lable at ScienceDirect

Toxicon 114 (2016) 65e74

Contents lists avai

Toxicon

journal homepage: www.elsevier .com/locate/ toxicon

A novel orellanine containing mushroom Cortinarius armillatus

Dahai Shao a, Shusheng Tang b, Rosanne A. Healy c, 1, Paula M. Imerman a,Dwayne E. Schrunk a, Wilson K. Rumbeiha a, *

a Department of Veterinary Diagnostic and Production Animal Medicine, Iowa State University, 50011, Ames, IA, USAb College of Veterinary Medicine, China Agriculture University, 100193, Beijing, Chinac Department of Organismic and Evolutionary Biology, Harvard University, 02138, Cambridge, MA, USA

a r t i c l e i n f o

Article history:Received 4 October 2015Received in revised form5 February 2016Accepted 11 February 2016Available online 23 February 2016

Keywords:ChromatographyMass spectrometryOrellanine

* Corresponding author. Department of VeterinaryAnimal Medicine, Iowa State University, 50011, Ames

E-mail address: rumbeiha@iastate.edu (W.K. Rumb1 Current address: Department of Plant Pathology,

Gainesville, FL, USA.

http://dx.doi.org/10.1016/j.toxicon.2016.02.0100041-0101/© 2016 Elsevier Ltd. All rights reserved.

a b s t r a c t

Orellanine (3,30 ,4,40-tetrahydroxy-2,20-bipyridine-1,10-dioxide) is a tetrahydroxylated di-N-oxidizedbipyridine compound. The toxin, present in certain species of Cortinarius mushrooms, is structurallysimilar to herbicides Paraquat and Diquat. Cortinarius orellanus and Cortinarius rubellus are the majororellanine-containing mushrooms. Cortinarius mushrooms are widely reported in Europe where theyhave caused human poisoning and deaths through accidental ingestion of the poisonous speciesmistaken for the edible ones. In North America, Cortinarius orellanosus mushroom poisoning was recentlyreported to cause renal failure in a Michigan patient. Cortinarius mushroom poisoning is characterized bydelayed acute renal failure, with some cases progressing to end-stage kidney disease. There is debatewhether other Cortinarius mushroom contain orellanine or not, especially in North America. Currently,there are no veterinary diagnostic laboratories in North America with established test methods fordetection and quantitation of orellanine. We have developed two diagnostic test methods based on HPLCand LC-MSMS for identification and quantitation of orellanine in mushrooms. Using these methods, wehave identified Cortinarius armillatus as a novel orellanine-containing mushroom in North America. Themean toxin concentration of 145 ug/g was <1% of that of the more toxic C. rubellus. The HPLC method candetect orellanine at 17 mg g�1 while the LC-MSMS method is almost 2000 times more sensitive and candetect orellanine at 30 ng g�1. Both tests are quantitative, selective and are now available for veterinarydiagnostic applications.

© 2016 Elsevier Ltd. All rights reserved.

1. Introduction

Fruiting bodies in the genus Cortinarius include both edible andtoxic mushrooms. Toxic species are in the subgenus Cortinarius,section Orellani (formerly subgenus Leprocybe, section OrellaniBresinsky and Besl, 1990). Toxic species contain orellanine(3,30,4,40-tetrahydroxy-2,20-bipyridine-1,10-dioxide), a tetrahy-droxylated di-N-oxidized bipyridine molecule, which is a highlypotent nephrotoxin. This toxin is similar in molecular structure toherbicides Paraquat and Diquat, and to the dopaminergic neuro-toxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). ToxicCortinarius mushrooms have been widely reported in Europe.

