndash)113ndash143 Q 5 125 hyaline in 25 KOH sublgobose toellipsoid ornamentation amyloid of narrow ridgesand a few isolated warts forming an incomplete reticu-lum to 03 mm high Basidia 737ndash889 6 102ndash127 mm four-spored Pleuromacrocystidia 762ndash10166 76ndash114 mm subfusiform to fusiform thin-walledwith rounded to acute to moniliform apex scarceCheilomacrocystidia 686ndash9146 76ndash102 mm subfusi-form to fusiform thin-walled with acute to rounded tomoniliform apex scattered to abundant Pileipellis anixocutis

Distribution and habitat Lactarius salicis-reticulatae firstwas described from alpine tundra with Salix on MtLaringktatjaringkka Sweden (Kuumlhner 1975) Phylogenetic ana-lyses including material from the type locality confirmthis species from alpine areas on the Beartooth Plateauwith Salix reticulata and shrubby Salix spp and in arctic-alpine areas of Alaska Svalbard and Finland OverallL salicis-reticulatae appears largely confined to arctic-alpine habitats Colorado collections with S reticulataand S planifolia may represent a distinct taxonalthough support is low

Previous arctic-alpine reports include Greenland(Lamoure et al 1982 Heilmann-Clausen et al1998 Borgen et al 2006) the Rocky Mountainalpine zone (Cripps and Horak 2008 Cripps andBarge 2013) the Alps (Kuumlhner 1975 Kuumlhner andLamoure 1986 Jamoni 1995 2008 Bon 1998 Heil-mann-Clausen et al 1998) the Pyrenees (Ballaraacute1997) Fennoscandia (Kuumlhner 1975 Heilmann-Clau-sen et al 1998 Gulden 2005 Knudsen and

BARGE ET AL ROCKY MOUNTAIN ALPINE LACTARIUS 433

Vesterholt 2008) and Siberia (Nezdoiminogo 1997Karatygin et al 1999) previous reports of L aspi-deoides Burl from arctic-alpine areas in Alaska (Laur-sen and Ammirati 1982) Greenland (Knudsen andBorgen 1982) and the British Isles (Watling 1987)may refer to this species

Specimens examined FINLAND Near Utsjoki 28 Aug 2012CLC 2885 (MONT) SWEDEN Mt Laringktatjaringkka 12 Aug1972 K72-104 (Holotype G) 15 Aug 1999 JV15133 (Typelocality TURA) USA ALASKA Kenai Peninsula County Pal-mer Creek Road 7 Aug 2011 CLC 2745 (MONT) MON-TANA Carbon County Beartooth Plateau Birch Site 17Aug 2011 CLC 2776 (MONT) Carbon County BeartoothPlateau Highline Trail 8 Aug 1998 CLC 1211 (MONT)WYOMING Park County Beartooth Plateau GardnerLake 16 Aug 2014 EB0057-14 (MONT)

Other specimens examined FINLAND Koillismaa 31 Aug2005 JV23334 (L flavopalustris TURA) GREENLAND Sis-miut 19 Aug 2000 CLC 1536 (L salicis-herbaceae MONT)USA COLORADO Pitkin County Sawatch Range Indepen-dence Pass 13 Aug 2001 CLC 1741 (L aff salicis-reticulataeMONT) San Juan County San Juan Range CinnamonPass 10 Aug 2001 CLC 1710 (L aff salicis-reticulataeMONT) San Juan County San Juan Range Maggie Gulch11 Aug 2011 EB0036 (L aff salicis-reticulatae MONT)San Juan County San Juan Range Stony Pass 9 Aug 2001CLC 1689 (L aff salicis-reticulatae MONT) 13 Aug 2011EB0039 (L aff salicis-reticulatae MONT) MICHIGANEmmet County 10 Aug 1973 RL Shaffer 6957 (L aspi-deoides MICH)

Comments Some taxonomic confusion has beencaused by the fact that Kuumlhner (1975) originallynamed this species L aspideoides Kuumlhner but subse-quently renamed it because the name had been pre-viously applied L aspideoides Burl (1907) is generallylarger with a somewhat zonate pileus more crowdedlamellae that lack salmon-colored hues slightly smallerbasidiospores with broader ridges a bitter to slightlyacrid flavor and a subalpine ecology apparently ineastern and western North America (pers obs of RLShaffer 6957 Hesler and Smith 1979) Lactarius fla-vopalustris is also similar however it has a largermore brightly yellow pileus with faint zonationmore crowded lamellae a weakly scrobiculate stipe(sometimes) smaller basidiospores with thickerridges and a subalpine ecology (Kytoumlvuori 2009)Phylogenetic analyses (FIG 5) reveal three collec-tions of L flavopalustris (two from near the typelocality in Finland) nested in a well-supported cladewith L aspideoides from a subalpine forest in north-ern Michigan suggesting the possible conspecificityof these taxa Neither is reported from the RockyMountains Two other collections identified as L fla-vopalustris (from Estonia) were resolved as distinctfrom the L aspideoidesflavopalustris clade (FIG 5)their identity is unclear

Lactarius aspideus is differentiated from L salicis-reti-culate by its smaller basidiospores with denser reticula-tion more crowded creamier-colored lamellae andhabitat mainly in subalpine areas (Heilmann-Clausenet al 1998) although an environmental sequence iso-lated from an S polaris root tip from arctic Svalbardclusters with L aspideus in phylogenetic analyses (FIG5) Lactarius dryadophilus is often larger with a cream-colored pileus that has a tomentose to bearded margin(Heilmann-Clausen et al 1998) Lactarius flavidus is lar-ger with a sparsely zonate pileus more crowdedcream-colored lamellae darker violet-staining contextbasidiospores with more prominent ornamentationand a distally amyloid plage and subalpine ecology(Heilmann-Clausen et al 1998) Lactarius flavoaspideusKytoumlv has slightly smaller basidiospores on averagewith a denser reticulum and subalpine ecology withSalix (Kytoumlvuori 2009) it has been reported onlyfrom northern Europe Lactarius salicis-herbaceae asimilar arctic-alpine Salix associate has smaller (typi-cally) darker yellow to yellow-brown sporocarpspale cream to grayish buff lamellae and more denselyreticulate basidiospores (pers obs Heilmann-Clausenet al 1998) It is fairly widespread in arctic-alpineareas but is not reported from alpine areas in the cen-tral and southern Rocky Mountains Lactarius aff sali-cis-reticulatae from Colorado may be a distinct taxonand sporocarps can obtain a larger size (pileus to 90mm diam) but otherwise morphological featuresstrongly overlap

KEY TO ROCKY MOUNTAIN ALPINE LACTARIUS SPECIES

1 Latex white turning violet to dull lilac or flesh stain-ing violet to dull lilac 2

1 Latex watery to white unchanging and flesh notstaining violet to dull lilac 42 Fruiting body robust pileus 60ndash100 mm diam

yellow-brown to orange-brown hairy especiallynear margin lamellae cream to pale-yellow stipestout cream to pale-yellow often with numerousyellow-brown pits odor spicy-floral flavor mildlyacrid basidiospores 8ndash105 6 6ndash85 mm puta-tively associated with Betula and Picea (possiblyalso Salix) in Rocky Mountain alpine zone andsubalpine and arctic-alpine areas in North Amer-ica and Eurasia L repraesentaneus

2 Fruiting body smaller 20ndash50 mm diam pileus andmargin smooth 3

3 Pileus pale yellow to pale ocher lamellae cream paleyellow or pale orange-ocher stipe cream to pale yel-low odor mild to slightly sweet flavor mild basidios-pores 85ndash115 6 8ndash10 mm in Rocky Mountain alpinezone and other arctic-alpine areas in North Americaand Eurasia with Salix L salicis-reticulatae

3 Pileus blotchy light tan to light brown to gray-brownmargin lighter lamellae white to pale yellow-cream

434 MYCOLOGIA

stipe white to cream odor mild flavor mild basi-

diospores 8ndash10 6 65ndash8 mm in low alpine areas inColorado with Salix planifolia and possibly Wyom-

ing with Salix glauca L pallidomarginatus4 Pileus bright orange to orange-brown 10ndash45

mm diam margin smooth lamellae pale creamto pale orange stipe pale orange odor mildflavor mild in Rocky Mountain alpine zonearctic-alpine Alaska (possibly also subalpine)to Svalbard with Salix L lanceolatus

4 Pileus pinkish buff gray-brown brown ormauve 5

5 With odor of coconuts pileus 15ndash50 mm diam palepinkish buff to pale gray-brown with glaucous coat-ing lamellae cream to pale yellow-orange stipe buffor pale salmon flavor mild basidiospores 7ndash9 6 5ndash7mm in Rocky Mountain alpine zone and subalpinearctic-alpine areas in North America and Eurasiawith Betula L glyciosmus

5 Without odor of coconuts pileus 10ndash50 mm diamgray-brown to liver-brown with whitish glaucous coat-ing lamellae cream to light tan sometimes with pink-ish tinge discoloring to dingy tan where damagedStipe pale apricot to dingy cream-tan flavor mild toslightly acrid basidiospores 7ndash105 6 5ndash8 mm inRocky Mountain alpine zone and arctic-alpine areasin Eurasia and likely other arctic-alpine areas in NorthAmerica with Salix L nanus

DISCUSSION

The number of Lactarius species reported from theRocky Mountain alpine zone represents only a fractionof the total number of Lactarius species reported else-where from arctic-alpine areas however nearly halfof the core arctic-alpine species are now confirmedfor continental North America Core species oftencited as being restricted to arctic-alpine areas withdwarf and shrubby Salix andor Dryas species includeL brunneoviolaceus L dryadophilus L lanceolatus Lnanus L pseudouvidus L salicis-herbaceae and L salicis-reticulatae (Knudsen and Borgen 1982 Heilmann-Clau-sen et al 1998 Basso 1999 Knudsen and Vesterholt2008) Phylogenetic analyses and morphological exam-ination confirm three out of seven of these species forthe Rocky Mountain alpine zone (L lanceolatus Lnanus L salicis-reticulatae) and support the hypothesisthat these species are largely restricted to arctic-alpineareas with Salix One definitive new species L pallido-marginatus (with Salix) was revealed along with a possi-ble cryptic species L aff salicis-reticulatae (also withSalix) Many Lactarius species previously reportedfrom arctic-alpine areas are also commonly reportedfrom subalpine areas with Betulaceae andor Pinaceae

as well as with other plant families (Heilmann-Clausenet al 1998) From this set of species we report L gly-ciosmus which associates with Betula glandulosa in theRocky Mountain alpine zone and Betula spp acrossthe rest of its distribution and L repraesentaneus whichis commonly reported with Betula and Picea (Heslerand Smith 1979 Heilmann-Clausen et al 1998) andappears to associate with Salix glauca andor perhapskrummholz Picea in the Rocky Mountain alpine zone

Several taxa of interest were not molecularly distinctIn dataset A L lanceolatus did not form a monophyleticgroup but was paraphyletic with respect to Lactarius sp2 the L luculentus group and L aurantiacus In datasetB L nanus and L hysginoides were not clearly distinctIn dataset C L salicis-reticulatae did not form a clearmonophyletic group and L pseudouvidus L brunneovio-laceus and L luridus were not clearly distinguishedfrom each other especially at the RPB2 locus poormolecular resolution also has been encountered forthe latter three taxa by Das et al (2015)

Overall there was little genetic variation distinguish-ing closely related species in this study These findingsare similar to reports from other researchers who sug-gest that there is in general relatively little molecularvariation yet high morphological variation in genusLactarius (Verbeken and Nuytinck 2013) While manyof the species could be morphologically distinguishedall of the Rocky Mountain alpine taxa delineated inthis study are 97 or more similar at the ITS regionto one or more species For example L repraesentaneusand L dryadophilus are as much as 994 similar L sal-icis-reticulatae is approximately 98 similar to L salicis-herbaceae and L flavopalustris L pallidomarginatus isapproximately 97ndash98 similar to L aff brunneoviola-ceus L luridus L montanus and members of the Laff pseudouvidus group and approximately 99 similarto L aspideus L glyciosmus is approximately 98 simi-lar to L mammosus L nanus is approximately 98similar to L hysginoides and L lanceolatus is approxi-mately 97 similar to L substriatussubflammeus L aur-antiacus L luculentus v laetus and Lactarius sp 1 and 2and approximately 98 similar to L luculentus Thishigh degree of similarity could be problematic forenvironmental sequencing studies that use a strict97 ITS region similarity cutoff for recognizing spe-cies (eg Geml et al 2012 Timling et al 2012 2014)In addition Tedersoo et al (2014) suggested that theITS region in the sebacina lineage evolves moreslowly in high latitude areas which could be true forother genera The RPB2 locus proved better for differ-entiating some species and overall phylogenetic ana-lyses revealed several taxonomic issues that we werenot able to resolve A search for additional phylogen-etically informative loci for Lactarius should beinitiated along with more species-level phylogenetic

BARGE ET AL ROCKY MOUNTAIN ALPINE LACTARIUS 435

studies to provide a high quality molecular databasefor ecological studies Furthermore improvement ofmethods for sequencing older material such as typespecimens is critical

Most of the Rocky Mountain alpine Lactarius speciesdelineated in this study (except L pallidomarginatusand L aff salicis-reticulatae for which further studycould reveal broader ranges) appear to have broadintercontinental distributions in North America andEurasia The current distributions of these specieslikely were shaped by a number of biotic and abioticfactors that include host dispersal and distribution cli-mate glaciation and geography Because the genusLactarius is obligately ectomycorrhizal it is likely thatthe distribution of each fungal species was shaped lar-gely by the distribution of suitable host plants manyof which also have broad intercontinental distribu-tions Long-distance dispersal is hypothesized to haveplayed an important role in structuring present distri-bution patterns of arctic-alpine plants although manyappear clonal (Abbott and Brochmann 2003 Alsoset al 2007) and ectomycorrhizal fungi (Geml et al2012 Timling et al 2014) It also has been suggestedthat long-distance dispersal mechanisms have beenselected for in arctic flora due to repeated glaciationsand the need to track potential niches (Alsos et al2007) Pollen data from Blacktail Pond YellowstoneNational Park suggest that montane mid-elevationsin the northern greater Yellowstone ecosystem (Mon-tana Wyoming) were occupied by alpine tundra(Betula Salix many herbaceous taxa) similar to thatof the present Rocky Mountain alpine vegetation dur-ing the last glacial period (14 000ndash11 500 calibratedyears before present) (Krause and Whitlock 2013)Alpine vegetation also appears to have dominatedareas in the southern Rocky Mountains (WyomingColorado) 17 500ndash12 600 calibrated years before pre-sent (Minckley 2014) Both studies suggest the pre-sence of broad low-elevation glacial refugia for arctic-alpine life in the Rocky Mountains along the glacialfront and along with this study lend support to theidea that arctic-alpine Lactarius species (and manyother ectomycorrhizal fungi) may have occupied tun-dra areas south of the icesheets during cooler weatherduring the last glacial maximum and then dispersedupward in elevation and northward as the glaciersand ectomycorrhizal host plants retreated Broaderecological tolerances of taxa such as L glyciosmus andL repraesentaneus which are not restricted to arctic-alpine habitats likely let these species persist in tundraand non-tundra areas during the last glacial maximumas well as to track arctic-alpine vegetation as it movedupward in elevation and northward following theretreat of the icesheets For these species to haveachieved and maintained such broad distributions

gene flow must have been possible over long distancesduring the Pleistocene perhaps mediated by long-dis-tance dispersal (Geml et al 2012) range shifts due toclimate change and glacial expansion and retreat(Birks 2008) glacial and interglacial refugia (Billings1974) and the Bering land bridge (Shapiro et al2004 Geml et al 2006)

ACKNOWLEDGMENTS

We thank Dr Ellen Larsson Dr Pierre-Arthur Moreau JukkaVauras Dr Ursula Peintner Dr Egon Horak and herbaria GIB TU O GB TURA C LIP MICH DBG VPI and NY forloan of specimens Carol Johnson and Dr Matt Lavin areacknowledged for reviewing early stages of the manuscriptWe thank Don Bachman Marcy Barge and Charissa Bujakfor assistance with field work We also gratefully acknowledgesupport from the Montana Institute on Ecosystems graduateenhancement fund

LITERATURE CITED

Abbott RJ Brochmann C 2003 History and evolution of thearctic flora in the footsteps of Eric Hulteacuten Mol Ecol12299ndash313 doi101046j1365-294X200301731x

Alsos IG Eidesen PB Ehrich D Skrede I Westergaard KJacobsen GH Landvik JY Taberlet P Brochmann C2007 Frequent long-distance plant colonization in thechanging arctic Science 3161606ndash1609 doi101126science1139178

Ballaraacute J 1997 Nou estudi drsquoespegravecies fuacutengiques interessantsdels estatges alpiacute i subalpiacute dels Pirineus catalans RevCatalana Micol 201ndash24

Barge E 2015 Systematics of Lactarius in the Rocky Moun-tain alpine zone (masterrsquos thesis) Bozeman MontanaMontana State University 265 p

Basso MT 1999 Lactarius Pers Fungi Europaei 7 AlassioMykoflora (Italy) 845 p

Beker HJ Eberhardt U Vesterholt J 2010 Hebeloma hiemaleBres In arcticalpine habitats N Am Fungi 551ndash65

Bessette AE Harris DB Bessette AR 2009 Milk mushroomsof North America a field identification guide to thegenus Lactarius Syracuse New York Syracuse UniversityPress 297 p

Billings WD 1974 Adaptations and origins of alpine plantsArctic Alpine Res 6129ndash142 doi1023071550081

mdashmdashmdash 1988 Alpine vegetation In Barbour MG BillingsWD eds North American terrestrial vegetation NewYork Cambridge University Press p 392ndash420

Birks HH 2008 The late-quaternary history of arctic andalpine plants Plant Ecol Divers 1135ndash146 doi10108017550870802328652

Bogar LM Kennedy PG 2013 New wrinkles in an old para-digm neighborhood effects can modify the structureand specificity of Alnus-associated ectomycorrhizal fun-gal communities FEMS Microbiol Ecol 83767ndash777doi1011111574-694112032

Bon M 1985a Stage mycologie alpine Lanslebourg (Savoie)du 1 aug 3 sep 1984 Bull Feacuted Mycol Daupineacute-Savoie9619ndash25

436 MYCOLOGIA

associates with members of the Pinaceae however italso has been reported with Betula mainly in subalpineareas (Heilmann-Clausen et al 1998) Lactarius vietus(Fr) Fr is also similar but has a distinct pale zonenear the stipe apex latex that dries greenish graylonger pleuromacrocystidia and a mild to slightly fruityodor (Heilmann-Clausen et al 1998) It has not beenreported from alpine areas in the Rocky Mountainsalthough it has been reported from elsewhere (mostlynorthern areas) in North America with Betula (Heslerand Smith 1979) The similar-looking L nanus alsooccurs in alpine areas of the Rocky Mountains andhas larger basidiospores larger and differently shapedmacrocystidia lacks an odor of coconut and associateswith Salix Lactarius spinosulus is phylogenetically closeto L glyciosmus (as well as L nanus) (FIG 4) howevermorphologically they are very different Lactarius spino-sulus has a distinctly zoned coarsely squamulose pink-ish salmon- or brick-colored pileus with a finely hairymargin and fruity odor (Heilmann-Clausen et al1998) it has not been reported from North AmericaNo definitive morphological differences were detectedbetween L glyciosmus and L aff glyciosmus and the lat-ter is not reported from the Rocky Mountainalpine zone

Lactarius pallidomarginatus Barge amp CL Cripps spnov FIGS 6 7Mycobank MB812246

Typification USA COLORADO San Juan CountySan Juan Range Stony Pass scattered solitary onsoil under Salix planifolia 13 Aug 2011 EB0041 (holo-type MONT) GenBank KR090940 (ITS) andKR091010 (RPB2)