Diagnostic and Production, IA. USA.eiha).University of Florida, 32611,

Among these, Cortinarius orellanus and Cortinarius rubellus arerecognized as the most frequent causes of human poisoning. Someof these human poisoning cases have resulted in mortality (Denalet al., 2001; Prast et al., 1988). Toxic Cortinarius mushrooms aremistaken for edible species Cortinarius caperatus and Cortinariuspraestans because species in Cortinarius are notoriously difficult tomorphologically identify with certainty (Niskanen et al., 2009,2011; Peintner et al., 2004). They have also been mistaken formore easily distinguished edible species such as chantherelles dueto lack of knowledge and experience on the part of amateur har-vesters (Lincoff, 2011; Phillips, 2006; Waring, 2007). So far, studieshave confirmed C. orellanus (Cantin et al., 1989; Koller et al., 2002;Oubrahim et al., 1997; Richard et al., 1988), C. rubellus (Cantin et al.,1989; Koller et al., 2002; Judge et al., 2010; Oubrahim et al., 1997),Cortinarius orellanosus (Judge et al., 2010), and Cortinarius rain-ierensis (Oubrahim et al., 1997) as some of the species with sig-nificant amounts of orellanine. In the opinion of Robertson et al.(2006). C. rainierensis is a synonym of C. rubellus. Other synonyms

D. Shao et al. / Toxicon 114 (2016) 65e7466

include Cortinarius orellanoides and varieties thereof, Cortinariusspeciosissimus and varieties thereof, Cortinarius speciosus J. Favre,and Dermocybe orellanoides (Kirk, 2016).

In North America, C. rubellus is found in Northern areas of theeast and west coasts, while C. orellanosus has been reported inMichigan. There is one reported case of human poisoning in NorthAmerica involving Cortinarius orellanosus, in which a definitiveidentification of Cortinarius mushrooms was made (Judge et al.,2010). Although there are other cases previously reported inNorth America, it is debatable whether the mushrooms involved inthose cases were actually Cortinarius since neither a definitiveidentification of species, nor a demonstration of presence of orel-lanine was done (Bronstein et al., 2008, 2010; Denal et al., 2001;Judge et al., 2010; Karlson-Stiber and Persson, 2003; Litovitzet al., 1996; Moore et al., 1991; Raff et al., 1992). Themix up is due inpart to misidentifications, a poor understanding of speciesgeographic limits of the fungi, and a failure to conduct confirmatoryanalytical tests for orellanine in suspect mushrooms. Until recently,North America and Europe were thought to share the same speciesof fungi. While it is true that there are some species that are broadlydispersed among continents, molecular phylogenetic analyses havedemonstrated that some fungi, particularly ectomycorrhizal fungilike Cortinarius, are endemic (Bonito et al., 2013; Matheny et al.,2009; O'Donnell et al., 2011).

Lack of awareness of species complexes have also contributed toerroneous identifications. Outdated mushroom field guides use asingle name to erroneously label multiple species, and yet thesespecies may not share the same genetic, chemical or ecologicalproperties (Harrington and Wingfield, 1995; Jaklitsch et al., 2013;Zhang et al., 2015). Mycologists are now more aware of these po-tential problems, and as a result, many groups of mushrooms arecurrently undergoing revision, with new species being discovered(Folz et al., 2013; Justo et al., 2014; Liimatainen et al., 2014).Therefore, it is important not to assume that toxins or lack thereofin European species are similarly present or absent in NorthAmerican species, even if they appear morphologically similar.Moreover, because some Cortinarius mushrooms are edible, it isadvisable to conduct additional studies to identify other Cortinariusspecies that may contain the toxin. Lastly, failure to test suspectmushrooms to confirm presence of the orellanine toxin has alsocontributed to erroneously attributing intoxication to orellanine.Chemical confirmation of the presence of orellanine is a better wayof diagnosing intoxication and adds value to morphologicalidentification.

Mushroom-induced toxicity is also commonly encountered inpets, particularly dogs that eat raw mushrooms in yards. The mostwidely recognized mushroom-induced toxicity in pets is theamatoxin-induced hepatotoxicity (Puschner et al., 2007; Puschnerand Wegenast, 2012). There are no reports of Cortinarius mush-room poisoning reported in pets. However, because these mush-rooms are present in North America and poisoning has beenreported in humans, we suspect that animal poisoning by thesemushrooms occurs. A lack of awareness among veterinarians aboutthe toxicity of these mushrooms and coupled with a lack ofconfirmatory diagnostic tests for orellanine may limit diagnosis ofpoisoning by these mushrooms. Until now, there was no veterinarydiagnostic laboratories set up for detection and quantitation oforellanine in mushrooms for confirmation of orellanine poisoningin people and pets in North America.