Etymology Referring to the pale pileus marginDiagnosis Lactarius pallidomarginatus is recognized by

its small to medium size blotchy light tan to lightbrown to gray-brown pileus which is typically lightertoward the margin white to pale yellow-cream lamel-lae pale cream stipe violet staining tissue mild odorand flavor and association with Salix Morphologicallyit is close to L pseudouvidus Kuumlhner but differs by hav-ing smaller basidiospores with an ornamentation ofthinner ridges as well as macrocystidia that becomemore lanceolate (as opposed to subfusiform) and atte-nuated Phylogenetically it is close to L aspideus (Fr)Fr but differs consistently in two nucleotide positionsin the rDNA ITS locus and at eight nucleotide posi-tions in the RPB2 locus morphologically L aspideushas larger yellow basidiomata

Pileus 20ndash50 mm diam convex to broadly convexbecoming broadly convex to plane with or without adepressed center smooth dry to slightly viscid azo-nate blotchy light tan to light brown to gray-browndeveloping violet stains lighter (to cream) toward mar-gin margin incurved to downturned when youngremaining downturned or becoming nearly straightwhen mature Lamellae adnate to subdecurrent subdi-stant to slightly crowded white to pale yellow-creamstaining violet where damaged Stipe 10ndash40 6 5ndash10mm equal to slightly clavate central smooth drywhite to cream staining violet where damaged hollowLatex scarce watery staining tissue violet Context ofpileus and stipe white to cream staining violet wheredamaged Odor mild Flavor mild Basidiosporedeposit not observed Basidiospores 8ndash10 6 65ndash8mm av 5 87 6 71 mm Q 5 114ndash136 Q5 123 hya-line in 25 KOH broadly ellipsoid to ellipsoid orna-mentation amyloid of thin ridges and a few isolatedwarts forming an incomplete to dense reticulum to05 mm high Basidia 508ndash711 6 102ndash127 mm four-spored Pleuromacrocystidia 813ndash1118 6 89ndash102mm cylindrical to lanceolate with acute to moniliformapex scarce to scattered Cheilomacrocystidia 483ndash1016 6 76ndash127 mm cylindrical to lanceolate withacute to moniliform apex scattered Pileipellis an(ixo-)cutis with some ascending hyphae embedded ina faint gelatinous layer

Distribution and habitat Rare in low alpine areas withSalix planifolia reported from Independence Pass inthe Sawatch Range and Stony Pass in the San JuanRange in Colorado An additional collection(ZT5229) from Union Peak in the Wind River Rangeof Wyoming with Salix glauca is likely conspecific how-ever this hypothesis has not been confirmed withmolecular data Due to confusion with L pseudouvidusit is possible that L pallidomarginatus is more wide-spread than confirmed here

Specimens examined USA COLORADO Pitkin CountySawatch Range Independence Pass 6 Aug 2000 CLC 1470(MONT) 9 Aug 2000 ZT9093 (MONT) San Juan CountySan Juan Range Stony Pass 13 Aug 2011 EB0041 (HolotypeMONT) WYOMING Sublette County Wind River RangeUnion Peak 22 Aug 1994 ZT5229 (MONT)

Other specimens examined AUSTRIA Tyrol 3 July 2007 UPeintner 20070035 (L aff pseudouvidus from near type local-ity IB) FINLAND Inarin Lappi 16 Aug 1995 JV10468 (Laff pseudouvidus TURA) Koillismaa JV28448F 30 Aug 2011(L aff pseudouvidus originally identified as L brunneoviola-ceus TURA) FRANCE Isegravere 13 Aug 1971 Va 7 1971 (Holo-type of L pseudouvidus G) ITALY Trentino 20 Aug 2004 U

r(K72-104 HOLOTYPE no cheilocystidia drawn) I L glyciosmus (TWO269) J L repraesentaneus (CLC1747) Bars 5 10 mm forbasidiospores and 20 mm for cystidia Drawings by E Barge

BARGE ET AL ROCKY MOUNTAIN ALPINE LACTARIUS 431

Peintner 20040156 (L aff pseudouvidus from near type local-ity originally identified as L robertianus IB) NORWAYFinse 10 Aug 2005 CLC 2133 (Lactarius aff brunneoviolaceusMONT) Stordal 26 Aug 2006 P Larsen 361395 (L aff brun-neoviolaceus originally identified as L pseudouvidus O) Sval-bard 18 Aug 2002 TWO809 (L aff pseudouvidus MONT)20 Aug 2002 CLC 1910 (Lactarius sp 5 MONT) Ulvik 11Aug 2011 E Soslashyland 73867 (L aff pseudouvidus O) SWE-DEN Latnjajaure 4 Aug 2011 EL101-11 (L aff pseudouvidusGB) Latnjavagge 17 Aug 2010 EL63-10 (L aff pseudouvi-dus GB)

Comments Morphological features of L pallidomar-ginatus fit the holotype description of L pseudouvidusand both are described as lighter near the pileus mar-gin (Kuumlhner 1975 Basso 1999) however basidios-pores of L pallidomarginatus are slightly smaller (8ndash10 6 65ndash8 mm as opposed to 75ndash12 6 65ndash8(85)mm) and have narrower ridges that form a less densereticulum and macrocystidia take on a more lanceo-late form with greater attenuation of the apex (FIG7) In phylogenetic analyses exsiccati identified as Lpseudouvidus from Europe (including specimensfrom near the type locality) appear as distinct fromRocky Mountain alpine material and may representa species complex Lactarius sp 3 (CLC1910) col-lected in Svalbard has darker more vinaceous pileithan L pallidomarginatus and may represent anotherundescribed species although only one collectionwas morphologically examined Members of the Laff brunneoviolaceus clade (FIG 5) that were exam-ined differ from L pallidomarginatus in having dar-ker more vinaceous pilei larger more elongatebasidiospores (to 13 mm) and green staining of exsic-cati in 25 KOH a feature not mentioned previouslyfor L brunneoviolaceus but which could be of taxo-nomic interest Based on taxa included in the phylo-genetic analyses (FIG 5) Lactarius aspideus appearsclosely related to L pallidomarginatus however theformer has more yellow coloration and typically lar-ger sporocarps (pileus 10ndash70 mm diam) and smallerbasidiospores (67ndash95 6 56ndash78 mm) traditionallyL aspideus has been placed with other yellow violet-staining taxa such as L flavidus Boud L salicis-reticu-latae and L salicis-herbaceae (Heilmann-Clausen et al1998) but it is separated here

Lactarius montanus another violet-staining speciesoriginally described from the Rocky Mountains dif-fers from L pallidomarginatus by its larger staturedarker grayish vinaceous to vinaceous-brown pileusand green staining flesh in 25 KOH it is typicallyfound in subalpine habitats with conifers but alsooccurs in the krummholz zone The violet-stainingLactarius pallescens Hesler amp AH Sm occurs in theRocky Mountains but its sporocarps are larger andlighter in color and it is reported from subalpineconifer forests (Hesler and Smith 1979) Other

gray-brown to brownish violet-staining species suchas L luridus L uvidus (Fr) Fr L violascens (JOtto) Fr L pallescens v palmerensis Hesler amp AHSm and L cordovaensis Hesler amp AH Sm are alsolarger and have a subalpine ecology none havebeen reported from the central or southern RockyMountains (Hesler and Smith 1979 Heilmann-Clau-sen et al 1998) Lactarius cistophilus Bon amp Trimbachtypically has larger basidiomata larger basidiospores(10ndash14 6 72ndash9 mm) and occurs under Cistus inthe Mediterranean region (Basso 1999) The smallpale gray-brown European alpine taxon L uvidus falpigines Jamoni amp Bon has microscopic featuresthat match those of L uvidus (Jamoni and Bon1991) but not L pallidomarginatus The small non-violet staining L nanus has basidiospores withthicker more jagged ridges and differently shapedmacrocystidia (often with more rounded apices)(FIG 7)

Lactarius repraesentaneus Britzelm FIGS 6 7Short description Lactarius repraesentaneus is recog-

nized by its medium to large size orange-brown to yel-low-brown hairy pileus with a heavily bearded margincream to pale-yellow lamellae stout cream to pale-yel-low often pitted stipe violet staining latex and tissuespicy-floral odor slightly acrid flavor and associationwith Betula Picea and possibly shrubby Salix speciessuch as S glauca

Pileus 60ndash100 mm diam convex to plano-convexusually with a depressed center and sometimes with apapilla smooth at center becoming increasingly hairyto bearded toward margin dry to viscid azonate sur-face orange-brown at center becoming pale yellow-brown to cream toward margin hairs pale yellow-brown to red-brown margin strongly incurvedremaining so or becoming nearly straight whenmature Lamellae adnate to decurrent crowdedcream to pale-yellow staining violet where damagedStipe 30ndash70 6 15ndash35 mm stout equal to clavate cen-tral dry to viscid cream to pale yellow surface oftenwith numerous dingy yellow golden yellow yellow-brown or light orange-brown pits hollow Latex scarceto abundant white becoming violet Context of pileusand stipe white staining violet where damaged Odorspicy-floral Flavor slightly acrid Basidiospore depositnot observed Basidiospores 8ndash105 6 6ndash85 mm av5 93 6 73 mm Q 5 113ndash143 Q5 127 hyaline in25 KOH broadly ellipsoid to ellipsoid ornamenta-tion amyloid of narrow to broad ridges and isolatedwarts forming an incomplete to dense reticulum to1 mm high Basidia 610ndash762 6 102ndash114 mm four-spored Pleuromacrocystidia 787ndash1448 6 89ndash127 mm subfusiform to lanceolate thin-walled withacute to moniliform apex scattered to abundant

432 MYCOLOGIA

Cheilomacrocystidia 559ndash1397 6 76ndash127 mm subfu-siform to lanceolate thin-walled with acute to monili-form apex scattered to abundant Pileipellis anixotrichoderm with well-developed gelatinous layer

Distribution and habitat Lactarius repraesentaneus firstwas described from Germany (Britzelmayr 1885) Phy-logenetic analyses including material from Germanyconfirm L repraesentaneus from alpine areas of theRocky Mountains and overall reinforce the traditionalconcept that this species has a broad ecological andgeographic range in arctic-alpine and subalpine habi-tats in North America northern central and southernEurope In alpine areas of the Rocky Mountains itassociates with Salix glauca however krummholz Piceaalso is typically (maybe always) present it is reportedwith Picea and Betula elsewhere

Previous reports from arctic-alpine areas mainlywith Betula include Alaska (Laursen and Ammirati1982 Geml et al 2009) the Rocky Mountain alpinezone (Cripps and Horak 2008 Cripps and Barge2013) Greenland (Knudsen and Borgen 1982 Watling1983 Elbourne and Knudsen 1990 Borgen 2006 Bor-gen et al 2006) Iceland (Eyjolfsdottir pers comm)Fennoscandia (Kallio and Kankainen 1964 Kuumlhner1975 Bon 1998) Siberia (Karatygin et al 1999) andthe Altai Mountains (Gorbunova 2010)

Specimens examined FINLAND Enontakiouml 16 Aug 2004JV21671 (TURA) Varsinais-Suomi 9 Aug 1998 JV13837F(TURA) SWEDEN Latnjavagge 12 Aug 2007 EL92-07(GB) USA COLORADO Chaffee County Sawatch RangeCottonwood Pass 4 Aug 2001 CLC 1643 (MONT) 16 Aug2011 EB0048 (MONT) Pitkin County Sawatch Range Inde-pendence Pass 14 Aug 2001 CLC 1747 (MONT) ZT9837(MONT) 15 Aug 2001 ZT9537 (MONT) MONTANA Car-bon County Beartooth Plateau Birch Site 12 Aug 2002CLC 1971 (MONT) 13 Aug 2007 CLC 2318 (MONT) 18Aug 2013 EB107-13 (MONT) WYOMING Park CountyBeartooth Plateau Frozen Lakes 21 Aug 1999 CLC1394 (MONT)

Other specimens examined SWEDEN Latnjavagge 14 Aug2010 EL57-10 (L dryadophilus GB) USA ALASKA 5 Aug2011 CLC 2729 (L dryadophilus MONT) Kenai PeninsulaCounty Palmer Creek Road 7 Aug 2011 CLC 2744 (L drya-dophilus MONT)

Comments The combination of a large pale yellow-brown to orange-brown pileus with a bearded marginand violet staining latex clearly separates L repraesenta-neus from other Lactarius species of the central andsouthern Rocky Mountain alpine zone Phylogeneti-cally close the arctic-alpine taxon L dryadophilus ismorphologically similar but has a cream to yellowsmoother pileus with hairs restricted to near the mar-gin basidiospore ornamentation consisting of sparsethinner ridges and ecological associations with Dryasand Salix It has not been reported from the RockyMountains

Lactarius salicis-reticulatae Kuumlhner FIGS 6 7Short description Lactarius salicis-reticulatae is recog-

nized by its small to medium cream pale yellow orpale ocher sporocarps with pale yellowish well-spacedlamellae often with a pinkish buff tint cream to pale yel-low stipe violet staining latex and tissue mild to slightlysweet odor mild flavor and association with dwarf (Sreticulata) and possibly also shrubby Salix species

Pileus 20ndash40 mm diam convex to broadly convexusually with a depressed center smooth dry to viscidazonate cream pale yellow or pale ocher sometimesdarker toward center staining violet where damagedmargin invcurved remaining so or becoming down-turned when mature Lamellae adnate to subdecur-rent fairly well-spaced cream pale yellow or paleorange-ocher often with a pinkish-buff tint stainingviolet where damaged Stipe 15ndash20 6 10ndash15 mmequal to clavate central dry to viscid cream to pale-yellow staining violet where damaged hollow Latexscarce watery-white becoming violet Context ofpileus and stipe white staining violet where damagedOdor mild to slightly sweet Flavor mild Basidiosporedeposit not observed Basidiospores (7ndash)85ndash115 6(7ndash)8ndash10 mm av 5 100 6 88 mm Q 5 (100ndash)113ndash143 Q 5 125 hyaline in 25 KOH sublgobose toellipsoid ornamentation amyloid of narrow ridgesand a few isolated warts forming an incomplete reticu-lum to 03 mm high Basidia 737ndash889 6 102ndash127 mm four-spored Pleuromacrocystidia 762ndash10166 76ndash114 mm subfusiform to fusiform thin-walledwith rounded to acute to moniliform apex scarceCheilomacrocystidia 686ndash9146 76ndash102 mm subfusi-form to fusiform thin-walled with acute to rounded tomoniliform apex scattered to abundant Pileipellis anixocutis

Distribution and habitat Lactarius salicis-reticulatae firstwas described from alpine tundra with Salix on MtLaringktatjaringkka Sweden (Kuumlhner 1975) Phylogenetic ana-lyses including material from the type locality confirmthis species from alpine areas on the Beartooth Plateauwith Salix reticulata and shrubby Salix spp and in arctic-alpine areas of Alaska Svalbard and Finland OverallL salicis-reticulatae appears largely confined to arctic-alpine habitats Colorado collections with S reticulataand S planifolia may represent a distinct taxonalthough support is low

Previous arctic-alpine reports include Greenland(Lamoure et al 1982 Heilmann-Clausen et al1998 Borgen et al 2006) the Rocky Mountainalpine zone (Cripps and Horak 2008 Cripps andBarge 2013) the Alps (Kuumlhner 1975 Kuumlhner andLamoure 1986 Jamoni 1995 2008 Bon 1998 Heil-mann-Clausen et al 1998) the Pyrenees (Ballaraacute1997) Fennoscandia (Kuumlhner 1975 Heilmann-Clau-sen et al 1998 Gulden 2005 Knudsen and

BARGE ET AL ROCKY MOUNTAIN ALPINE LACTARIUS 433

Vesterholt 2008) and Siberia (Nezdoiminogo 1997Karatygin et al 1999) previous reports of L aspi-deoides Burl from arctic-alpine areas in Alaska (Laur-sen and Ammirati 1982) Greenland (Knudsen andBorgen 1982) and the British Isles (Watling 1987)may refer to this species

Specimens examined FINLAND Near Utsjoki 28 Aug 2012CLC 2885 (MONT) SWEDEN Mt Laringktatjaringkka 12 Aug1972 K72-104 (Holotype G) 15 Aug 1999 JV15133 (Typelocality TURA) USA ALASKA Kenai Peninsula County Pal-mer Creek Road 7 Aug 2011 CLC 2745 (MONT) MON-TANA Carbon County Beartooth Plateau Birch Site 17Aug 2011 CLC 2776 (MONT) Carbon County BeartoothPlateau Highline Trail 8 Aug 1998 CLC 1211 (MONT)WYOMING Park County Beartooth Plateau GardnerLake 16 Aug 2014 EB0057-14 (MONT)

Other specimens examined FINLAND Koillismaa 31 Aug2005 JV23334 (L flavopalustris TURA) GREENLAND Sis-miut 19 Aug 2000 CLC 1536 (L salicis-herbaceae MONT)USA COLORADO Pitkin County Sawatch Range Indepen-dence Pass 13 Aug 2001 CLC 1741 (L aff salicis-reticulataeMONT) San Juan County San Juan Range CinnamonPass 10 Aug 2001 CLC 1710 (L aff salicis-reticulataeMONT) San Juan County San Juan Range Maggie Gulch11 Aug 2011 EB0036 (L aff salicis-reticulatae MONT)San Juan County San Juan Range Stony Pass 9 Aug 2001CLC 1689 (L aff salicis-reticulatae MONT) 13 Aug 2011EB0039 (L aff salicis-reticulatae MONT) MICHIGANEmmet County 10 Aug 1973 RL Shaffer 6957 (L aspi-deoides MICH)

Comments Some taxonomic confusion has beencaused by the fact that Kuumlhner (1975) originallynamed this species L aspideoides Kuumlhner but subse-quently renamed it because the name had been pre-viously applied L aspideoides Burl (1907) is generallylarger with a somewhat zonate pileus more crowdedlamellae that lack salmon-colored hues slightly smallerbasidiospores with broader ridges a bitter to slightlyacrid flavor and a subalpine ecology apparently ineastern and western North America (pers obs of RLShaffer 6957 Hesler and Smith 1979) Lactarius fla-vopalustris is also similar however it has a largermore brightly yellow pileus with faint zonationmore crowded lamellae a weakly scrobiculate stipe(sometimes) smaller basidiospores with thickerridges and a subalpine ecology (Kytoumlvuori 2009)Phylogenetic analyses (FIG 5) reveal three collec-tions of L flavopalustris (two from near the typelocality in Finland) nested in a well-supported cladewith L aspideoides from a subalpine forest in north-ern Michigan suggesting the possible conspecificityof these taxa Neither is reported from the RockyMountains Two other collections identified as L fla-vopalustris (from Estonia) were resolved as distinctfrom the L aspideoidesflavopalustris clade (FIG 5)their identity is unclear

Lactarius aspideus is differentiated from L salicis-reti-culate by its smaller basidiospores with denser reticula-tion more crowded creamier-colored lamellae andhabitat mainly in subalpine areas (Heilmann-Clausenet al 1998) although an environmental sequence iso-lated from an S polaris root tip from arctic Svalbardclusters with L aspideus in phylogenetic analyses (FIG5) Lactarius dryadophilus is often larger with a cream-colored pileus that has a tomentose to bearded margin(Heilmann-Clausen et al 1998) Lactarius flavidus is lar-ger with a sparsely zonate pileus more crowdedcream-colored lamellae darker violet-staining contextbasidiospores with more prominent ornamentationand a distally amyloid plage and subalpine ecology(Heilmann-Clausen et al 1998) Lactarius flavoaspideusKytoumlv has slightly smaller basidiospores on averagewith a denser reticulum and subalpine ecology withSalix (Kytoumlvuori 2009) it has been reported onlyfrom northern Europe Lactarius salicis-herbaceae asimilar arctic-alpine Salix associate has smaller (typi-cally) darker yellow to yellow-brown sporocarpspale cream to grayish buff lamellae and more denselyreticulate basidiospores (pers obs Heilmann-Clausenet al 1998) It is fairly widespread in arctic-alpineareas but is not reported from alpine areas in the cen-tral and southern Rocky Mountains Lactarius aff sali-cis-reticulatae from Colorado may be a distinct taxonand sporocarps can obtain a larger size (pileus to 90mm diam) but otherwise morphological featuresstrongly overlap