There were two objectives for this study: 1) to develop selective,sensitive and quantitative in-house diagnostic test methods fordetection and quantitation of orellanine in mushrooms. Thesemethods will be used for definitive confirmation of the presence oforellanine for quick unequivocal diagnosis of orellanine poisoningin animals. The test can also be used for research purposes; and 2)

to test Cortinarius mushroom specimens collected from NorthAmerica, for presence of orellanine. Confirmation of mushroomspecies was done by genetic methods.

2. Materials and methods

2.1. Chemicals, reagents, and materials

High performance liquid chromatography (HPLC)-grade Meth-anol, HPLC-grade acetonitrile, concentrated hydrochloric acid,ammonium hydroxide, ammonium acetate, o-phosphoric acid(85%), formic acid, and 0.45 mm nylon syringe filters were pur-chased from Thermo Fisher Scientific (Waltham, MA). Orellaninestandard (95%) was purchased from Ramidus AB (Lund, Scania.Sweden). All aqueous solutions were prepared in 18.2 MU $cm de-ionized (DI) water (Aries High Purity Water System, Aries FilterNetwork, West Berlin, NJ). Bond Elut Jr Mycotoxin clean-up col-umns were purchased from Agilent Technologies (Santa Clara, CA).A solution of 3M hydrochloric acid was prepared by adding 24.9 mLconcentrated hydrochloric acid into DI water and bringing thevolume to 100 mL by water. A solution of 3 M hydrochloric acid inmethanol was prepared by adding 24.9 mL concentrated hydro-chloric acid into HPLC-grade methanol and bringing the volume to100 mL by methanol. A 4 mM ammonium acetate aqueous solutionwas prepared by dissolving 0.308 g ammonium acetate in DI waterand bringing the volume to 1 L. A 1% formic acid aqueous solutionwas prepared by diluting 5 mL formic acid to a total volume of500 mL with DI water. Orellanine standards were prepared bydissolving a corresponding amount of orellanine pure standard in3M hydrochloric acid. They were stored in aluminum-foil-wrappedvials for protection from photo-decomposition.

2.2. Cortinarius mushrooms analyzed for orellanine

All mushrooms studied are from the genus Cortinarius. Corti-narius distans, C.alboviolaceus, C. glaucopus, C. cinnabarinus, and C.castaneus were provided by the Ada Hayden Herbarium (ISC) ofIowa State University (Ames, IA). Cortinarius alboviolaceus, C. cor-rugatus, C. coalescens, and C. armillatus were collected in HarvardForest, Petersham, Massachusetts and were accessioned at theFarlow Herbarium (FH) of the Harvard University Herbaria (Cam-bridge, MA). Cortinarius rubellus, a positive control specimenknown to contain significant quantities of orellanine, was a giftfrom Dr. Otto Miettinen, Finnish Museum of Natural History e

LUOMUS Botanical Museum, University of Helsinki (Helsinki,Finland). (Accession number: H 6835:3623, H 6694-310, H 6693-310, H 6869 446, H 6689 398, H 6705 352, and H 6900 335.) Anedible mushroom, known to be free of orellanine, Shiitake (Lenti-nula edodes), was purchased from a local grocery store in Ames, IAand served as a negative control sample. The photographs of theabove-mentioned mushrooms are shown in Figs. 1 and 2. Allvoucher specimens used in this study are listed along with theircollector numbers, date of collection, and collection sites in Table 1,except for C. rubellus for which collection site information was notavailable.

2.3. Sample preparation

The sample preparation procedure was adapted from a pub-lished method (Herrmann et al., 2012). Two major changes weremade. A clean-up step was added to further remove the in-terferences from orellanine. This cleanup step gave good selectivityfor orellanine. Because the pH of mobile phases was essential instabilizing orellanine in the HPLC analysis, we replaced formic acidwith o-phosphoric acid because the latter is less volatile and able to

Fig. 1. A. Cortinarius armillatus RH1535; B. Cortinarius armillatus RH1557; C. Cortinarius armillatus RH1557; D. Cortinarius cf distans (recorded as C. brunneus, ISC 437745); E. Cor-tinarius cinnabarinus (ISC 443350); F. Cortinarius rubellus drawing by Belinda Y Mahama. All bars ¼ 2 cm.