KEY TO ROCKY MOUNTAIN ALPINE LACTARIUS SPECIES

1 Latex white turning violet to dull lilac or flesh stain-ing violet to dull lilac 2

1 Latex watery to white unchanging and flesh notstaining violet to dull lilac 42 Fruiting body robust pileus 60ndash100 mm diam

yellow-brown to orange-brown hairy especiallynear margin lamellae cream to pale-yellow stipestout cream to pale-yellow often with numerousyellow-brown pits odor spicy-floral flavor mildlyacrid basidiospores 8ndash105 6 6ndash85 mm puta-tively associated with Betula and Picea (possiblyalso Salix) in Rocky Mountain alpine zone andsubalpine and arctic-alpine areas in North Amer-ica and Eurasia L repraesentaneus

2 Fruiting body smaller 20ndash50 mm diam pileus andmargin smooth 3

3 Pileus pale yellow to pale ocher lamellae cream paleyellow or pale orange-ocher stipe cream to pale yel-low odor mild to slightly sweet flavor mild basidios-pores 85ndash115 6 8ndash10 mm in Rocky Mountain alpinezone and other arctic-alpine areas in North Americaand Eurasia with Salix L salicis-reticulatae

3 Pileus blotchy light tan to light brown to gray-brownmargin lighter lamellae white to pale yellow-cream

434 MYCOLOGIA

stipe white to cream odor mild flavor mild basi-

diospores 8ndash10 6 65ndash8 mm in low alpine areas inColorado with Salix planifolia and possibly Wyom-

ing with Salix glauca L pallidomarginatus4 Pileus bright orange to orange-brown 10ndash45

mm diam margin smooth lamellae pale creamto pale orange stipe pale orange odor mildflavor mild in Rocky Mountain alpine zonearctic-alpine Alaska (possibly also subalpine)to Svalbard with Salix L lanceolatus

4 Pileus pinkish buff gray-brown brown ormauve 5

5 With odor of coconuts pileus 15ndash50 mm diam palepinkish buff to pale gray-brown with glaucous coat-ing lamellae cream to pale yellow-orange stipe buffor pale salmon flavor mild basidiospores 7ndash9 6 5ndash7mm in Rocky Mountain alpine zone and subalpinearctic-alpine areas in North America and Eurasiawith Betula L glyciosmus

5 Without odor of coconuts pileus 10ndash50 mm diamgray-brown to liver-brown with whitish glaucous coat-ing lamellae cream to light tan sometimes with pink-ish tinge discoloring to dingy tan where damagedStipe pale apricot to dingy cream-tan flavor mild toslightly acrid basidiospores 7ndash105 6 5ndash8 mm inRocky Mountain alpine zone and arctic-alpine areasin Eurasia and likely other arctic-alpine areas in NorthAmerica with Salix L nanus

DISCUSSION

The number of Lactarius species reported from theRocky Mountain alpine zone represents only a fractionof the total number of Lactarius species reported else-where from arctic-alpine areas however nearly halfof the core arctic-alpine species are now confirmedfor continental North America Core species oftencited as being restricted to arctic-alpine areas withdwarf and shrubby Salix andor Dryas species includeL brunneoviolaceus L dryadophilus L lanceolatus Lnanus L pseudouvidus L salicis-herbaceae and L salicis-reticulatae (Knudsen and Borgen 1982 Heilmann-Clau-sen et al 1998 Basso 1999 Knudsen and Vesterholt2008) Phylogenetic analyses and morphological exam-ination confirm three out of seven of these species forthe Rocky Mountain alpine zone (L lanceolatus Lnanus L salicis-reticulatae) and support the hypothesisthat these species are largely restricted to arctic-alpineareas with Salix One definitive new species L pallido-marginatus (with Salix) was revealed along with a possi-ble cryptic species L aff salicis-reticulatae (also withSalix) Many Lactarius species previously reportedfrom arctic-alpine areas are also commonly reportedfrom subalpine areas with Betulaceae andor Pinaceae

as well as with other plant families (Heilmann-Clausenet al 1998) From this set of species we report L gly-ciosmus which associates with Betula glandulosa in theRocky Mountain alpine zone and Betula spp acrossthe rest of its distribution and L repraesentaneus whichis commonly reported with Betula and Picea (Heslerand Smith 1979 Heilmann-Clausen et al 1998) andappears to associate with Salix glauca andor perhapskrummholz Picea in the Rocky Mountain alpine zone

Several taxa of interest were not molecularly distinctIn dataset A L lanceolatus did not form a monophyleticgroup but was paraphyletic with respect to Lactarius sp2 the L luculentus group and L aurantiacus In datasetB L nanus and L hysginoides were not clearly distinctIn dataset C L salicis-reticulatae did not form a clearmonophyletic group and L pseudouvidus L brunneovio-laceus and L luridus were not clearly distinguishedfrom each other especially at the RPB2 locus poormolecular resolution also has been encountered forthe latter three taxa by Das et al (2015)

Overall there was little genetic variation distinguish-ing closely related species in this study These findingsare similar to reports from other researchers who sug-gest that there is in general relatively little molecularvariation yet high morphological variation in genusLactarius (Verbeken and Nuytinck 2013) While manyof the species could be morphologically distinguishedall of the Rocky Mountain alpine taxa delineated inthis study are 97 or more similar at the ITS regionto one or more species For example L repraesentaneusand L dryadophilus are as much as 994 similar L sal-icis-reticulatae is approximately 98 similar to L salicis-herbaceae and L flavopalustris L pallidomarginatus isapproximately 97ndash98 similar to L aff brunneoviola-ceus L luridus L montanus and members of the Laff pseudouvidus group and approximately 99 similarto L aspideus L glyciosmus is approximately 98 simi-lar to L mammosus L nanus is approximately 98similar to L hysginoides and L lanceolatus is approxi-mately 97 similar to L substriatussubflammeus L aur-antiacus L luculentus v laetus and Lactarius sp 1 and 2and approximately 98 similar to L luculentus Thishigh degree of similarity could be problematic forenvironmental sequencing studies that use a strict97 ITS region similarity cutoff for recognizing spe-cies (eg Geml et al 2012 Timling et al 2012 2014)In addition Tedersoo et al (2014) suggested that theITS region in the sebacina lineage evolves moreslowly in high latitude areas which could be true forother genera The RPB2 locus proved better for differ-entiating some species and overall phylogenetic ana-lyses revealed several taxonomic issues that we werenot able to resolve A search for additional phylogen-etically informative loci for Lactarius should beinitiated along with more species-level phylogenetic

BARGE ET AL ROCKY MOUNTAIN ALPINE LACTARIUS 435

studies to provide a high quality molecular databasefor ecological studies Furthermore improvement ofmethods for sequencing older material such as typespecimens is critical

Most of the Rocky Mountain alpine Lactarius speciesdelineated in this study (except L pallidomarginatusand L aff salicis-reticulatae for which further studycould reveal broader ranges) appear to have broadintercontinental distributions in North America andEurasia The current distributions of these specieslikely were shaped by a number of biotic and abioticfactors that include host dispersal and distribution cli-mate glaciation and geography Because the genusLactarius is obligately ectomycorrhizal it is likely thatthe distribution of each fungal species was shaped lar-gely by the distribution of suitable host plants manyof which also have broad intercontinental distribu-tions Long-distance dispersal is hypothesized to haveplayed an important role in structuring present distri-bution patterns of arctic-alpine plants although manyappear clonal (Abbott and Brochmann 2003 Alsoset al 2007) and ectomycorrhizal fungi (Geml et al2012 Timling et al 2014) It also has been suggestedthat long-distance dispersal mechanisms have beenselected for in arctic flora due to repeated glaciationsand the need to track potential niches (Alsos et al2007) Pollen data from Blacktail Pond YellowstoneNational Park suggest that montane mid-elevationsin the northern greater Yellowstone ecosystem (Mon-tana Wyoming) were occupied by alpine tundra(Betula Salix many herbaceous taxa) similar to thatof the present Rocky Mountain alpine vegetation dur-ing the last glacial period (14 000ndash11 500 calibratedyears before present) (Krause and Whitlock 2013)Alpine vegetation also appears to have dominatedareas in the southern Rocky Mountains (WyomingColorado) 17 500ndash12 600 calibrated years before pre-sent (Minckley 2014) Both studies suggest the pre-sence of broad low-elevation glacial refugia for arctic-alpine life in the Rocky Mountains along the glacialfront and along with this study lend support to theidea that arctic-alpine Lactarius species (and manyother ectomycorrhizal fungi) may have occupied tun-dra areas south of the icesheets during cooler weatherduring the last glacial maximum and then dispersedupward in elevation and northward as the glaciersand ectomycorrhizal host plants retreated Broaderecological tolerances of taxa such as L glyciosmus andL repraesentaneus which are not restricted to arctic-alpine habitats likely let these species persist in tundraand non-tundra areas during the last glacial maximumas well as to track arctic-alpine vegetation as it movedupward in elevation and northward following theretreat of the icesheets For these species to haveachieved and maintained such broad distributions

gene flow must have been possible over long distancesduring the Pleistocene perhaps mediated by long-dis-tance dispersal (Geml et al 2012) range shifts due toclimate change and glacial expansion and retreat(Birks 2008) glacial and interglacial refugia (Billings1974) and the Bering land bridge (Shapiro et al2004 Geml et al 2006)

ACKNOWLEDGMENTS

We thank Dr Ellen Larsson Dr Pierre-Arthur Moreau JukkaVauras Dr Ursula Peintner Dr Egon Horak and herbaria GIB TU O GB TURA C LIP MICH DBG VPI and NY forloan of specimens Carol Johnson and Dr Matt Lavin areacknowledged for reviewing early stages of the manuscriptWe thank Don Bachman Marcy Barge and Charissa Bujakfor assistance with field work We also gratefully acknowledgesupport from the Montana Institute on Ecosystems graduateenhancement fund

LITERATURE CITED

Abbott RJ Brochmann C 2003 History and evolution of thearctic flora in the footsteps of Eric Hulteacuten Mol Ecol12299ndash313 doi101046j1365-294X200301731x

Alsos IG Eidesen PB Ehrich D Skrede I Westergaard KJacobsen GH Landvik JY Taberlet P Brochmann C2007 Frequent long-distance plant colonization in thechanging arctic Science 3161606ndash1609 doi101126science1139178

Ballaraacute J 1997 Nou estudi drsquoespegravecies fuacutengiques interessantsdels estatges alpiacute i subalpiacute dels Pirineus catalans RevCatalana Micol 201ndash24

Barge E 2015 Systematics of Lactarius in the Rocky Moun-tain alpine zone (masterrsquos thesis) Bozeman MontanaMontana State University 265 p

Basso MT 1999 Lactarius Pers Fungi Europaei 7 AlassioMykoflora (Italy) 845 p

Beker HJ Eberhardt U Vesterholt J 2010 Hebeloma hiemaleBres In arcticalpine habitats N Am Fungi 551ndash65

Bessette AE Harris DB Bessette AR 2009 Milk mushroomsof North America a field identification guide to thegenus Lactarius Syracuse New York Syracuse UniversityPress 297 p

Billings WD 1974 Adaptations and origins of alpine plantsArctic Alpine Res 6129ndash142 doi1023071550081

mdashmdashmdash 1988 Alpine vegetation In Barbour MG BillingsWD eds North American terrestrial vegetation NewYork Cambridge University Press p 392ndash420

Birks HH 2008 The late-quaternary history of arctic andalpine plants Plant Ecol Divers 1135ndash146 doi10108017550870802328652

Bogar LM Kennedy PG 2013 New wrinkles in an old para-digm neighborhood effects can modify the structureand specificity of Alnus-associated ectomycorrhizal fun-gal communities FEMS Microbiol Ecol 83767ndash777doi1011111574-694112032

Bon M 1985a Stage mycologie alpine Lanslebourg (Savoie)du 1 aug 3 sep 1984 Bull Feacuted Mycol Daupineacute-Savoie9619ndash25

436 MYCOLOGIA

Peintner 20040156 (L aff pseudouvidus from near type local-ity originally identified as L robertianus IB) NORWAYFinse 10 Aug 2005 CLC 2133 (Lactarius aff brunneoviolaceusMONT) Stordal 26 Aug 2006 P Larsen 361395 (L aff brun-neoviolaceus originally identified as L pseudouvidus O) Sval-bard 18 Aug 2002 TWO809 (L aff pseudouvidus MONT)20 Aug 2002 CLC 1910 (Lactarius sp 5 MONT) Ulvik 11Aug 2011 E Soslashyland 73867 (L aff pseudouvidus O) SWE-DEN Latnjajaure 4 Aug 2011 EL101-11 (L aff pseudouvidusGB) Latnjavagge 17 Aug 2010 EL63-10 (L aff pseudouvi-dus GB)

Comments Morphological features of L pallidomar-ginatus fit the holotype description of L pseudouvidusand both are described as lighter near the pileus mar-gin (Kuumlhner 1975 Basso 1999) however basidios-pores of L pallidomarginatus are slightly smaller (8ndash10 6 65ndash8 mm as opposed to 75ndash12 6 65ndash8(85)mm) and have narrower ridges that form a less densereticulum and macrocystidia take on a more lanceo-late form with greater attenuation of the apex (FIG7) In phylogenetic analyses exsiccati identified as Lpseudouvidus from Europe (including specimensfrom near the type locality) appear as distinct fromRocky Mountain alpine material and may representa species complex Lactarius sp 3 (CLC1910) col-lected in Svalbard has darker more vinaceous pileithan L pallidomarginatus and may represent anotherundescribed species although only one collectionwas morphologically examined Members of the Laff brunneoviolaceus clade (FIG 5) that were exam-ined differ from L pallidomarginatus in having dar-ker more vinaceous pilei larger more elongatebasidiospores (to 13 mm) and green staining of exsic-cati in 25 KOH a feature not mentioned previouslyfor L brunneoviolaceus but which could be of taxo-nomic interest Based on taxa included in the phylo-genetic analyses (FIG 5) Lactarius aspideus appearsclosely related to L pallidomarginatus however theformer has more yellow coloration and typically lar-ger sporocarps (pileus 10ndash70 mm diam) and smallerbasidiospores (67ndash95 6 56ndash78 mm) traditionallyL aspideus has been placed with other yellow violet-staining taxa such as L flavidus Boud L salicis-reticu-latae and L salicis-herbaceae (Heilmann-Clausen et al1998) but it is separated here

Lactarius montanus another violet-staining speciesoriginally described from the Rocky Mountains dif-fers from L pallidomarginatus by its larger staturedarker grayish vinaceous to vinaceous-brown pileusand green staining flesh in 25 KOH it is typicallyfound in subalpine habitats with conifers but alsooccurs in the krummholz zone The violet-stainingLactarius pallescens Hesler amp AH Sm occurs in theRocky Mountains but its sporocarps are larger andlighter in color and it is reported from subalpineconifer forests (Hesler and Smith 1979) Other

gray-brown to brownish violet-staining species suchas L luridus L uvidus (Fr) Fr L violascens (JOtto) Fr L pallescens v palmerensis Hesler amp AHSm and L cordovaensis Hesler amp AH Sm are alsolarger and have a subalpine ecology none havebeen reported from the central or southern RockyMountains (Hesler and Smith 1979 Heilmann-Clau-sen et al 1998) Lactarius cistophilus Bon amp Trimbachtypically has larger basidiomata larger basidiospores(10ndash14 6 72ndash9 mm) and occurs under Cistus inthe Mediterranean region (Basso 1999) The smallpale gray-brown European alpine taxon L uvidus falpigines Jamoni amp Bon has microscopic featuresthat match those of L uvidus (Jamoni and Bon1991) but not L pallidomarginatus The small non-violet staining L nanus has basidiospores withthicker more jagged ridges and differently shapedmacrocystidia (often with more rounded apices)(FIG 7)

Lactarius repraesentaneus Britzelm FIGS 6 7Short description Lactarius repraesentaneus is recog-

nized by its medium to large size orange-brown to yel-low-brown hairy pileus with a heavily bearded margincream to pale-yellow lamellae stout cream to pale-yel-low often pitted stipe violet staining latex and tissuespicy-floral odor slightly acrid flavor and associationwith Betula Picea and possibly shrubby Salix speciessuch as S glauca

Pileus 60ndash100 mm diam convex to plano-convexusually with a depressed center and sometimes with apapilla smooth at center becoming increasingly hairyto bearded toward margin dry to viscid azonate sur-face orange-brown at center becoming pale yellow-brown to cream toward margin hairs pale yellow-brown to red-brown margin strongly incurvedremaining so or becoming nearly straight whenmature Lamellae adnate to decurrent crowdedcream to pale-yellow staining violet where damagedStipe 30ndash70 6 15ndash35 mm stout equal to clavate cen-tral dry to viscid cream to pale yellow surface oftenwith numerous dingy yellow golden yellow yellow-brown or light orange-brown pits hollow Latex scarceto abundant white becoming violet Context of pileusand stipe white staining violet where damaged Odorspicy-floral Flavor slightly acrid Basidiospore depositnot observed Basidiospores 8ndash105 6 6ndash85 mm av5 93 6 73 mm Q 5 113ndash143 Q5 127 hyaline in25 KOH broadly ellipsoid to ellipsoid ornamenta-tion amyloid of narrow to broad ridges and isolatedwarts forming an incomplete to dense reticulum to1 mm high Basidia 610ndash762 6 102ndash114 mm four-spored Pleuromacrocystidia 787ndash1448 6 89ndash127 mm subfusiform to lanceolate thin-walled withacute to moniliform apex scattered to abundant

432 MYCOLOGIA

Cheilomacrocystidia 559ndash1397 6 76ndash127 mm subfu-siform to lanceolate thin-walled with acute to monili-form apex scattered to abundant Pileipellis anixotrichoderm with well-developed gelatinous layer

Distribution and habitat Lactarius repraesentaneus firstwas described from Germany (Britzelmayr 1885) Phy-logenetic analyses including material from Germanyconfirm L repraesentaneus from alpine areas of theRocky Mountains and overall reinforce the traditionalconcept that this species has a broad ecological andgeographic range in arctic-alpine and subalpine habi-tats in North America northern central and southernEurope In alpine areas of the Rocky Mountains itassociates with Salix glauca however krummholz Piceaalso is typically (maybe always) present it is reportedwith Picea and Betula elsewhere

Previous reports from arctic-alpine areas mainlywith Betula include Alaska (Laursen and Ammirati1982 Geml et al 2009) the Rocky Mountain alpinezone (Cripps and Horak 2008 Cripps and Barge2013) Greenland (Knudsen and Borgen 1982 Watling1983 Elbourne and Knudsen 1990 Borgen 2006 Bor-gen et al 2006) Iceland (Eyjolfsdottir pers comm)Fennoscandia (Kallio and Kankainen 1964 Kuumlhner1975 Bon 1998) Siberia (Karatygin et al 1999) andthe Altai Mountains (Gorbunova 2010)

Specimens examined FINLAND Enontakiouml 16 Aug 2004JV21671 (TURA) Varsinais-Suomi 9 Aug 1998 JV13837F(TURA) SWEDEN Latnjavagge 12 Aug 2007 EL92-07(GB) USA COLORADO Chaffee County Sawatch RangeCottonwood Pass 4 Aug 2001 CLC 1643 (MONT) 16 Aug2011 EB0048 (MONT) Pitkin County Sawatch Range Inde-pendence Pass 14 Aug 2001 CLC 1747 (MONT) ZT9837(MONT) 15 Aug 2001 ZT9537 (MONT) MONTANA Car-bon County Beartooth Plateau Birch Site 12 Aug 2002CLC 1971 (MONT) 13 Aug 2007 CLC 2318 (MONT) 18Aug 2013 EB107-13 (MONT) WYOMING Park CountyBeartooth Plateau Frozen Lakes 21 Aug 1999 CLC1394 (MONT)

Other specimens examined SWEDEN Latnjavagge 14 Aug2010 EL57-10 (L dryadophilus GB) USA ALASKA 5 Aug2011 CLC 2729 (L dryadophilus MONT) Kenai PeninsulaCounty Palmer Creek Road 7 Aug 2011 CLC 2744 (L drya-dophilus MONT)

Comments The combination of a large pale yellow-brown to orange-brown pileus with a bearded marginand violet staining latex clearly separates L repraesenta-neus from other Lactarius species of the central andsouthern Rocky Mountain alpine zone Phylogeneti-cally close the arctic-alpine taxon L dryadophilus ismorphologically similar but has a cream to yellowsmoother pileus with hairs restricted to near the mar-gin basidiospore ornamentation consisting of sparsethinner ridges and ecological associations with Dryasand Salix It has not been reported from the RockyMountains

Lactarius salicis-reticulatae Kuumlhner FIGS 6 7Short description Lactarius salicis-reticulatae is recog-

nized by its small to medium cream pale yellow orpale ocher sporocarps with pale yellowish well-spacedlamellae often with a pinkish buff tint cream to pale yel-low stipe violet staining latex and tissue mild to slightlysweet odor mild flavor and association with dwarf (Sreticulata) and possibly also shrubby Salix species

Pileus 20ndash40 mm diam convex to broadly convexusually with a depressed center smooth dry to viscidazonate cream pale yellow or pale ocher sometimesdarker toward center staining violet where damagedmargin invcurved remaining so or becoming down-turned when mature Lamellae adnate to subdecur-rent fairly well-spaced cream pale yellow or paleorange-ocher often with a pinkish-buff tint stainingviolet where damaged Stipe 15ndash20 6 10ndash15 mmequal to clavate central dry to viscid cream to pale-yellow staining violet where damaged hollow Latexscarce watery-white becoming violet Context ofpileus and stipe white staining violet where damagedOdor mild to slightly sweet Flavor mild Basidiosporedeposit not observed Basidiospores (7ndash)85ndash115 6(7ndash)8ndash10 mm av 5 100 6 88 mm Q 5 (100ndash)113ndash143 Q 5 125 hyaline in 25 KOH sublgobose toellipsoid ornamentation amyloid of narrow ridgesand a few isolated warts forming an incomplete reticu-lum to 03 mm high Basidia 737ndash889 6 102ndash127 mm four-spored Pleuromacrocystidia 762ndash10166 76ndash114 mm subfusiform to fusiform thin-walledwith rounded to acute to moniliform apex scarceCheilomacrocystidia 686ndash9146 76ndash102 mm subfusi-form to fusiform thin-walled with acute to rounded tomoniliform apex scattered to abundant Pileipellis anixocutis

Distribution and habitat Lactarius salicis-reticulatae firstwas described from alpine tundra with Salix on MtLaringktatjaringkka Sweden (Kuumlhner 1975) Phylogenetic ana-lyses including material from the type locality confirmthis species from alpine areas on the Beartooth Plateauwith Salix reticulata and shrubby Salix spp and in arctic-alpine areas of Alaska Svalbard and Finland OverallL salicis-reticulatae appears largely confined to arctic-alpine habitats Colorado collections with S reticulataand S planifolia may represent a distinct taxonalthough support is low

Previous arctic-alpine reports include Greenland(Lamoure et al 1982 Heilmann-Clausen et al1998 Borgen et al 2006) the Rocky Mountainalpine zone (Cripps and Horak 2008 Cripps andBarge 2013) the Alps (Kuumlhner 1975 Kuumlhner andLamoure 1986 Jamoni 1995 2008 Bon 1998 Heil-mann-Clausen et al 1998) the Pyrenees (Ballaraacute1997) Fennoscandia (Kuumlhner 1975 Heilmann-Clau-sen et al 1998 Gulden 2005 Knudsen and

BARGE ET AL ROCKY MOUNTAIN ALPINE LACTARIUS 433

Vesterholt 2008) and Siberia (Nezdoiminogo 1997Karatygin et al 1999) previous reports of L aspi-deoides Burl from arctic-alpine areas in Alaska (Laur-sen and Ammirati 1982) Greenland (Knudsen andBorgen 1982) and the British Isles (Watling 1987)may refer to this species

Specimens examined FINLAND Near Utsjoki 28 Aug 2012CLC 2885 (MONT) SWEDEN Mt Laringktatjaringkka 12 Aug1972 K72-104 (Holotype G) 15 Aug 1999 JV15133 (Typelocality TURA) USA ALASKA Kenai Peninsula County Pal-mer Creek Road 7 Aug 2011 CLC 2745 (MONT) MON-TANA Carbon County Beartooth Plateau Birch Site 17Aug 2011 CLC 2776 (MONT) Carbon County BeartoothPlateau Highline Trail 8 Aug 1998 CLC 1211 (MONT)WYOMING Park County Beartooth Plateau GardnerLake 16 Aug 2014 EB0057-14 (MONT)

Other specimens examined FINLAND Koillismaa 31 Aug2005 JV23334 (L flavopalustris TURA) GREENLAND Sis-miut 19 Aug 2000 CLC 1536 (L salicis-herbaceae MONT)USA COLORADO Pitkin County Sawatch Range Indepen-dence Pass 13 Aug 2001 CLC 1741 (L aff salicis-reticulataeMONT) San Juan County San Juan Range CinnamonPass 10 Aug 2001 CLC 1710 (L aff salicis-reticulataeMONT) San Juan County San Juan Range Maggie Gulch11 Aug 2011 EB0036 (L aff salicis-reticulatae MONT)San Juan County San Juan Range Stony Pass 9 Aug 2001CLC 1689 (L aff salicis-reticulatae MONT) 13 Aug 2011EB0039 (L aff salicis-reticulatae MONT) MICHIGANEmmet County 10 Aug 1973 RL Shaffer 6957 (L aspi-deoides MICH)

Comments Some taxonomic confusion has beencaused by the fact that Kuumlhner (1975) originallynamed this species L aspideoides Kuumlhner but subse-quently renamed it because the name had been pre-viously applied L aspideoides Burl (1907) is generallylarger with a somewhat zonate pileus more crowdedlamellae that lack salmon-colored hues slightly smallerbasidiospores with broader ridges a bitter to slightlyacrid flavor and a subalpine ecology apparently ineastern and western North America (pers obs of RLShaffer 6957 Hesler and Smith 1979) Lactarius fla-vopalustris is also similar however it has a largermore brightly yellow pileus with faint zonationmore crowded lamellae a weakly scrobiculate stipe(sometimes) smaller basidiospores with thickerridges and a subalpine ecology (Kytoumlvuori 2009)Phylogenetic analyses (FIG 5) reveal three collec-tions of L flavopalustris (two from near the typelocality in Finland) nested in a well-supported cladewith L aspideoides from a subalpine forest in north-ern Michigan suggesting the possible conspecificityof these taxa Neither is reported from the RockyMountains Two other collections identified as L fla-vopalustris (from Estonia) were resolved as distinctfrom the L aspideoidesflavopalustris clade (FIG 5)their identity is unclear

Lactarius aspideus is differentiated from L salicis-reti-culate by its smaller basidiospores with denser reticula-tion more crowded creamier-colored lamellae andhabitat mainly in subalpine areas (Heilmann-Clausenet al 1998) although an environmental sequence iso-lated from an S polaris root tip from arctic Svalbardclusters with L aspideus in phylogenetic analyses (FIG5) Lactarius dryadophilus is often larger with a cream-colored pileus that has a tomentose to bearded margin(Heilmann-Clausen et al 1998) Lactarius flavidus is lar-ger with a sparsely zonate pileus more crowdedcream-colored lamellae darker violet-staining contextbasidiospores with more prominent ornamentationand a distally amyloid plage and subalpine ecology(Heilmann-Clausen et al 1998) Lactarius flavoaspideusKytoumlv has slightly smaller basidiospores on averagewith a denser reticulum and subalpine ecology withSalix (Kytoumlvuori 2009) it has been reported onlyfrom northern Europe Lactarius salicis-herbaceae asimilar arctic-alpine Salix associate has smaller (typi-cally) darker yellow to yellow-brown sporocarpspale cream to grayish buff lamellae and more denselyreticulate basidiospores (pers obs Heilmann-Clausenet al 1998) It is fairly widespread in arctic-alpineareas but is not reported from alpine areas in the cen-tral and southern Rocky Mountains Lactarius aff sali-cis-reticulatae from Colorado may be a distinct taxonand sporocarps can obtain a larger size (pileus to 90mm diam) but otherwise morphological featuresstrongly overlap

KEY TO ROCKY MOUNTAIN ALPINE LACTARIUS SPECIES

1 Latex white turning violet to dull lilac or flesh stain-ing violet to dull lilac 2

1 Latex watery to white unchanging and flesh notstaining violet to dull lilac 42 Fruiting body robust pileus 60ndash100 mm diam

yellow-brown to orange-brown hairy especiallynear margin lamellae cream to pale-yellow stipestout cream to pale-yellow often with numerousyellow-brown pits odor spicy-floral flavor mildlyacrid basidiospores 8ndash105 6 6ndash85 mm puta-tively associated with Betula and Picea (possiblyalso Salix) in Rocky Mountain alpine zone andsubalpine and arctic-alpine areas in North Amer-ica and Eurasia L repraesentaneus

2 Fruiting body smaller 20ndash50 mm diam pileus andmargin smooth 3

3 Pileus pale yellow to pale ocher lamellae cream paleyellow or pale orange-ocher stipe cream to pale yel-low odor mild to slightly sweet flavor mild basidios-pores 85ndash115 6 8ndash10 mm in Rocky Mountain alpinezone and other arctic-alpine areas in North Americaand Eurasia with Salix L salicis-reticulatae

3 Pileus blotchy light tan to light brown to gray-brownmargin lighter lamellae white to pale yellow-cream

434 MYCOLOGIA

stipe white to cream odor mild flavor mild basi-

diospores 8ndash10 6 65ndash8 mm in low alpine areas inColorado with Salix planifolia and possibly Wyom-

ing with Salix glauca L pallidomarginatus4 Pileus bright orange to orange-brown 10ndash45

mm diam margin smooth lamellae pale creamto pale orange stipe pale orange odor mildflavor mild in Rocky Mountain alpine zonearctic-alpine Alaska (possibly also subalpine)to Svalbard with Salix L lanceolatus

4 Pileus pinkish buff gray-brown brown ormauve 5

5 With odor of coconuts pileus 15ndash50 mm diam palepinkish buff to pale gray-brown with glaucous coat-ing lamellae cream to pale yellow-orange stipe buffor pale salmon flavor mild basidiospores 7ndash9 6 5ndash7mm in Rocky Mountain alpine zone and subalpinearctic-alpine areas in North America and Eurasiawith Betula L glyciosmus

5 Without odor of coconuts pileus 10ndash50 mm diamgray-brown to liver-brown with whitish glaucous coat-ing lamellae cream to light tan sometimes with pink-ish tinge discoloring to dingy tan where damagedStipe pale apricot to dingy cream-tan flavor mild toslightly acrid basidiospores 7ndash105 6 5ndash8 mm inRocky Mountain alpine zone and arctic-alpine areasin Eurasia and likely other arctic-alpine areas in NorthAmerica with Salix L nanus

DISCUSSION

The number of Lactarius species reported from theRocky Mountain alpine zone represents only a fractionof the total number of Lactarius species reported else-where from arctic-alpine areas however nearly halfof the core arctic-alpine species are now confirmedfor continental North America Core species oftencited as being restricted to arctic-alpine areas withdwarf and shrubby Salix andor Dryas species includeL brunneoviolaceus L dryadophilus L lanceolatus Lnanus L pseudouvidus L salicis-herbaceae and L salicis-reticulatae (Knudsen and Borgen 1982 Heilmann-Clau-sen et al 1998 Basso 1999 Knudsen and Vesterholt2008) Phylogenetic analyses and morphological exam-ination confirm three out of seven of these species forthe Rocky Mountain alpine zone (L lanceolatus Lnanus L salicis-reticulatae) and support the hypothesisthat these species are largely restricted to arctic-alpineareas with Salix One definitive new species L pallido-marginatus (with Salix) was revealed along with a possi-ble cryptic species L aff salicis-reticulatae (also withSalix) Many Lactarius species previously reportedfrom arctic-alpine areas are also commonly reportedfrom subalpine areas with Betulaceae andor Pinaceae

as well as with other plant families (Heilmann-Clausenet al 1998) From this set of species we report L gly-ciosmus which associates with Betula glandulosa in theRocky Mountain alpine zone and Betula spp acrossthe rest of its distribution and L repraesentaneus whichis commonly reported with Betula and Picea (Heslerand Smith 1979 Heilmann-Clausen et al 1998) andappears to associate with Salix glauca andor perhapskrummholz Picea in the Rocky Mountain alpine zone

Several taxa of interest were not molecularly distinctIn dataset A L lanceolatus did not form a monophyleticgroup but was paraphyletic with respect to Lactarius sp2 the L luculentus group and L aurantiacus In datasetB L nanus and L hysginoides were not clearly distinctIn dataset C L salicis-reticulatae did not form a clearmonophyletic group and L pseudouvidus L brunneovio-laceus and L luridus were not clearly distinguishedfrom each other especially at the RPB2 locus poormolecular resolution also has been encountered forthe latter three taxa by Das et al (2015)

Overall there was little genetic variation distinguish-ing closely related species in this study These findingsare similar to reports from other researchers who sug-gest that there is in general relatively little molecularvariation yet high morphological variation in genusLactarius (Verbeken and Nuytinck 2013) While manyof the species could be morphologically distinguishedall of the Rocky Mountain alpine taxa delineated inthis study are 97 or more similar at the ITS regionto one or more species For example L repraesentaneusand L dryadophilus are as much as 994 similar L sal-icis-reticulatae is approximately 98 similar to L salicis-herbaceae and L flavopalustris L pallidomarginatus isapproximately 97ndash98 similar to L aff brunneoviola-ceus L luridus L montanus and members of the Laff pseudouvidus group and approximately 99 similarto L aspideus L glyciosmus is approximately 98 simi-lar to L mammosus L nanus is approximately 98similar to L hysginoides and L lanceolatus is approxi-mately 97 similar to L substriatussubflammeus L aur-antiacus L luculentus v laetus and Lactarius sp 1 and 2and approximately 98 similar to L luculentus Thishigh degree of similarity could be problematic forenvironmental sequencing studies that use a strict97 ITS region similarity cutoff for recognizing spe-cies (eg Geml et al 2012 Timling et al 2012 2014)In addition Tedersoo et al (2014) suggested that theITS region in the sebacina lineage evolves moreslowly in high latitude areas which could be true forother genera The RPB2 locus proved better for differ-entiating some species and overall phylogenetic ana-lyses revealed several taxonomic issues that we werenot able to resolve A search for additional phylogen-etically informative loci for Lactarius should beinitiated along with more species-level phylogenetic

BARGE ET AL ROCKY MOUNTAIN ALPINE LACTARIUS 435

studies to provide a high quality molecular databasefor ecological studies Furthermore improvement ofmethods for sequencing older material such as typespecimens is critical

Most of the Rocky Mountain alpine Lactarius speciesdelineated in this study (except L pallidomarginatusand L aff salicis-reticulatae for which further studycould reveal broader ranges) appear to have broadintercontinental distributions in North America andEurasia The current distributions of these specieslikely were shaped by a number of biotic and abioticfactors that include host dispersal and distribution cli-mate glaciation and geography Because the genusLactarius is obligately ectomycorrhizal it is likely thatthe distribution of each fungal species was shaped lar-gely by the distribution of suitable host plants manyof which also have broad intercontinental distribu-tions Long-distance dispersal is hypothesized to haveplayed an important role in structuring present distri-bution patterns of arctic-alpine plants although manyappear clonal (Abbott and Brochmann 2003 Alsoset al 2007) and ectomycorrhizal fungi (Geml et al2012 Timling et al 2014) It also has been suggestedthat long-distance dispersal mechanisms have beenselected for in arctic flora due to repeated glaciationsand the need to track potential niches (Alsos et al2007) Pollen data from Blacktail Pond YellowstoneNational Park suggest that montane mid-elevationsin the northern greater Yellowstone ecosystem (Mon-tana Wyoming) were occupied by alpine tundra(Betula Salix many herbaceous taxa) similar to thatof the present Rocky Mountain alpine vegetation dur-ing the last glacial period (14 000ndash11 500 calibratedyears before present) (Krause and Whitlock 2013)Alpine vegetation also appears to have dominatedareas in the southern Rocky Mountains (WyomingColorado) 17 500ndash12 600 calibrated years before pre-sent (Minckley 2014) Both studies suggest the pre-sence of broad low-elevation glacial refugia for arctic-alpine life in the Rocky Mountains along the glacialfront and along with this study lend support to theidea that arctic-alpine Lactarius species (and manyother ectomycorrhizal fungi) may have occupied tun-dra areas south of the icesheets during cooler weatherduring the last glacial maximum and then dispersedupward in elevation and northward as the glaciersand ectomycorrhizal host plants retreated Broaderecological tolerances of taxa such as L glyciosmus andL repraesentaneus which are not restricted to arctic-alpine habitats likely let these species persist in tundraand non-tundra areas during the last glacial maximumas well as to track arctic-alpine vegetation as it movedupward in elevation and northward following theretreat of the icesheets For these species to haveachieved and maintained such broad distributions

gene flow must have been possible over long distancesduring the Pleistocene perhaps mediated by long-dis-tance dispersal (Geml et al 2012) range shifts due toclimate change and glacial expansion and retreat(Birks 2008) glacial and interglacial refugia (Billings1974) and the Bering land bridge (Shapiro et al2004 Geml et al 2006)

ACKNOWLEDGMENTS

We thank Dr Ellen Larsson Dr Pierre-Arthur Moreau JukkaVauras Dr Ursula Peintner Dr Egon Horak and herbaria GIB TU O GB TURA C LIP MICH DBG VPI and NY forloan of specimens Carol Johnson and Dr Matt Lavin areacknowledged for reviewing early stages of the manuscriptWe thank Don Bachman Marcy Barge and Charissa Bujakfor assistance with field work We also gratefully acknowledgesupport from the Montana Institute on Ecosystems graduateenhancement fund

LITERATURE CITED

Abbott RJ Brochmann C 2003 History and evolution of thearctic flora in the footsteps of Eric Hulteacuten Mol Ecol12299ndash313 doi101046j1365-294X200301731x

Alsos IG Eidesen PB Ehrich D Skrede I Westergaard KJacobsen GH Landvik JY Taberlet P Brochmann C2007 Frequent long-distance plant colonization in thechanging arctic Science 3161606ndash1609 doi101126science1139178

Ballaraacute J 1997 Nou estudi drsquoespegravecies fuacutengiques interessantsdels estatges alpiacute i subalpiacute dels Pirineus catalans RevCatalana Micol 201ndash24