D. Shao et al. / Toxicon 114 (2016) 65e74 67

maintain the pH for an extended period of time.Briefly, a 15 mg mushroom sample was mixed with 4 mL 3 M

hydrochloric acid and then extracted for 3 h with magnetic stirring.The mixture was then centrifuged at 3000 rpm for 30 min(1744 � g). The pH of the supernatant was neutralized with 1 mLammonium hydroxide and then added onto clean-up columns.After washing the columns with 20 mL DI water and 20 mLmethanol (both adjusted to pH 1.5 with formic acid), the orellaninewas eluted with 5 mL 3 M hydrochloric acid in methanol at a flowrate of 10e20 mL/min. The eluent was concentrated to 0.2 mL bynitrogen gas.

The negative control mushroom, Shiitake (L. edodes) was forti-fied with orellanine at a level of 50 mg mL�1. The extracts wereprepared in the same way as described above and subjected toanalysis by HPLC or liquid chromatography-tandem mass spec-trometry (LC-MSMS). This was the approach used for determina-tion of spike recovery. The orellanine-positive control mushroom,C. rubellus was also analyzed as a reference. All mushroom extractswere quantified by the HPLC method and those that tested positivefor orellanine were subject to LC-MSMS confirmation. In all testprocedures, a positive control C. rubellus extract and a negative

control, and a negative control orellanine-spiked mushroom sam-ple were included. A calibration curve was established with 0 mg,0.25 mg, 0.50 mg, 1.0 mg, 1.5 mg, 2.0 mg, and 2.5 mg of orellanine insolvent for HPLC analyses. Because the LC-MSMSmethod was moresensitive than the HPLC method, a calibration curve with 0 mg,0.1 mg, and 0.2 mg of orellanine in solvent was used for LC-MSMSanalyses. These extracts were used for both HPLC and LC-MSMSanalyses. In order to protect orellanine from photo-decomposition, orellanine-containing solutions were kept inaluminum-foil-wrapped tubes throughout the procedure.

2.4. HPLC conditions

Measurements were performed with an Ultimate 3000 HPLCsystem (Dionex, now Thermo Fisher Scientific, Waltham, MA)equippedwith a Chromeleon program for the systemmanipulation,data acquisition and analysis, a photodiode array UVeVis detector,and an HPLC column (PLRP-S C18, 3 mm,150 mm � 4.6 mm, AgilentTechnologies, Santa Clara, CA). The mobile phase consisting of4 mM ammonium acetate aqueous solution (A) and methanol (B)(Herrmann et al., 2012) was pumped at a flow rate of 0.3 mL min�1.

Fig. 2. AeB Cortinarius corrugatus FH RH1522; C-D. Cortinarius cf alboviolaceus FH RH1546; E-F Cortinarius cf coalescens FH RH1552; All bars ¼ 2 cm.

Table 1Voucher information for Cortinarius tested for orellanine in this study.

Species Herbarium Accession no. Collector no. Locality Date

armillatus FH RH1535 RH1535 USA, MA: Harvard Forest 5-Sep-13armillatus FH RH1577 RH1577 USA, MA: Harvard Forest 6-Sep-13cf coalescens FH RH1552 RH1552 USA, MA: Harvard Forest 5-Sep-13corrugatus FH RH1522 RH1522 USA, MA: Harvard Forest 5-Sep-13corrugatus FH RH1545 RH1545 USA, MA: Harvard Forest 5-Sep-13cf alboviolaceus FH RH1546 RH1546 USA, MA: Harvard Forest 5-Sep-13cf alboviolaceus FH RH1574 RH1574 USA, MA: Harvard Forest 6-Sep-13cf distans ISC 437745 RH and LHT sn USA, IA: Mann Wilderness Area State Preserve 13-Jul-04alboviolaceus, ISC 437743 RH sn USA, IA: Mann Wilderness Area State Preserve 18-Aug-04glaucopus ISC 438732 HD Thiers 53,156 USA, CA: Lincoln Creek Campground, Sierra County 5-June-90cinnabarinus ISC 443350 RH66 USA, IA: Woodman Hollow State Preserve 30-Aug-06castaneus ISC 438439 RH sn USA, IA: Fallen Rock State Preserve 6-Nov-04rubellus H 6835:3623 2009rubellus H 6694-310 2000rubellus H 6693-310 2000rubellus H 6869 446 1996rubellus H 6689 398 2000rubellus H 6705 352 3-Oct-03rubellus H 6900 335 1997