Barge E 2015 Systematics of Lactarius in the Rocky Moun-tain alpine zone (masterrsquos thesis) Bozeman MontanaMontana State University 265 p

Basso MT 1999 Lactarius Pers Fungi Europaei 7 AlassioMykoflora (Italy) 845 p

Beker HJ Eberhardt U Vesterholt J 2010 Hebeloma hiemaleBres In arcticalpine habitats N Am Fungi 551ndash65

Bessette AE Harris DB Bessette AR 2009 Milk mushroomsof North America a field identification guide to thegenus Lactarius Syracuse New York Syracuse UniversityPress 297 p

Billings WD 1974 Adaptations and origins of alpine plantsArctic Alpine Res 6129ndash142 doi1023071550081

mdashmdashmdash 1988 Alpine vegetation In Barbour MG BillingsWD eds North American terrestrial vegetation NewYork Cambridge University Press p 392ndash420

Birks HH 2008 The late-quaternary history of arctic andalpine plants Plant Ecol Divers 1135ndash146 doi10108017550870802328652

Bogar LM Kennedy PG 2013 New wrinkles in an old para-digm neighborhood effects can modify the structureand specificity of Alnus-associated ectomycorrhizal fun-gal communities FEMS Microbiol Ecol 83767ndash777doi1011111574-694112032

Bon M 1985a Stage mycologie alpine Lanslebourg (Savoie)du 1 aug 3 sep 1984 Bull Feacuted Mycol Daupineacute-Savoie9619ndash25

436 MYCOLOGIA

Cheilomacrocystidia 559ndash1397 6 76ndash127 mm subfu-siform to lanceolate thin-walled with acute to monili-form apex scattered to abundant Pileipellis anixotrichoderm with well-developed gelatinous layer

Distribution and habitat Lactarius repraesentaneus firstwas described from Germany (Britzelmayr 1885) Phy-logenetic analyses including material from Germanyconfirm L repraesentaneus from alpine areas of theRocky Mountains and overall reinforce the traditionalconcept that this species has a broad ecological andgeographic range in arctic-alpine and subalpine habi-tats in North America northern central and southernEurope In alpine areas of the Rocky Mountains itassociates with Salix glauca however krummholz Piceaalso is typically (maybe always) present it is reportedwith Picea and Betula elsewhere

Previous reports from arctic-alpine areas mainlywith Betula include Alaska (Laursen and Ammirati1982 Geml et al 2009) the Rocky Mountain alpinezone (Cripps and Horak 2008 Cripps and Barge2013) Greenland (Knudsen and Borgen 1982 Watling1983 Elbourne and Knudsen 1990 Borgen 2006 Bor-gen et al 2006) Iceland (Eyjolfsdottir pers comm)Fennoscandia (Kallio and Kankainen 1964 Kuumlhner1975 Bon 1998) Siberia (Karatygin et al 1999) andthe Altai Mountains (Gorbunova 2010)

Specimens examined FINLAND Enontakiouml 16 Aug 2004JV21671 (TURA) Varsinais-Suomi 9 Aug 1998 JV13837F(TURA) SWEDEN Latnjavagge 12 Aug 2007 EL92-07(GB) USA COLORADO Chaffee County Sawatch RangeCottonwood Pass 4 Aug 2001 CLC 1643 (MONT) 16 Aug2011 EB0048 (MONT) Pitkin County Sawatch Range Inde-pendence Pass 14 Aug 2001 CLC 1747 (MONT) ZT9837(MONT) 15 Aug 2001 ZT9537 (MONT) MONTANA Car-bon County Beartooth Plateau Birch Site 12 Aug 2002CLC 1971 (MONT) 13 Aug 2007 CLC 2318 (MONT) 18Aug 2013 EB107-13 (MONT) WYOMING Park CountyBeartooth Plateau Frozen Lakes 21 Aug 1999 CLC1394 (MONT)

Other specimens examined SWEDEN Latnjavagge 14 Aug2010 EL57-10 (L dryadophilus GB) USA ALASKA 5 Aug2011 CLC 2729 (L dryadophilus MONT) Kenai PeninsulaCounty Palmer Creek Road 7 Aug 2011 CLC 2744 (L drya-dophilus MONT)

Comments The combination of a large pale yellow-brown to orange-brown pileus with a bearded marginand violet staining latex clearly separates L repraesenta-neus from other Lactarius species of the central andsouthern Rocky Mountain alpine zone Phylogeneti-cally close the arctic-alpine taxon L dryadophilus ismorphologically similar but has a cream to yellowsmoother pileus with hairs restricted to near the mar-gin basidiospore ornamentation consisting of sparsethinner ridges and ecological associations with Dryasand Salix It has not been reported from the RockyMountains

Lactarius salicis-reticulatae Kuumlhner FIGS 6 7Short description Lactarius salicis-reticulatae is recog-

nized by its small to medium cream pale yellow orpale ocher sporocarps with pale yellowish well-spacedlamellae often with a pinkish buff tint cream to pale yel-low stipe violet staining latex and tissue mild to slightlysweet odor mild flavor and association with dwarf (Sreticulata) and possibly also shrubby Salix species

Pileus 20ndash40 mm diam convex to broadly convexusually with a depressed center smooth dry to viscidazonate cream pale yellow or pale ocher sometimesdarker toward center staining violet where damagedmargin invcurved remaining so or becoming down-turned when mature Lamellae adnate to subdecur-rent fairly well-spaced cream pale yellow or paleorange-ocher often with a pinkish-buff tint stainingviolet where damaged Stipe 15ndash20 6 10ndash15 mmequal to clavate central dry to viscid cream to pale-yellow staining violet where damaged hollow Latexscarce watery-white becoming violet Context ofpileus and stipe white staining violet where damagedOdor mild to slightly sweet Flavor mild Basidiosporedeposit not observed Basidiospores (7ndash)85ndash115 6(7ndash)8ndash10 mm av 5 100 6 88 mm Q 5 (100ndash)113ndash143 Q 5 125 hyaline in 25 KOH sublgobose toellipsoid ornamentation amyloid of narrow ridgesand a few isolated warts forming an incomplete reticu-lum to 03 mm high Basidia 737ndash889 6 102ndash127 mm four-spored Pleuromacrocystidia 762ndash10166 76ndash114 mm subfusiform to fusiform thin-walledwith rounded to acute to moniliform apex scarceCheilomacrocystidia 686ndash9146 76ndash102 mm subfusi-form to fusiform thin-walled with acute to rounded tomoniliform apex scattered to abundant Pileipellis anixocutis

Distribution and habitat Lactarius salicis-reticulatae firstwas described from alpine tundra with Salix on MtLaringktatjaringkka Sweden (Kuumlhner 1975) Phylogenetic ana-lyses including material from the type locality confirmthis species from alpine areas on the Beartooth Plateauwith Salix reticulata and shrubby Salix spp and in arctic-alpine areas of Alaska Svalbard and Finland OverallL salicis-reticulatae appears largely confined to arctic-alpine habitats Colorado collections with S reticulataand S planifolia may represent a distinct taxonalthough support is low

Previous arctic-alpine reports include Greenland(Lamoure et al 1982 Heilmann-Clausen et al1998 Borgen et al 2006) the Rocky Mountainalpine zone (Cripps and Horak 2008 Cripps andBarge 2013) the Alps (Kuumlhner 1975 Kuumlhner andLamoure 1986 Jamoni 1995 2008 Bon 1998 Heil-mann-Clausen et al 1998) the Pyrenees (Ballaraacute1997) Fennoscandia (Kuumlhner 1975 Heilmann-Clau-sen et al 1998 Gulden 2005 Knudsen and

BARGE ET AL ROCKY MOUNTAIN ALPINE LACTARIUS 433

Vesterholt 2008) and Siberia (Nezdoiminogo 1997Karatygin et al 1999) previous reports of L aspi-deoides Burl from arctic-alpine areas in Alaska (Laur-sen and Ammirati 1982) Greenland (Knudsen andBorgen 1982) and the British Isles (Watling 1987)may refer to this species

Specimens examined FINLAND Near Utsjoki 28 Aug 2012CLC 2885 (MONT) SWEDEN Mt Laringktatjaringkka 12 Aug1972 K72-104 (Holotype G) 15 Aug 1999 JV15133 (Typelocality TURA) USA ALASKA Kenai Peninsula County Pal-mer Creek Road 7 Aug 2011 CLC 2745 (MONT) MON-TANA Carbon County Beartooth Plateau Birch Site 17Aug 2011 CLC 2776 (MONT) Carbon County BeartoothPlateau Highline Trail 8 Aug 1998 CLC 1211 (MONT)WYOMING Park County Beartooth Plateau GardnerLake 16 Aug 2014 EB0057-14 (MONT)

Other specimens examined FINLAND Koillismaa 31 Aug2005 JV23334 (L flavopalustris TURA) GREENLAND Sis-miut 19 Aug 2000 CLC 1536 (L salicis-herbaceae MONT)USA COLORADO Pitkin County Sawatch Range Indepen-dence Pass 13 Aug 2001 CLC 1741 (L aff salicis-reticulataeMONT) San Juan County San Juan Range CinnamonPass 10 Aug 2001 CLC 1710 (L aff salicis-reticulataeMONT) San Juan County San Juan Range Maggie Gulch11 Aug 2011 EB0036 (L aff salicis-reticulatae MONT)San Juan County San Juan Range Stony Pass 9 Aug 2001CLC 1689 (L aff salicis-reticulatae MONT) 13 Aug 2011EB0039 (L aff salicis-reticulatae MONT) MICHIGANEmmet County 10 Aug 1973 RL Shaffer 6957 (L aspi-deoides MICH)

Comments Some taxonomic confusion has beencaused by the fact that Kuumlhner (1975) originallynamed this species L aspideoides Kuumlhner but subse-quently renamed it because the name had been pre-viously applied L aspideoides Burl (1907) is generallylarger with a somewhat zonate pileus more crowdedlamellae that lack salmon-colored hues slightly smallerbasidiospores with broader ridges a bitter to slightlyacrid flavor and a subalpine ecology apparently ineastern and western North America (pers obs of RLShaffer 6957 Hesler and Smith 1979) Lactarius fla-vopalustris is also similar however it has a largermore brightly yellow pileus with faint zonationmore crowded lamellae a weakly scrobiculate stipe(sometimes) smaller basidiospores with thickerridges and a subalpine ecology (Kytoumlvuori 2009)Phylogenetic analyses (FIG 5) reveal three collec-tions of L flavopalustris (two from near the typelocality in Finland) nested in a well-supported cladewith L aspideoides from a subalpine forest in north-ern Michigan suggesting the possible conspecificityof these taxa Neither is reported from the RockyMountains Two other collections identified as L fla-vopalustris (from Estonia) were resolved as distinctfrom the L aspideoidesflavopalustris clade (FIG 5)their identity is unclear

Lactarius aspideus is differentiated from L salicis-reti-culate by its smaller basidiospores with denser reticula-tion more crowded creamier-colored lamellae andhabitat mainly in subalpine areas (Heilmann-Clausenet al 1998) although an environmental sequence iso-lated from an S polaris root tip from arctic Svalbardclusters with L aspideus in phylogenetic analyses (FIG5) Lactarius dryadophilus is often larger with a cream-colored pileus that has a tomentose to bearded margin(Heilmann-Clausen et al 1998) Lactarius flavidus is lar-ger with a sparsely zonate pileus more crowdedcream-colored lamellae darker violet-staining contextbasidiospores with more prominent ornamentationand a distally amyloid plage and subalpine ecology(Heilmann-Clausen et al 1998) Lactarius flavoaspideusKytoumlv has slightly smaller basidiospores on averagewith a denser reticulum and subalpine ecology withSalix (Kytoumlvuori 2009) it has been reported onlyfrom northern Europe Lactarius salicis-herbaceae asimilar arctic-alpine Salix associate has smaller (typi-cally) darker yellow to yellow-brown sporocarpspale cream to grayish buff lamellae and more denselyreticulate basidiospores (pers obs Heilmann-Clausenet al 1998) It is fairly widespread in arctic-alpineareas but is not reported from alpine areas in the cen-tral and southern Rocky Mountains Lactarius aff sali-cis-reticulatae from Colorado may be a distinct taxonand sporocarps can obtain a larger size (pileus to 90mm diam) but otherwise morphological featuresstrongly overlap

KEY TO ROCKY MOUNTAIN ALPINE LACTARIUS SPECIES

1 Latex white turning violet to dull lilac or flesh stain-ing violet to dull lilac 2

1 Latex watery to white unchanging and flesh notstaining violet to dull lilac 42 Fruiting body robust pileus 60ndash100 mm diam

yellow-brown to orange-brown hairy especiallynear margin lamellae cream to pale-yellow stipestout cream to pale-yellow often with numerousyellow-brown pits odor spicy-floral flavor mildlyacrid basidiospores 8ndash105 6 6ndash85 mm puta-tively associated with Betula and Picea (possiblyalso Salix) in Rocky Mountain alpine zone andsubalpine and arctic-alpine areas in North Amer-ica and Eurasia L repraesentaneus

2 Fruiting body smaller 20ndash50 mm diam pileus andmargin smooth 3

3 Pileus pale yellow to pale ocher lamellae cream paleyellow or pale orange-ocher stipe cream to pale yel-low odor mild to slightly sweet flavor mild basidios-pores 85ndash115 6 8ndash10 mm in Rocky Mountain alpinezone and other arctic-alpine areas in North Americaand Eurasia with Salix L salicis-reticulatae

3 Pileus blotchy light tan to light brown to gray-brownmargin lighter lamellae white to pale yellow-cream

434 MYCOLOGIA

stipe white to cream odor mild flavor mild basi-

diospores 8ndash10 6 65ndash8 mm in low alpine areas inColorado with Salix planifolia and possibly Wyom-

ing with Salix glauca L pallidomarginatus4 Pileus bright orange to orange-brown 10ndash45

mm diam margin smooth lamellae pale creamto pale orange stipe pale orange odor mildflavor mild in Rocky Mountain alpine zonearctic-alpine Alaska (possibly also subalpine)to Svalbard with Salix L lanceolatus

4 Pileus pinkish buff gray-brown brown ormauve 5

5 With odor of coconuts pileus 15ndash50 mm diam palepinkish buff to pale gray-brown with glaucous coat-ing lamellae cream to pale yellow-orange stipe buffor pale salmon flavor mild basidiospores 7ndash9 6 5ndash7mm in Rocky Mountain alpine zone and subalpinearctic-alpine areas in North America and Eurasiawith Betula L glyciosmus

5 Without odor of coconuts pileus 10ndash50 mm diamgray-brown to liver-brown with whitish glaucous coat-ing lamellae cream to light tan sometimes with pink-ish tinge discoloring to dingy tan where damagedStipe pale apricot to dingy cream-tan flavor mild toslightly acrid basidiospores 7ndash105 6 5ndash8 mm inRocky Mountain alpine zone and arctic-alpine areasin Eurasia and likely other arctic-alpine areas in NorthAmerica with Salix L nanus

DISCUSSION

The number of Lactarius species reported from theRocky Mountain alpine zone represents only a fractionof the total number of Lactarius species reported else-where from arctic-alpine areas however nearly halfof the core arctic-alpine species are now confirmedfor continental North America Core species oftencited as being restricted to arctic-alpine areas withdwarf and shrubby Salix andor Dryas species includeL brunneoviolaceus L dryadophilus L lanceolatus Lnanus L pseudouvidus L salicis-herbaceae and L salicis-reticulatae (Knudsen and Borgen 1982 Heilmann-Clau-sen et al 1998 Basso 1999 Knudsen and Vesterholt2008) Phylogenetic analyses and morphological exam-ination confirm three out of seven of these species forthe Rocky Mountain alpine zone (L lanceolatus Lnanus L salicis-reticulatae) and support the hypothesisthat these species are largely restricted to arctic-alpineareas with Salix One definitive new species L pallido-marginatus (with Salix) was revealed along with a possi-ble cryptic species L aff salicis-reticulatae (also withSalix) Many Lactarius species previously reportedfrom arctic-alpine areas are also commonly reportedfrom subalpine areas with Betulaceae andor Pinaceae

as well as with other plant families (Heilmann-Clausenet al 1998) From this set of species we report L gly-ciosmus which associates with Betula glandulosa in theRocky Mountain alpine zone and Betula spp acrossthe rest of its distribution and L repraesentaneus whichis commonly reported with Betula and Picea (Heslerand Smith 1979 Heilmann-Clausen et al 1998) andappears to associate with Salix glauca andor perhapskrummholz Picea in the Rocky Mountain alpine zone

Several taxa of interest were not molecularly distinctIn dataset A L lanceolatus did not form a monophyleticgroup but was paraphyletic with respect to Lactarius sp2 the L luculentus group and L aurantiacus In datasetB L nanus and L hysginoides were not clearly distinctIn dataset C L salicis-reticulatae did not form a clearmonophyletic group and L pseudouvidus L brunneovio-laceus and L luridus were not clearly distinguishedfrom each other especially at the RPB2 locus poormolecular resolution also has been encountered forthe latter three taxa by Das et al (2015)

Overall there was little genetic variation distinguish-ing closely related species in this study These findingsare similar to reports from other researchers who sug-gest that there is in general relatively little molecularvariation yet high morphological variation in genusLactarius (Verbeken and Nuytinck 2013) While manyof the species could be morphologically distinguishedall of the Rocky Mountain alpine taxa delineated inthis study are 97 or more similar at the ITS regionto one or more species For example L repraesentaneusand L dryadophilus are as much as 994 similar L sal-icis-reticulatae is approximately 98 similar to L salicis-herbaceae and L flavopalustris L pallidomarginatus isapproximately 97ndash98 similar to L aff brunneoviola-ceus L luridus L montanus and members of the Laff pseudouvidus group and approximately 99 similarto L aspideus L glyciosmus is approximately 98 simi-lar to L mammosus L nanus is approximately 98similar to L hysginoides and L lanceolatus is approxi-mately 97 similar to L substriatussubflammeus L aur-antiacus L luculentus v laetus and Lactarius sp 1 and 2and approximately 98 similar to L luculentus Thishigh degree of similarity could be problematic forenvironmental sequencing studies that use a strict97 ITS region similarity cutoff for recognizing spe-cies (eg Geml et al 2012 Timling et al 2012 2014)In addition Tedersoo et al (2014) suggested that theITS region in the sebacina lineage evolves moreslowly in high latitude areas which could be true forother genera The RPB2 locus proved better for differ-entiating some species and overall phylogenetic ana-lyses revealed several taxonomic issues that we werenot able to resolve A search for additional phylogen-etically informative loci for Lactarius should beinitiated along with more species-level phylogenetic

BARGE ET AL ROCKY MOUNTAIN ALPINE LACTARIUS 435

studies to provide a high quality molecular databasefor ecological studies Furthermore improvement ofmethods for sequencing older material such as typespecimens is critical

Most of the Rocky Mountain alpine Lactarius speciesdelineated in this study (except L pallidomarginatusand L aff salicis-reticulatae for which further studycould reveal broader ranges) appear to have broadintercontinental distributions in North America andEurasia The current distributions of these specieslikely were shaped by a number of biotic and abioticfactors that include host dispersal and distribution cli-mate glaciation and geography Because the genusLactarius is obligately ectomycorrhizal it is likely thatthe distribution of each fungal species was shaped lar-gely by the distribution of suitable host plants manyof which also have broad intercontinental distribu-tions Long-distance dispersal is hypothesized to haveplayed an important role in structuring present distri-bution patterns of arctic-alpine plants although manyappear clonal (Abbott and Brochmann 2003 Alsoset al 2007) and ectomycorrhizal fungi (Geml et al2012 Timling et al 2014) It also has been suggestedthat long-distance dispersal mechanisms have beenselected for in arctic flora due to repeated glaciationsand the need to track potential niches (Alsos et al2007) Pollen data from Blacktail Pond YellowstoneNational Park suggest that montane mid-elevationsin the northern greater Yellowstone ecosystem (Mon-tana Wyoming) were occupied by alpine tundra(Betula Salix many herbaceous taxa) similar to thatof the present Rocky Mountain alpine vegetation dur-ing the last glacial period (14 000ndash11 500 calibratedyears before present) (Krause and Whitlock 2013)Alpine vegetation also appears to have dominatedareas in the southern Rocky Mountains (WyomingColorado) 17 500ndash12 600 calibrated years before pre-sent (Minckley 2014) Both studies suggest the pre-sence of broad low-elevation glacial refugia for arctic-alpine life in the Rocky Mountains along the glacialfront and along with this study lend support to theidea that arctic-alpine Lactarius species (and manyother ectomycorrhizal fungi) may have occupied tun-dra areas south of the icesheets during cooler weatherduring the last glacial maximum and then dispersedupward in elevation and northward as the glaciersand ectomycorrhizal host plants retreated Broaderecological tolerances of taxa such as L glyciosmus andL repraesentaneus which are not restricted to arctic-alpine habitats likely let these species persist in tundraand non-tundra areas during the last glacial maximumas well as to track arctic-alpine vegetation as it movedupward in elevation and northward following theretreat of the icesheets For these species to haveachieved and maintained such broad distributions

gene flow must have been possible over long distancesduring the Pleistocene perhaps mediated by long-dis-tance dispersal (Geml et al 2012) range shifts due toclimate change and glacial expansion and retreat(Birks 2008) glacial and interglacial refugia (Billings1974) and the Bering land bridge (Shapiro et al2004 Geml et al 2006)

ACKNOWLEDGMENTS

We thank Dr Ellen Larsson Dr Pierre-Arthur Moreau JukkaVauras Dr Ursula Peintner Dr Egon Horak and herbaria GIB TU O GB TURA C LIP MICH DBG VPI and NY forloan of specimens Carol Johnson and Dr Matt Lavin areacknowledged for reviewing early stages of the manuscriptWe thank Don Bachman Marcy Barge and Charissa Bujakfor assistance with field work We also gratefully acknowledgesupport from the Montana Institute on Ecosystems graduateenhancement fund

LITERATURE CITED

Abbott RJ Brochmann C 2003 History and evolution of thearctic flora in the footsteps of Eric Hulteacuten Mol Ecol12299ndash313 doi101046j1365-294X200301731x

Alsos IG Eidesen PB Ehrich D Skrede I Westergaard KJacobsen GH Landvik JY Taberlet P Brochmann C2007 Frequent long-distance plant colonization in thechanging arctic Science 3161606ndash1609 doi101126science1139178

Ballaraacute J 1997 Nou estudi drsquoespegravecies fuacutengiques interessantsdels estatges alpiacute i subalpiacute dels Pirineus catalans RevCatalana Micol 201ndash24

Barge E 2015 Systematics of Lactarius in the Rocky Moun-tain alpine zone (masterrsquos thesis) Bozeman MontanaMontana State University 265 p

Basso MT 1999 Lactarius Pers Fungi Europaei 7 AlassioMykoflora (Italy) 845 p

Beker HJ Eberhardt U Vesterholt J 2010 Hebeloma hiemaleBres In arcticalpine habitats N Am Fungi 551ndash65

Bessette AE Harris DB Bessette AR 2009 Milk mushroomsof North America a field identification guide to thegenus Lactarius Syracuse New York Syracuse UniversityPress 297 p

Billings WD 1974 Adaptations and origins of alpine plantsArctic Alpine Res 6129ndash142 doi1023071550081

mdashmdashmdash 1988 Alpine vegetation In Barbour MG BillingsWD eds North American terrestrial vegetation NewYork Cambridge University Press p 392ndash420

Birks HH 2008 The late-quaternary history of arctic andalpine plants Plant Ecol Divers 1135ndash146 doi10108017550870802328652

Bogar LM Kennedy PG 2013 New wrinkles in an old para-digm neighborhood effects can modify the structureand specificity of Alnus-associated ectomycorrhizal fun-gal communities FEMS Microbiol Ecol 83767ndash777doi1011111574-694112032

Bon M 1985a Stage mycologie alpine Lanslebourg (Savoie)du 1 aug 3 sep 1984 Bull Feacuted Mycol Daupineacute-Savoie9619ndash25

436 MYCOLOGIA

Vesterholt 2008) and Siberia (Nezdoiminogo 1997Karatygin et al 1999) previous reports of L aspi-deoides Burl from arctic-alpine areas in Alaska (Laur-sen and Ammirati 1982) Greenland (Knudsen andBorgen 1982) and the British Isles (Watling 1987)may refer to this species

Specimens examined FINLAND Near Utsjoki 28 Aug 2012CLC 2885 (MONT) SWEDEN Mt Laringktatjaringkka 12 Aug1972 K72-104 (Holotype G) 15 Aug 1999 JV15133 (Typelocality TURA) USA ALASKA Kenai Peninsula County Pal-mer Creek Road 7 Aug 2011 CLC 2745 (MONT) MON-TANA Carbon County Beartooth Plateau Birch Site 17Aug 2011 CLC 2776 (MONT) Carbon County BeartoothPlateau Highline Trail 8 Aug 1998 CLC 1211 (MONT)WYOMING Park County Beartooth Plateau GardnerLake 16 Aug 2014 EB0057-14 (MONT)

Other specimens examined FINLAND Koillismaa 31 Aug2005 JV23334 (L flavopalustris TURA) GREENLAND Sis-miut 19 Aug 2000 CLC 1536 (L salicis-herbaceae MONT)USA COLORADO Pitkin County Sawatch Range Indepen-dence Pass 13 Aug 2001 CLC 1741 (L aff salicis-reticulataeMONT) San Juan County San Juan Range CinnamonPass 10 Aug 2001 CLC 1710 (L aff salicis-reticulataeMONT) San Juan County San Juan Range Maggie Gulch11 Aug 2011 EB0036 (L aff salicis-reticulatae MONT)San Juan County San Juan Range Stony Pass 9 Aug 2001CLC 1689 (L aff salicis-reticulatae MONT) 13 Aug 2011EB0039 (L aff salicis-reticulatae MONT) MICHIGANEmmet County 10 Aug 1973 RL Shaffer 6957 (L aspi-deoides MICH)

Comments Some taxonomic confusion has beencaused by the fact that Kuumlhner (1975) originallynamed this species L aspideoides Kuumlhner but subse-quently renamed it because the name had been pre-viously applied L aspideoides Burl (1907) is generallylarger with a somewhat zonate pileus more crowdedlamellae that lack salmon-colored hues slightly smallerbasidiospores with broader ridges a bitter to slightlyacrid flavor and a subalpine ecology apparently ineastern and western North America (pers obs of RLShaffer 6957 Hesler and Smith 1979) Lactarius fla-vopalustris is also similar however it has a largermore brightly yellow pileus with faint zonationmore crowded lamellae a weakly scrobiculate stipe(sometimes) smaller basidiospores with thickerridges and a subalpine ecology (Kytoumlvuori 2009)Phylogenetic analyses (FIG 5) reveal three collec-tions of L flavopalustris (two from near the typelocality in Finland) nested in a well-supported cladewith L aspideoides from a subalpine forest in north-ern Michigan suggesting the possible conspecificityof these taxa Neither is reported from the RockyMountains Two other collections identified as L fla-vopalustris (from Estonia) were resolved as distinctfrom the L aspideoidesflavopalustris clade (FIG 5)their identity is unclear

Lactarius aspideus is differentiated from L salicis-reti-culate by its smaller basidiospores with denser reticula-tion more crowded creamier-colored lamellae andhabitat mainly in subalpine areas (Heilmann-Clausenet al 1998) although an environmental sequence iso-lated from an S polaris root tip from arctic Svalbardclusters with L aspideus in phylogenetic analyses (FIG5) Lactarius dryadophilus is often larger with a cream-colored pileus that has a tomentose to bearded margin(Heilmann-Clausen et al 1998) Lactarius flavidus is lar-ger with a sparsely zonate pileus more crowdedcream-colored lamellae darker violet-staining contextbasidiospores with more prominent ornamentationand a distally amyloid plage and subalpine ecology(Heilmann-Clausen et al 1998) Lactarius flavoaspideusKytoumlv has slightly smaller basidiospores on averagewith a denser reticulum and subalpine ecology withSalix (Kytoumlvuori 2009) it has been reported onlyfrom northern Europe Lactarius salicis-herbaceae asimilar arctic-alpine Salix associate has smaller (typi-cally) darker yellow to yellow-brown sporocarpspale cream to grayish buff lamellae and more denselyreticulate basidiospores (pers obs Heilmann-Clausenet al 1998) It is fairly widespread in arctic-alpineareas but is not reported from alpine areas in the cen-tral and southern Rocky Mountains Lactarius aff sali-cis-reticulatae from Colorado may be a distinct taxonand sporocarps can obtain a larger size (pileus to 90mm diam) but otherwise morphological featuresstrongly overlap

KEY TO ROCKY MOUNTAIN ALPINE LACTARIUS SPECIES

1 Latex white turning violet to dull lilac or flesh stain-ing violet to dull lilac 2

1 Latex watery to white unchanging and flesh notstaining violet to dull lilac 42 Fruiting body robust pileus 60ndash100 mm diam

yellow-brown to orange-brown hairy especiallynear margin lamellae cream to pale-yellow stipestout cream to pale-yellow often with numerousyellow-brown pits odor spicy-floral flavor mildlyacrid basidiospores 8ndash105 6 6ndash85 mm puta-tively associated with Betula and Picea (possiblyalso Salix) in Rocky Mountain alpine zone andsubalpine and arctic-alpine areas in North Amer-ica and Eurasia L repraesentaneus

2 Fruiting body smaller 20ndash50 mm diam pileus andmargin smooth 3

3 Pileus pale yellow to pale ocher lamellae cream paleyellow or pale orange-ocher stipe cream to pale yel-low odor mild to slightly sweet flavor mild basidios-pores 85ndash115 6 8ndash10 mm in Rocky Mountain alpinezone and other arctic-alpine areas in North Americaand Eurasia with Salix L salicis-reticulatae

3 Pileus blotchy light tan to light brown to gray-brownmargin lighter lamellae white to pale yellow-cream

434 MYCOLOGIA

stipe white to cream odor mild flavor mild basi-

diospores 8ndash10 6 65ndash8 mm in low alpine areas inColorado with Salix planifolia and possibly Wyom-

ing with Salix glauca L pallidomarginatus4 Pileus bright orange to orange-brown 10ndash45

mm diam margin smooth lamellae pale creamto pale orange stipe pale orange odor mildflavor mild in Rocky Mountain alpine zonearctic-alpine Alaska (possibly also subalpine)to Svalbard with Salix L lanceolatus

4 Pileus pinkish buff gray-brown brown ormauve 5

5 With odor of coconuts pileus 15ndash50 mm diam palepinkish buff to pale gray-brown with glaucous coat-ing lamellae cream to pale yellow-orange stipe buffor pale salmon flavor mild basidiospores 7ndash9 6 5ndash7mm in Rocky Mountain alpine zone and subalpinearctic-alpine areas in North America and Eurasiawith Betula L glyciosmus

5 Without odor of coconuts pileus 10ndash50 mm diamgray-brown to liver-brown with whitish glaucous coat-ing lamellae cream to light tan sometimes with pink-ish tinge discoloring to dingy tan where damagedStipe pale apricot to dingy cream-tan flavor mild toslightly acrid basidiospores 7ndash105 6 5ndash8 mm inRocky Mountain alpine zone and arctic-alpine areasin Eurasia and likely other arctic-alpine areas in NorthAmerica with Salix L nanus

DISCUSSION

The number of Lactarius species reported from theRocky Mountain alpine zone represents only a fractionof the total number of Lactarius species reported else-where from arctic-alpine areas however nearly halfof the core arctic-alpine species are now confirmedfor continental North America Core species oftencited as being restricted to arctic-alpine areas withdwarf and shrubby Salix andor Dryas species includeL brunneoviolaceus L dryadophilus L lanceolatus Lnanus L pseudouvidus L salicis-herbaceae and L salicis-reticulatae (Knudsen and Borgen 1982 Heilmann-Clau-sen et al 1998 Basso 1999 Knudsen and Vesterholt2008) Phylogenetic analyses and morphological exam-ination confirm three out of seven of these species forthe Rocky Mountain alpine zone (L lanceolatus Lnanus L salicis-reticulatae) and support the hypothesisthat these species are largely restricted to arctic-alpineareas with Salix One definitive new species L pallido-marginatus (with Salix) was revealed along with a possi-ble cryptic species L aff salicis-reticulatae (also withSalix) Many Lactarius species previously reportedfrom arctic-alpine areas are also commonly reportedfrom subalpine areas with Betulaceae andor Pinaceae

as well as with other plant families (Heilmann-Clausenet al 1998) From this set of species we report L gly-ciosmus which associates with Betula glandulosa in theRocky Mountain alpine zone and Betula spp acrossthe rest of its distribution and L repraesentaneus whichis commonly reported with Betula and Picea (Heslerand Smith 1979 Heilmann-Clausen et al 1998) andappears to associate with Salix glauca andor perhapskrummholz Picea in the Rocky Mountain alpine zone

Several taxa of interest were not molecularly distinctIn dataset A L lanceolatus did not form a monophyleticgroup but was paraphyletic with respect to Lactarius sp2 the L luculentus group and L aurantiacus In datasetB L nanus and L hysginoides were not clearly distinctIn dataset C L salicis-reticulatae did not form a clearmonophyletic group and L pseudouvidus L brunneovio-laceus and L luridus were not clearly distinguishedfrom each other especially at the RPB2 locus poormolecular resolution also has been encountered forthe latter three taxa by Das et al (2015)

Overall there was little genetic variation distinguish-ing closely related species in this study These findingsare similar to reports from other researchers who sug-gest that there is in general relatively little molecularvariation yet high morphological variation in genusLactarius (Verbeken and Nuytinck 2013) While manyof the species could be morphologically distinguishedall of the Rocky Mountain alpine taxa delineated inthis study are 97 or more similar at the ITS regionto one or more species For example L repraesentaneusand L dryadophilus are as much as 994 similar L sal-icis-reticulatae is approximately 98 similar to L salicis-herbaceae and L flavopalustris L pallidomarginatus isapproximately 97ndash98 similar to L aff brunneoviola-ceus L luridus L montanus and members of the Laff pseudouvidus group and approximately 99 similarto L aspideus L glyciosmus is approximately 98 simi-lar to L mammosus L nanus is approximately 98similar to L hysginoides and L lanceolatus is approxi-mately 97 similar to L substriatussubflammeus L aur-antiacus L luculentus v laetus and Lactarius sp 1 and 2and approximately 98 similar to L luculentus Thishigh degree of similarity could be problematic forenvironmental sequencing studies that use a strict97 ITS region similarity cutoff for recognizing spe-cies (eg Geml et al 2012 Timling et al 2012 2014)In addition Tedersoo et al (2014) suggested that theITS region in the sebacina lineage evolves moreslowly in high latitude areas which could be true forother genera The RPB2 locus proved better for differ-entiating some species and overall phylogenetic ana-lyses revealed several taxonomic issues that we werenot able to resolve A search for additional phylogen-etically informative loci for Lactarius should beinitiated along with more species-level phylogenetic

BARGE ET AL ROCKY MOUNTAIN ALPINE LACTARIUS 435

studies to provide a high quality molecular databasefor ecological studies Furthermore improvement ofmethods for sequencing older material such as typespecimens is critical

Most of the Rocky Mountain alpine Lactarius speciesdelineated in this study (except L pallidomarginatusand L aff salicis-reticulatae for which further studycould reveal broader ranges) appear to have broadintercontinental distributions in North America andEurasia The current distributions of these specieslikely were shaped by a number of biotic and abioticfactors that include host dispersal and distribution cli-mate glaciation and geography Because the genusLactarius is obligately ectomycorrhizal it is likely thatthe distribution of each fungal species was shaped lar-gely by the distribution of suitable host plants manyof which also have broad intercontinental distribu-tions Long-distance dispersal is hypothesized to haveplayed an important role in structuring present distri-bution patterns of arctic-alpine plants although manyappear clonal (Abbott and Brochmann 2003 Alsoset al 2007) and ectomycorrhizal fungi (Geml et al2012 Timling et al 2014) It also has been suggestedthat long-distance dispersal mechanisms have beenselected for in arctic flora due to repeated glaciationsand the need to track potential niches (Alsos et al2007) Pollen data from Blacktail Pond YellowstoneNational Park suggest that montane mid-elevationsin the northern greater Yellowstone ecosystem (Mon-tana Wyoming) were occupied by alpine tundra(Betula Salix many herbaceous taxa) similar to thatof the present Rocky Mountain alpine vegetation dur-ing the last glacial period (14 000ndash11 500 calibratedyears before present) (Krause and Whitlock 2013)Alpine vegetation also appears to have dominatedareas in the southern Rocky Mountains (WyomingColorado) 17 500ndash12 600 calibrated years before pre-sent (Minckley 2014) Both studies suggest the pre-sence of broad low-elevation glacial refugia for arctic-alpine life in the Rocky Mountains along the glacialfront and along with this study lend support to theidea that arctic-alpine Lactarius species (and manyother ectomycorrhizal fungi) may have occupied tun-dra areas south of the icesheets during cooler weatherduring the last glacial maximum and then dispersedupward in elevation and northward as the glaciersand ectomycorrhizal host plants retreated Broaderecological tolerances of taxa such as L glyciosmus andL repraesentaneus which are not restricted to arctic-alpine habitats likely let these species persist in tundraand non-tundra areas during the last glacial maximumas well as to track arctic-alpine vegetation as it movedupward in elevation and northward following theretreat of the icesheets For these species to haveachieved and maintained such broad distributions

gene flow must have been possible over long distancesduring the Pleistocene perhaps mediated by long-dis-tance dispersal (Geml et al 2012) range shifts due toclimate change and glacial expansion and retreat(Birks 2008) glacial and interglacial refugia (Billings1974) and the Bering land bridge (Shapiro et al2004 Geml et al 2006)

ACKNOWLEDGMENTS

We thank Dr Ellen Larsson Dr Pierre-Arthur Moreau JukkaVauras Dr Ursula Peintner Dr Egon Horak and herbaria GIB TU O GB TURA C LIP MICH DBG VPI and NY forloan of specimens Carol Johnson and Dr Matt Lavin areacknowledged for reviewing early stages of the manuscriptWe thank Don Bachman Marcy Barge and Charissa Bujakfor assistance with field work We also gratefully acknowledgesupport from the Montana Institute on Ecosystems graduateenhancement fund

LITERATURE CITED

Abbott RJ Brochmann C 2003 History and evolution of thearctic flora in the footsteps of Eric Hulteacuten Mol Ecol12299ndash313 doi101046j1365-294X200301731x

Alsos IG Eidesen PB Ehrich D Skrede I Westergaard KJacobsen GH Landvik JY Taberlet P Brochmann C2007 Frequent long-distance plant colonization in thechanging arctic Science 3161606ndash1609 doi101126science1139178

Ballaraacute J 1997 Nou estudi drsquoespegravecies fuacutengiques interessantsdels estatges alpiacute i subalpiacute dels Pirineus catalans RevCatalana Micol 201ndash24

Barge E 2015 Systematics of Lactarius in the Rocky Moun-tain alpine zone (masterrsquos thesis) Bozeman MontanaMontana State University 265 p