D. Shao et al. / Toxicon 114 (2016) 65e7468

Table 3A summary of LC-MSMS specifications for orellanine analysis.

Q1, m/z Q3, m/z Capillary voltage, V Collision voltage, V Dwell time, ms

253 163 36 20 0.5253 191 36 25.5 0.5253 219 36 20 0.5253 236 36 14 0.5

D. Shao et al. / Toxicon 114 (2016) 65e74 69

Both mobile phases were adjusted to pH 1.5 with o-phosphoricacid. A gradient elution was used to give an optimum separation.The details of the gradient protocol were tabulated in Table 2. Atotal running time of 20 min was used. The injection volume was50 mL. The separation was performed at room temperature. Orel-lanine was detected spectroscopically at the wavelength 295 nm inthe UVeVis detector.

2.5. LC-MSMS conditions

Analyses were performed using a triple-quadruple Varian 310LC-MSMS system (Agilent Technologies, Santa Clara, CA) with anelectrospray ionization (ESI) chamber in the positive mode, a 410Varian Prostar autosampler (Agilent Technologies, Santa Clara, CA),and two 210 Varian Prostar HPLC pumps (Agilent Technologies,Santa Clara, CA). Needle voltage was�3000 V, CID gas pressurewas0.20 Pa, drying gas temperature was 150 �C, nebulizer gas pressurewas 3.45 � 105 Pa, drying gas pressure was 6.89 � 104 Pa, detectorwas biased at 1500 V, and acquisition time was 10 min. The mobilephase consisted of solvent A: 1% formic acid, and solvent B:acetonitrile, at an isocratic elution with a ratio of 5:95, v:v. Themobile phasewasmodified from a study (Herrmann et al., 2012) foruse with PRP-1 column (Hamilton PRP-1, 10 mm, 250 � 4.1 mm I.D.purchased from Phenomenex, Torrance, CA). The flow rate was0.4 mL/min. This also was modified to speed up the analysis.Orellanine was detected using the following parent to daughter iontransitions: 253 > 163, 253 > 191, 253 > 219, 253 > 236 which wereobtained by direct infusion of orellanine. A detailed summary of LC-MSMS parameters is listed in Table 3. Samples were diluted 1:10 in90:10, v:v, methanol: 3MHCl, and 20 ml was injected on the column(Herrmann et al., 2012).

2.6. Genetic confirmation of Cortinarius armillatus

Cortinarius armillatusmushrooms (Fig. 1 AeB) were collected onSept 6e7, 2013 in Harvard Forest, in Petersham, located in centralMassachusetts. This was part of a study of ectomycorrhizal(mutually beneficial root symbionts) fungi in the Harvard Forest.Collections used in this study included RH1577 and RH1593, whichcame from the Prospect Hill tract, and RH1535, which came fromthe Tom's Swamp tract. These tracts were moist, well drained siteswith acidic soil (pH 3.6) supporting a mixed forest dominated by acanopy of red oak, paper birch, white pine, red maple, and easternhemlock with an understory of American beech. All Cortinariusbasidiomata were collected under red oak tree canopies, but C.armillatus was sequenced from birch root tips. Spores were exam-ined and measured in a water mount, and color change noted withMelzer's reagent (iodine solution). Images of C. armillatus used inthis study are shown in Fig. 1 A and B.