Basso MT 1999 Lactarius Pers Fungi Europaei 7 AlassioMykoflora (Italy) 845 p

Beker HJ Eberhardt U Vesterholt J 2010 Hebeloma hiemaleBres In arcticalpine habitats N Am Fungi 551ndash65

Bessette AE Harris DB Bessette AR 2009 Milk mushroomsof North America a field identification guide to thegenus Lactarius Syracuse New York Syracuse UniversityPress 297 p

Billings WD 1974 Adaptations and origins of alpine plantsArctic Alpine Res 6129ndash142 doi1023071550081

mdashmdashmdash 1988 Alpine vegetation In Barbour MG BillingsWD eds North American terrestrial vegetation NewYork Cambridge University Press p 392ndash420

Birks HH 2008 The late-quaternary history of arctic andalpine plants Plant Ecol Divers 1135ndash146 doi10108017550870802328652

Bogar LM Kennedy PG 2013 New wrinkles in an old para-digm neighborhood effects can modify the structureand specificity of Alnus-associated ectomycorrhizal fun-gal communities FEMS Microbiol Ecol 83767ndash777doi1011111574-694112032

Bon M 1985a Stage mycologie alpine Lanslebourg (Savoie)du 1 aug 3 sep 1984 Bull Feacuted Mycol Daupineacute-Savoie9619ndash25

436 MYCOLOGIA

stipe white to cream odor mild flavor mild basi-

diospores 8ndash10 6 65ndash8 mm in low alpine areas inColorado with Salix planifolia and possibly Wyom-

ing with Salix glauca L pallidomarginatus4 Pileus bright orange to orange-brown 10ndash45

mm diam margin smooth lamellae pale creamto pale orange stipe pale orange odor mildflavor mild in Rocky Mountain alpine zonearctic-alpine Alaska (possibly also subalpine)to Svalbard with Salix L lanceolatus

4 Pileus pinkish buff gray-brown brown ormauve 5

5 With odor of coconuts pileus 15ndash50 mm diam palepinkish buff to pale gray-brown with glaucous coat-ing lamellae cream to pale yellow-orange stipe buffor pale salmon flavor mild basidiospores 7ndash9 6 5ndash7mm in Rocky Mountain alpine zone and subalpinearctic-alpine areas in North America and Eurasiawith Betula L glyciosmus

5 Without odor of coconuts pileus 10ndash50 mm diamgray-brown to liver-brown with whitish glaucous coat-ing lamellae cream to light tan sometimes with pink-ish tinge discoloring to dingy tan where damagedStipe pale apricot to dingy cream-tan flavor mild toslightly acrid basidiospores 7ndash105 6 5ndash8 mm inRocky Mountain alpine zone and arctic-alpine areasin Eurasia and likely other arctic-alpine areas in NorthAmerica with Salix L nanus

DISCUSSION

The number of Lactarius species reported from theRocky Mountain alpine zone represents only a fractionof the total number of Lactarius species reported else-where from arctic-alpine areas however nearly halfof the core arctic-alpine species are now confirmedfor continental North America Core species oftencited as being restricted to arctic-alpine areas withdwarf and shrubby Salix andor Dryas species includeL brunneoviolaceus L dryadophilus L lanceolatus Lnanus L pseudouvidus L salicis-herbaceae and L salicis-reticulatae (Knudsen and Borgen 1982 Heilmann-Clau-sen et al 1998 Basso 1999 Knudsen and Vesterholt2008) Phylogenetic analyses and morphological exam-ination confirm three out of seven of these species forthe Rocky Mountain alpine zone (L lanceolatus Lnanus L salicis-reticulatae) and support the hypothesisthat these species are largely restricted to arctic-alpineareas with Salix One definitive new species L pallido-marginatus (with Salix) was revealed along with a possi-ble cryptic species L aff salicis-reticulatae (also withSalix) Many Lactarius species previously reportedfrom arctic-alpine areas are also commonly reportedfrom subalpine areas with Betulaceae andor Pinaceae

as well as with other plant families (Heilmann-Clausenet al 1998) From this set of species we report L gly-ciosmus which associates with Betula glandulosa in theRocky Mountain alpine zone and Betula spp acrossthe rest of its distribution and L repraesentaneus whichis commonly reported with Betula and Picea (Heslerand Smith 1979 Heilmann-Clausen et al 1998) andappears to associate with Salix glauca andor perhapskrummholz Picea in the Rocky Mountain alpine zone

Several taxa of interest were not molecularly distinctIn dataset A L lanceolatus did not form a monophyleticgroup but was paraphyletic with respect to Lactarius sp2 the L luculentus group and L aurantiacus In datasetB L nanus and L hysginoides were not clearly distinctIn dataset C L salicis-reticulatae did not form a clearmonophyletic group and L pseudouvidus L brunneovio-laceus and L luridus were not clearly distinguishedfrom each other especially at the RPB2 locus poormolecular resolution also has been encountered forthe latter three taxa by Das et al (2015)

Overall there was little genetic variation distinguish-ing closely related species in this study These findingsare similar to reports from other researchers who sug-gest that there is in general relatively little molecularvariation yet high morphological variation in genusLactarius (Verbeken and Nuytinck 2013) While manyof the species could be morphologically distinguishedall of the Rocky Mountain alpine taxa delineated inthis study are 97 or more similar at the ITS regionto one or more species For example L repraesentaneusand L dryadophilus are as much as 994 similar L sal-icis-reticulatae is approximately 98 similar to L salicis-herbaceae and L flavopalustris L pallidomarginatus isapproximately 97ndash98 similar to L aff brunneoviola-ceus L luridus L montanus and members of the Laff pseudouvidus group and approximately 99 similarto L aspideus L glyciosmus is approximately 98 simi-lar to L mammosus L nanus is approximately 98similar to L hysginoides and L lanceolatus is approxi-mately 97 similar to L substriatussubflammeus L aur-antiacus L luculentus v laetus and Lactarius sp 1 and 2and approximately 98 similar to L luculentus Thishigh degree of similarity could be problematic forenvironmental sequencing studies that use a strict97 ITS region similarity cutoff for recognizing spe-cies (eg Geml et al 2012 Timling et al 2012 2014)In addition Tedersoo et al (2014) suggested that theITS region in the sebacina lineage evolves moreslowly in high latitude areas which could be true forother genera The RPB2 locus proved better for differ-entiating some species and overall phylogenetic ana-lyses revealed several taxonomic issues that we werenot able to resolve A search for additional phylogen-etically informative loci for Lactarius should beinitiated along with more species-level phylogenetic

BARGE ET AL ROCKY MOUNTAIN ALPINE LACTARIUS 435

studies to provide a high quality molecular databasefor ecological studies Furthermore improvement ofmethods for sequencing older material such as typespecimens is critical

Most of the Rocky Mountain alpine Lactarius speciesdelineated in this study (except L pallidomarginatusand L aff salicis-reticulatae for which further studycould reveal broader ranges) appear to have broadintercontinental distributions in North America andEurasia The current distributions of these specieslikely were shaped by a number of biotic and abioticfactors that include host dispersal and distribution cli-mate glaciation and geography Because the genusLactarius is obligately ectomycorrhizal it is likely thatthe distribution of each fungal species was shaped lar-gely by the distribution of suitable host plants manyof which also have broad intercontinental distribu-tions Long-distance dispersal is hypothesized to haveplayed an important role in structuring present distri-bution patterns of arctic-alpine plants although manyappear clonal (Abbott and Brochmann 2003 Alsoset al 2007) and ectomycorrhizal fungi (Geml et al2012 Timling et al 2014) It also has been suggestedthat long-distance dispersal mechanisms have beenselected for in arctic flora due to repeated glaciationsand the need to track potential niches (Alsos et al2007) Pollen data from Blacktail Pond YellowstoneNational Park suggest that montane mid-elevationsin the northern greater Yellowstone ecosystem (Mon-tana Wyoming) were occupied by alpine tundra(Betula Salix many herbaceous taxa) similar to thatof the present Rocky Mountain alpine vegetation dur-ing the last glacial period (14 000ndash11 500 calibratedyears before present) (Krause and Whitlock 2013)Alpine vegetation also appears to have dominatedareas in the southern Rocky Mountains (WyomingColorado) 17 500ndash12 600 calibrated years before pre-sent (Minckley 2014) Both studies suggest the pre-sence of broad low-elevation glacial refugia for arctic-alpine life in the Rocky Mountains along the glacialfront and along with this study lend support to theidea that arctic-alpine Lactarius species (and manyother ectomycorrhizal fungi) may have occupied tun-dra areas south of the icesheets during cooler weatherduring the last glacial maximum and then dispersedupward in elevation and northward as the glaciersand ectomycorrhizal host plants retreated Broaderecological tolerances of taxa such as L glyciosmus andL repraesentaneus which are not restricted to arctic-alpine habitats likely let these species persist in tundraand non-tundra areas during the last glacial maximumas well as to track arctic-alpine vegetation as it movedupward in elevation and northward following theretreat of the icesheets For these species to haveachieved and maintained such broad distributions

gene flow must have been possible over long distancesduring the Pleistocene perhaps mediated by long-dis-tance dispersal (Geml et al 2012) range shifts due toclimate change and glacial expansion and retreat(Birks 2008) glacial and interglacial refugia (Billings1974) and the Bering land bridge (Shapiro et al2004 Geml et al 2006)

ACKNOWLEDGMENTS

We thank Dr Ellen Larsson Dr Pierre-Arthur Moreau JukkaVauras Dr Ursula Peintner Dr Egon Horak and herbaria GIB TU O GB TURA C LIP MICH DBG VPI and NY forloan of specimens Carol Johnson and Dr Matt Lavin areacknowledged for reviewing early stages of the manuscriptWe thank Don Bachman Marcy Barge and Charissa Bujakfor assistance with field work We also gratefully acknowledgesupport from the Montana Institute on Ecosystems graduateenhancement fund

LITERATURE CITED

Abbott RJ Brochmann C 2003 History and evolution of thearctic flora in the footsteps of Eric Hulteacuten Mol Ecol12299ndash313 doi101046j1365-294X200301731x

Alsos IG Eidesen PB Ehrich D Skrede I Westergaard KJacobsen GH Landvik JY Taberlet P Brochmann C2007 Frequent long-distance plant colonization in thechanging arctic Science 3161606ndash1609 doi101126science1139178

Ballaraacute J 1997 Nou estudi drsquoespegravecies fuacutengiques interessantsdels estatges alpiacute i subalpiacute dels Pirineus catalans RevCatalana Micol 201ndash24

Barge E 2015 Systematics of Lactarius in the Rocky Moun-tain alpine zone (masterrsquos thesis) Bozeman MontanaMontana State University 265 p

Basso MT 1999 Lactarius Pers Fungi Europaei 7 AlassioMykoflora (Italy) 845 p

Beker HJ Eberhardt U Vesterholt J 2010 Hebeloma hiemaleBres In arcticalpine habitats N Am Fungi 551ndash65

Bessette AE Harris DB Bessette AR 2009 Milk mushroomsof North America a field identification guide to thegenus Lactarius Syracuse New York Syracuse UniversityPress 297 p

Billings WD 1974 Adaptations and origins of alpine plantsArctic Alpine Res 6129ndash142 doi1023071550081

mdashmdashmdash 1988 Alpine vegetation In Barbour MG BillingsWD eds North American terrestrial vegetation NewYork Cambridge University Press p 392ndash420

Birks HH 2008 The late-quaternary history of arctic andalpine plants Plant Ecol Divers 1135ndash146 doi10108017550870802328652

Bogar LM Kennedy PG 2013 New wrinkles in an old para-digm neighborhood effects can modify the structureand specificity of Alnus-associated ectomycorrhizal fun-gal communities FEMS Microbiol Ecol 83767ndash777doi1011111574-694112032

Bon M 1985a Stage mycologie alpine Lanslebourg (Savoie)du 1 aug 3 sep 1984 Bull Feacuted Mycol Daupineacute-Savoie9619ndash25

436 MYCOLOGIA

studies to provide a high quality molecular databasefor ecological studies Furthermore improvement ofmethods for sequencing older material such as typespecimens is critical

Most of the Rocky Mountain alpine Lactarius speciesdelineated in this study (except L pallidomarginatusand L aff salicis-reticulatae for which further studycould reveal broader ranges) appear to have broadintercontinental distributions in North America andEurasia The current distributions of these specieslikely were shaped by a number of biotic and abioticfactors that include host dispersal and distribution cli-mate glaciation and geography Because the genusLactarius is obligately ectomycorrhizal it is likely thatthe distribution of each fungal species was shaped lar-gely by the distribution of suitable host plants manyof which also have broad intercontinental distribu-tions Long-distance dispersal is hypothesized to haveplayed an important role in structuring present distri-bution patterns of arctic-alpine plants although manyappear clonal (Abbott and Brochmann 2003 Alsoset al 2007) and ectomycorrhizal fungi (Geml et al2012 Timling et al 2014) It also has been suggestedthat long-distance dispersal mechanisms have beenselected for in arctic flora due to repeated glaciationsand the need to track potential niches (Alsos et al2007) Pollen data from Blacktail Pond YellowstoneNational Park suggest that montane mid-elevationsin the northern greater Yellowstone ecosystem (Mon-tana Wyoming) were occupied by alpine tundra(Betula Salix many herbaceous taxa) similar to thatof the present Rocky Mountain alpine vegetation dur-ing the last glacial period (14 000ndash11 500 calibratedyears before present) (Krause and Whitlock 2013)Alpine vegetation also appears to have dominatedareas in the southern Rocky Mountains (WyomingColorado) 17 500ndash12 600 calibrated years before pre-sent (Minckley 2014) Both studies suggest the pre-sence of broad low-elevation glacial refugia for arctic-alpine life in the Rocky Mountains along the glacialfront and along with this study lend support to theidea that arctic-alpine Lactarius species (and manyother ectomycorrhizal fungi) may have occupied tun-dra areas south of the icesheets during cooler weatherduring the last glacial maximum and then dispersedupward in elevation and northward as the glaciersand ectomycorrhizal host plants retreated Broaderecological tolerances of taxa such as L glyciosmus andL repraesentaneus which are not restricted to arctic-alpine habitats likely let these species persist in tundraand non-tundra areas during the last glacial maximumas well as to track arctic-alpine vegetation as it movedupward in elevation and northward following theretreat of the icesheets For these species to haveachieved and maintained such broad distributions

gene flow must have been possible over long distancesduring the Pleistocene perhaps mediated by long-dis-tance dispersal (Geml et al 2012) range shifts due toclimate change and glacial expansion and retreat(Birks 2008) glacial and interglacial refugia (Billings1974) and the Bering land bridge (Shapiro et al2004 Geml et al 2006)

ACKNOWLEDGMENTS

We thank Dr Ellen Larsson Dr Pierre-Arthur Moreau JukkaVauras Dr Ursula Peintner Dr Egon Horak and herbaria GIB TU O GB TURA C LIP MICH DBG VPI and NY forloan of specimens Carol Johnson and Dr Matt Lavin areacknowledged for reviewing early stages of the manuscriptWe thank Don Bachman Marcy Barge and Charissa Bujakfor assistance with field work We also gratefully acknowledgesupport from the Montana Institute on Ecosystems graduateenhancement fund

LITERATURE CITED

Abbott RJ Brochmann C 2003 History and evolution of thearctic flora in the footsteps of Eric Hulteacuten Mol Ecol12299ndash313 doi101046j1365-294X200301731x

Alsos IG Eidesen PB Ehrich D Skrede I Westergaard KJacobsen GH Landvik JY Taberlet P Brochmann C2007 Frequent long-distance plant colonization in thechanging arctic Science 3161606ndash1609 doi101126science1139178

Ballaraacute J 1997 Nou estudi drsquoespegravecies fuacutengiques interessantsdels estatges alpiacute i subalpiacute dels Pirineus catalans RevCatalana Micol 201ndash24

Barge E 2015 Systematics of Lactarius in the Rocky Moun-tain alpine zone (masterrsquos thesis) Bozeman MontanaMontana State University 265 p

Basso MT 1999 Lactarius Pers Fungi Europaei 7 AlassioMykoflora (Italy) 845 p

Beker HJ Eberhardt U Vesterholt J 2010 Hebeloma hiemaleBres In arcticalpine habitats N Am Fungi 551ndash65

Bessette AE Harris DB Bessette AR 2009 Milk mushroomsof North America a field identification guide to thegenus Lactarius Syracuse New York Syracuse UniversityPress 297 p

Billings WD 1974 Adaptations and origins of alpine plantsArctic Alpine Res 6129ndash142 doi1023071550081

mdashmdashmdash 1988 Alpine vegetation In Barbour MG BillingsWD eds North American terrestrial vegetation NewYork Cambridge University Press p 392ndash420

Birks HH 2008 The late-quaternary history of arctic andalpine plants Plant Ecol Divers 1135ndash146 doi10108017550870802328652

Bogar LM Kennedy PG 2013 New wrinkles in an old para-digm neighborhood effects can modify the structureand specificity of Alnus-associated ectomycorrhizal fun-gal communities FEMS Microbiol Ecol 83767ndash777doi1011111574-694112032

Bon M 1985a Stage mycologie alpine Lanslebourg (Savoie)du 1 aug 3 sep 1984 Bull Feacuted Mycol Daupineacute-Savoie9619ndash25

436 MYCOLOGIA

mdashmdashmdash 1985b Quelques nouveaux taxons de la flore myco-logique alpine Bull Feacuted Mycol Daupineacute-Savoie9723ndash30

mdashmdashmdash 1989 Quelques Agaricomycetes inteacuteressants de lazone alpine reacutecolteeacutes dans le Tessin Mycol Helvetica 3(3)315ndash330

mdashmdashmdash 1991 Inventaires des espeacuteces reacutecoltees au stage demycology alpine Bull Feacuted Mycol Dauphineacute-Savoie12225ndash28

mdashmdashmdash 1998 Cleacute monographique des lactaires alpines BullFeacuted Mycol Dauphineacute-Savoie 15015ndash22

mdashmdashmdash Ballaraacute J 1996 Aportacioacute a lrsquoestudi de la microfloraalpine dels Pirineus (2a part) Rev Catalana Mycol19139ndash153

Borgen T 2006 Distribution of selected basidiomycetes inoceanic dwarf-scrub heaths in Paamiut area low arcticsouth Greenland In Boertmann D Knudsen H edsArctic Alpine Mycol 6 Meddelelser om GroslashnlandBioscience Greenland Museum Tusulanum Press p25ndash36

mdashmdashmdash Elbourne SA Knudsen H 2006 A checklist of theGreenland basidiomycetes In Boertmann D KnudsenH eds Arctic Alpine Mycol 6 Meddelelser omGroslashnland Bioscience Greenland Museum TusulanumPress p 37ndash59

Britzelmayr M 1885 Hymenomyceten aus Suumldbayern Teil 69 Bericht des Naturhistorischen Vereins in Augsburg28119ndash160

Brouillet L Whetstone RD 1993 Climate and physiographyIn Flora of North America Editorial Committee edsFlora of North America north of Mexico OxfordOxford University Press p 15ndash46

Brown J Ferrians OJ Jr Heginbottom JA Melnikov ES 2001Circumarctic map of permafrost and ground iceconditions Boulder Colorado National Snow and IceData CenterWorld Data Center for Glaciology

Burlingham GS 1907 Some Lactarii from Windham Coun-try Vermont MemTorrey Bot Club 3485ndash95 doi1023072478917

Buyck B Hofstetter V Eberhardt U Verbeken A Kauff F2008 Walking the thin line between Russula and Lactar-ius the dilemma of Russula subsect Ochricompactae Fun-gal Divers 2815ndash40

mdashmdashmdash mdashmdashmdash Verbeken A Walleyn R 2010 Proposal toconserve Lactarius nom cons (Basidiomycota) with aconserved type Taxon 59295ndash296

Castresana J 2000 Selection of conserved blocks from multi-ple alignments for their use in phylogenetic analysisMol Biol Evol 17540ndash552 doi101093oxfordjournalsmolbeva026334

Chapin FS III Koumlrner C 1995 Patterns causes changes andconsequences of biodiversity in arctic and alpine ecosys-tems In Chapin FS Korner CH eds Arctic and Alpinebiodiversity patterns causes and ecosystem conse-quences Ecological studies 113 New York Springer-Verlag p 313ndash320

Cooper SV Lesica P Page-Dumroese D 1997 Plant commu-nity classification for alpine vegetation on the Beaver-head National Forest Montana Ogden Utah USDAForest Service Gen Tech Rep INT-GTR-362

Corriol G 2008 Checklist of Pyrenean alpine-stage macro-fungi Sommerfeltia 3129ndash99 doi102478v10208-011-0004-6

Cripps CL Barge E 2013 Notes on the genus Lactarius fromthe Rocky Mountain alpine zone in regard to Finnisharctic-alpine species Karstenia 5329ndash37

mdashmdashmdash Eddington LH 2005 Distribution of mycorrhizaltypes among alpine vascular plant families on the Bear-tooth Plateau Rocky Mountains USA in reference tolarge-scale patterns in arctic-alpine habitats Arctic Ant-arctic Alpine Res 37177ndash188 doi1016571523-0430(2005)037[0177DOMTAA]20CO2

mdashmdashmdash Horak E 2008 Checklist and ecology of the Agari-cales Russulales and Boletales in the alpine zone ofthe Rocky Mountains (Colorado Montana Wyoming)at 3000ndash4000 m ASL Sommerfeltia 31101ndash123doi102478v10208-011-0005-5

mdashmdashmdash mdashmdashmdash 2010 Amanita in the Rocky Mountain alpinezone USA new records for A nivalis and A groenlandicaN Am Fungi 59ndash21

mdashmdashmdash Larsson E Horak E 2010 Subgenus Mallocybe (Ino-cybe) in the Rocky Mountain alpine zone with molecularreference to European arctic-alpine material N AmFungi 597ndash126

Dahlberg A Buumlltmann H 2013 Fungi Chapter 10 In Mel-tofte H ed Arctic biodiversity assessmentmdashstatus andtrends in arctic biodiversity Akureyri Iceland Conser-vation of Arctic Flora and Fauna p 292ndash309

Das K Verbeken A Nuytinck J 2015 Morphology and phylo-geny of four new Lactarius species from HimalayanIndia Mycotaxon 130105ndash130 doi105248130105

Drummond AJ Ashton B Cheung M Heled J Kearse MMoir R Stones-Havas S Thierer T Wilson A 2009 Gen-eious 47httpwwwgeneiouscom

Durall DM Gamiet S Simard SM Kudrna L Sakakibara SM2006 Effects of clearcut logging and tree species compo-sition on the diversity and community composition ofepigeous fruit bodies formed by ectomycorrhizal fungiCan J Bot 84966ndash980 doi101139b06-045

Elborne SA Knudsen H 1990 Larger fungi associated withBetula pubescens in Greenland Meddelelser GroslashnlandBiosci 3377ndash80

Farris JS Kaumlllersjouml M Kluge AG Bult C 1995 Constructing asignificance test for incongruence Syst Biol 44570ndash572doi101093sysbio444570

Favre J 1955 Les Champignons supeacuterieurs de la zone alpinedu Parc National Suisse Resultats des recherches scien-tifiques enterprises au Parc National Suisse Ergebnisseder wissenschaftlichen Untersuchungen des schweizer-ischen National Parks 51ndash212

Fujiyoshi M Yoshitake S Watanabe K Murota K Tsuchiya YUchida M Nakatsubo T 2011 Successional changes inectomycorrhizal fungi associated with the polar willowSalix polaris in a deglaciated area in the high arctic Sval-bard Polar Biol 34667ndash673 doi101007s00300-010-0922-9

Gardes M Bruns T 1993 ITS primers with enhanced specifi-city for basidiomycetesmdashapplication to the identifica-tion of mycorrhizae and rusts Mol Ecol 2113ndash118doi101111j1365-294X1993tb00005x

BARGE ET AL ROCKY MOUNTAIN ALPINE LACTARIUS 437

Geml J Laursen GA OrsquoNeill K Nusbaum C Taylor DL 2006Beringian origins and cryptic speciation events in the flyagaric (Amanita muscaria) Mol Ecol 15225ndash239doi101111j1365-294X200502799x

mdashmdashmdash mdashmdashmdash Timling I McFarland JM Booth G LennonN Nusbaum C Taylor DL 2009 Molecular phyloge-netic biodiversity assessment of arctic and boreal ecto-mycorrhizal Lactarius Pers (Russulales Basidiomycota)in Alaska based on soil and sporocarp DNA Mol Ecol182213ndash2227 doi101111j1365-294X200904192x

mdashmdashmdash Timling I Robinson C Lennon N Nusbaum HCBrochmann C Noordeloos ME Taylor DL 2012 Anarctic community of symbiotic fungi assembled bylong-distance dispersers phylogenetic diversity of ecto-mycorrhizal basidiomycetes in Svalbard based on soiland sporocarp DNA J Biogeogr 3974ndash88

Gorbunova IA 2010 Macromycetes of alpine area of AltaiTurczaninowia 13125ndash134

mdashmdashmdash Taylakov AA 2011 New species of Russulaceae in themycobiota of south Siberia Turczaninowia 1487ndash90

Gulden G 2005 A preliminary guide to the macromycetes inthe Finse area Hardangervidda Norway Prepared forISAM VII Jul 2005 81 p

mdashmdashmdash Jenssen KM 1988 Arctic and alpine fungi 2 OsloSoppkonsulenten 58 p

mdashmdashmdash mdashmdashmdash Stordal J 1985 Arctic and alpine fungi1 Oslo Soppkonsulenten 62 p

mdashmdashmdash Torkelsen AE 1996 Fungi I Basidiomycota Agari-cales Aphyllophorales Exobasdiales Dacrymycetalesand Tremellales In Elvebakk A Prestrud P eds A cata-log of Svalbard plants fungi algae andcyanobacteria Norsk Polarinstitt Skrifter 198 p173ndash206

Heilmann-Clausen J Verbeken A Vesterholt J 1998 Thegenus Lactarius Denmark Danish Mycological Society287 p

Heinonsalo J Koskiahde I Sen R 2007 Scots pine bait seed-ling performance and root colonizing ectomycorrhizalfungal community dynamics before and during the 4years after forest clear-cut logging Can J For Res37415ndash429 doi101139x06-213

Hesler LR Smith AH 1979 North American species ofLactarius Ann Arbor Michigan University of MichiganPress 841 p

Jamoni PG 1995 Russulaceae della zona alpine Proposta dichiavi di determinazione per le specie crescent nellazona alpine delle Alpi Riv Micol 275ndash80

mdashmdashmdash 2008 Fungi alpini delle zone alpine superiori einferiori Trento Association Micologica BresadolaFondazione Centro Studi Micologici 543 p

mdashmdashmdash Bon M 1991 Note di micologia alpina repperti rari enuovi della zona alpina del Massiccio del Monte Rosa(1a parte) Assoc Micol Bresadola 3255ndash274

Johnson PL Billings WD 1962 The alpine vegetation of theBeartooth Plateau in relation to cryopedogenic pro-cesses and patterns Ecol Monogr 32105ndash135doi1023071942382

Kalamees K 2008 Some agarics in the subalpine and alpinebelts of the Altaj (Russia) and Tyanrsquo-Shanrsquo (Kyrgyzstan)

Mountains Sommerfeltia 31133ndash138 doi102478v10208-011-0007-3

mdashmdashmdash Vaasma M 1993 Mycobiota of alpine and subalpinesites of Kamchatka In Petrini O Laursen GA eds Arc-tic and Alpine Mycology 3ndash4 Berlin J Cramer p121ndash131

Kallio P Kankainen E 1964 Notes on the Macromycetes ofFinnish Lapland and adjacent Finnmark Reports fromthe Kevo Subarctic Research Station 1178ndash235

Karatygin EL Nezdoiminogo EL Novozhilov YK ZhurbenkoMP 1999 Russian arctic fungi Checklist (in Russian) StPetersburg Academy of Chemistry and Pharmacology

Kasuya T 2010 Lycoperdaceae (Agaricales) on the Bear-tooth Plateau Rocky Mountains USA N Am Fungi5159ndash171

Kjoslashller R Clemmensen KE 2009 Belowground ectomycor-rhizal fungal communities respond to liming in threesouthern Swedish coniferous forest stands For EcolManag 2572217ndash2225

Knudsen H Borgen T 1982 Russulaceae in Greenland InLaursen GA Ammirati JF eds Arctic and alpine mycol-ogy 1 Seattle Washington University of WashingtonPress p 216ndash244

mdashmdashmdash mdashmdashmdash 1994 The Lactarius torminosus-group inGreenland Mycol Helvetica 649ndash56

mdashmdashmdash Lamoure D 1993 Notes on Lactarius dryadophilusKuumlhner and L groenlandicus Terkelsen Biblioth Mycol150147ndash154

mdashmdashmdash Mukhin VA 1998 The arctic-alpine agaric elementin the polar Urals and Yamal Western Siberia InMukhin VA Knudsen H eds Arctic and alpine mycol-ogy 5 Russia Yekaterinburg Publications p 152ndash162

mdashmdashmdash Vesterholt J eds 2008 Funga Nordica AgaricoidBoletoid and Cyphelloid genera Copenhagen Nords-vamp 965 p

Kobayasi Y Hiratsuka N Otani Y Tubaki K Udagawa SSugiyama J Konno K 1971 Mycological studies of theAngmagssalik region of Greenland Bull Natl Sci MusTokyo 141ndash96

Krause TR Whitlock C 2013 Climate and vegetation changeduring the late-glacialearly-Holocene transitioninferred from multiple proxy records from BlacktailPond Yellowstone National Park USA Quaternary Res79391ndash402 doi101016jyqres201301005

Kuumlhner R 1975 Agaricales de la zone alpine Lactarius Bulltrimestriel Soc Mycol Fr 915ndash69

mdashmdashmdash Lamoure D 1986 Catalogue des Agaricales (Basidio-mycegravetes) de la zone alpine du Parc Nationale de laVanoise et des reacutegions limitrophes Traveaux Sci ParcNatl Vanoise 15103ndash187

Kytoumlvuori I 2009 Two new Lactarius species L flavopalustrisand L flavoaspideus in Fennoscandia Karstenia4919ndash31

Lamoure D Lange M Petersen PM 1982 Agaricales foundin the Godhavn area west Greenland Nord J Bot285ndash90 doi101111j1756-10511982tb01438x

Lange M 1957 Den Botaniske Ekspedition til Vestgroslashnland1946 Macromycetes III I Greenland Agaricales (pars)Macromycetes caeteri II Ecol Plant Geogr Stud Medde-lelser Groslashnland 1381ndash125

438 MYCOLOGIA

Larsson E Ammirati JF 1982 Lactarii in Alaskan arctic tun-dra In Laursen GA Ammirati JF Redhead SA eds Arc-tic and alpine mycology 1 Seattle WashingtonUniversity of Washington Press p 245ndash281

mdashmdashmdash Vauras J Cripps CL 2014 Inocybe leiocephala aspecies with an intercontinental distribution rangemdashdisentangling the I leiocephala-subbrunnea-catalunicamorphological species complex Karstenia 5415ndash39

Liu YL Whelen S Hall BD 1999 Phylogenetic relationshipsamong ascomycetes evidence from an RNA polymeraseII subunit Mol Biol Evol 161799ndash1808 doi101093oxfordjournalsmolbeva026092

Marshall DC 2010 Cryptic failure of partitioned Bayesianphylogenetic analyses lost in the land of long treesSyst Biol 59108ndash117

Matheny PB 2005 Improving phylogenetic inference ofmushrooms with RPB1 and RPB2 nucleotide sequences(Inocybe Agaricales) Mol Phylogenet Evol 351ndash20doi101016jympev200411014

Methven AS 1997 The Agaricales (gilled fungi) of Califor-nia 10 Russulaceae II Lactarius Eureka CaliforniaMad River Press 79 p

Miller OK Jr Laursen GA 1973 Arctic and alpine agaricsfrom Alaska and Canada Can J Bot 5143ndash49doi101139b73-007

Minckley TA 2014 Postglacial vegetation history of southes-tern Wyoming USA Rocky Mountain Geol 4961ndash74doi102113gsrocky49161

Moser MM McKnight K 1987 Fungi (Agaricales Russulales)from the alpine zone of Yellowstone National Park andBeartooth Mountains with emphasis on Cortinarius InLaursen GA Ammirati JF Redhead SA eds Arctic andalpine mycology 2 New York Plenum Press p 299ndash318

Nezdoiminogo EL 1997 Homobasidiomycetes of naturalreserves of the Russian arctic Mikol Fitopatol 3140ndash43

Nuytinck J Verbeken A Miller SL 2007 Worldwide phylo-geny of Lactarius section Deliciosi inferred from ITS andglyceraldehyde-3-phosphate dehydrogenase genesequences Mycologia 99820ndash832 doi103852mycologia996820

Nylander JAA 2004 MrModeltest 23Distributed by theauthor

mdashmdashmdash Wilgenbusch JC Warren DL Swofford DL 2008AWTY (are we there yet) a system for graphicalexploration of MCMC convergence in Bayesian phyloge-netics Bioinformatics 24581ndash583 doi101093bioinfor-maticsbtm388

mdashmdashmdash 1971 The larger fungi of Svalbard and their ecologyRep Kevo Subarct Res Stat 8122ndash147

Ohenoja E Ohenoja M 1993 Lactarii of the Franklin andKeewatin districts of the Northwest Territories arcticCanada In Petrini O Laursen GA eds Arctic andalpine mycology 3 4 Berlin J Cramer p 179ndash192

mdashmdashmdash mdashmdashmdash 2010 Larger fungi of the Canadian arctic NAm Fungi 585ndash96

Osmundson TW Cripps CL Mueller GM 2005 Morphologi-cal and molecular systematics of Rocky Mountain alpineLaccaria Mycologia 97949ndash972 doi103852mycologia975949

mdashmdashmdash Eyre CA Hayden KM Dhillon J Garbelotto MM2013 Back to basics an evaluation of NaOH and alter-native rapid DNA extraction protocols for DNA barcod-ing genotyping and disease diagnostics from fungal andoomycete samples Mol Ecol Res 1366ndash74 doi1011111755-099812031

Peintner U 2008 Cortinarius alpinus as an example for mor-phological and phylogenetic species concepts in ectomy-corrhizal fungi Sommerfeltia 31161ndash177 doi102478v10208-011-0009-1

Rambaut A 1995 Se-Al sequence alignment editor OxfordUK Oxford University

Ronikier A 2008 Contribution to the biogeography of arctic-alpine fungi first records in the southern Carpathians(Romania) Sommerfeltia 31191ndash211 doi102478v10208-011-0011-7

Ronquist F Huelsenbeck JP 2003 MrBayes 3 Bayesian phylo-genetic inference under mixed models Bioinformatics191572ndash1574 doi101093bioinformaticsbtg180

Schadt CW 2002 Studies on the fungal associations of thealpine sedge Kobresia myosuroides [doctoral dissertation]University of Colorado Boulder Press

Senn-Irlet B 1993 The mycoflora of Alpine mire commu-nities rich in Salix In Petrini O Laursen GA eds Arcticand alpine Mycology 3 4 Berlin J Cramer p 235ndash429

Shapiro B Drummond AJ Rambaut A Wilson MC MatheusPE Sher AV Pybus OG Gilbert MTP Barnes I Binla-den J Willerslev E Hansen AJ Baryshnikov GF BurnsFA Davydov S Driver JC Froese DG Harington CRKeddie G Kosintsev P Kunz ML Martin LD Stephen-son RO Storer J Tedford R Zimov S Cooper A 2004Rise and fall of the Beringian steppe bison Science3061561ndash1565 doi101126science1101074

Silvestro D Michalak I 2012 raxmlGUI a graphical front-end for RAxML Organisms Divers Evol 12335ndash337doi101007s13127-011-0056-0

Stamatakis A 2006 RAxML-VI-HPC maximum likelihood-based phylogenetic analyses with thousands of taxa andmixed models Bioinformatics 222688ndash2690 doi101093bioinformaticsbtl446

Stoumlver BC Muumlller KF 2010 TreeGraph 2 combining andvisualizing evidence from different phylogenetic ana-lyses BMC Bioinform 117

Stubbe D Nuytinck J Verbeken A 2010 Critical assessmentof the Lactarius gerardii species complex (Russulales) Fun-gal Biol 114271ndash283 doi101016jfunbio201001008

mdashmdashmdash Verbeken A 2012 Lactarius subg Plinthogalus theEuropean taxa and American varieties of L lignyotusre-evaluated Mycologia 1041490ndash1501

Stucky B 2012 SeqTrace a graphical tool for rapidly proces-sing DNA sequencing chromatograms J Biomol Tech2390ndash93 doi107171jbt12-2303-004

Swofford DL 2001 PAUP 40b (phylogenetic analysis usingparsimony) Sunderland Massachusetts Sinauer Associ-ates Inc 142 p

Tedersoo L Bahram M Ryberg M Otsing E Kotildeljalg U Abar-enkov K 2014 Global biogeography of the ectomycor-rhizal sebacina lineage (Fungi Sebacinales) as

BARGE ET AL ROCKY MOUNTAIN ALPINE LACTARIUS 439

revealed from comparative phylogenetic analyses MolEcol 234168ndash4183 doi101111mec12849

Terkelsen F 1956 Lactarius groenlandicus sp nov Friesia5417ndash419

Thiers B (continuously updated) Index herbariorum a glo-bal directory of public herbaria and associated staff NewYork Botanical Gardenrsquos Virtual Herbarium httpsweetgumnybgorgih

Timling I Dahlberg A Walker DA Gardes M Charcosset JYWelker JM Taylor DL 2012 Distribution and drivers ofectomycorrhizal fungal communities across the NorthAmerican arctic Ecosphere 31ndash25

mdashmdashmdash Walker DA Nusbaum C Lennon NJ Taylor DL2014 Rich and cold diversity distribution and driversof fungal communities in patterned-ground ecosystemsof the North American arctic Mol Ecol 233258ndash3272doi101111mec12743

Verbeken A Nuytinck J 2013 Not every milkcap is a Lactar-ius Scripta Bot Belgic 51162ndash168

mdashmdashmdash Stubbe D van de Putte K Eberhardt U NuytinckJ 2014 Tales of the unexpected angiocarpousrepresentatives of the Russulaceae in tropical southEast Asia Persoonia 3213ndash24

Vila J Llistosella J Llimona X 1997 Contribucio al coneixe-ment dels fongs de lrsquoestage alpi dels Pirineus de Catalu-nya Rev Catalana Mycol 20221ndash232

WatlingR 1983 Larger cold-climate fungi Sydowia 36308ndash325mdashmdashmdash 1987 Larger arctic-alpine fungi in Scotland In

Laursen G Ammirati JF Redhead SA eds Arctic alpinemycology 2 New York Plenum Press p 17ndash46

mdashmdashmdash 1997 Larger fungi from Greenland Astarte 1061ndash71mdashmdashmdash King R Riddiford N 2001 New and interesting

records of fungi from Shetland Bot J Scot 5357ndash64White TJ Bruns T Lee S Taylor J 1990 Amplification and

direct sequencing of fungal ribosomal RNA genes forphylogenetics In Innis MA Gelfand DH Sninsky JJWhite TJ eds PCR protocols a guide to methods andapplications London Academic Press Inc p 315ndash322

440 MYCOLOGIA