For a definitive identification of the mushrooms, the ITS locus,which is the barcoding region for fungi was amplified andsequenced. The ITS region of DNA is the official barcode locus forfungi (Schoch et al., 2009), and is a good indicator of speciesidentity for most fungi, including Cortinarius (Niskanen et al., 2009,2011). For isolation of genomic DNA, a 3 mm2 piece of clean gillwith spores was excised from each specimen using sterile

Table 2A summary of HPLC gradient for orellanine analysis.

Time, min 4 mM ammonium acetate aqu

0e2.0 982.0e4.0 154.0e20.0 15

technique, and each placed into 250 ml of 2x CTAB where it wasstored at 24 �C until extraction. Samples were pulverized to a finegrain in CTAB, and 250 ml additional 2x CTAB added, mixed well,and incubated at 60 �C for 1 h. DNA was extracted with an equalvolume of 24:1 chloroform:isoamyl alcohol followed by centrifu-gation at 13,000 rpm for 12min. Samples were incubatedwith 50 mlRNAase A at 37 �C for 30 min, followed by another extraction andcentrifugation as before. A 2/3 volume of isopropanol was added toeach extract, chilled at �20 �C overnight, then centrifuged at13,000 rpm for 7 min. Supernatant was removed and the pellet waswashed with chilled 70% ethanol followed by centrifugation at13,000 rpm for 5min, then ethanol pipetted off, and the pellet driedin a laminar flow hood for 1 h. DNA was reconstituted in 50 ml TEbuffer, and an aliquot diluted to 10x. The internal transcribed spacer(ITS) and 50 end of the 28S (LSU) regions of nuclear ribosomal DNAwere amplified using primer pairs ITS5/LR5 (Bertini et al., 1999;Vilgalys and Hester, 1990) with the following PCR cyclingschedule: 1) 94 �C for 2 min 2) 94 �C for 1 min 3) 50 �C for 45s 4)72 �C for 1.5 min 5) repeat steps 2e4 35 times 6) final elongation at72 �C for 5min. Sanger sequencingwas donewith the same primersat Beckman Coulter Genomics Facility. Sequences were viewed andtrimmed to include only high quality bases in Geneious Pro v 5.6.7(Drummond et al., 2012). Sequences were hand aligned with highlysimilar sequences in SeAl v 2.0a11 (Rambaut, 2007). MaximumLikelihood analysis was performed on the final alignment usingRAxML (Stamatakis, 2014) with a GTR þ gamma nucleotide sub-stitution model and 1000 bootstrap iterations on the Cipres Portal(Miller et al., 2010). Sequences have been deposited in GenBankunder the numbers KT750146-KT750155.

3. Results

3.1. HPLC

HPLC analysis was performed on all species of mushrooms listedin Section 2.2. Results show that of all the mushrooms analyzed,only C. armillatus and the well-known orellanine-containing spe-cies, C. rubellus, tested positive for orellanine. Since this is the firsttime orellanine was found in C. armillatus, this mushroom wassubsequently re-extracted and tested on a separate day for confir-mation of the presence and quantification of orellanine.

In Fig. 3, Chromatographs of orellanine are shown for (a) fromtop to bottom line, C. armillatus, negative control mushroom (L.edodes), and orellanine standard solution and (b) from top to bot-tom line, C. rubellus (positive control), negative control mushroom(L. edodes), and orellanine standard solution.

A summary of orellanine results for C. armillatus and C. rubellus

eous solution (A), % Methanol (B), %

28585

Fig. 3. Chromatographs of orellanine in: (a) Test mushroom C. armillatus, negative control mushroom (Lentinula edodes), and orellanine standard solution; (b) Positive control C.rubellus, negative control mushroom (Lentinula edodes), and orellanine standard solution.

D. Shao et al. / Toxicon 114 (2016) 65e7470

are shown in Table 4. The spike recovery results are shown as well.The chromatographs of these samples as well as the ones fornegative control Shiitake mushrooms (L. edodes) are shown inFig. 3. Calibration curves showed a good linearity of 0.996 and 0.999for the ranges of 0.25e2.5 mg and 1e10 mg respectively for cali-bration curves established when testing orellanine in C. armillatus.The dry-weight orellanine concentration was 153 mg g�1 and

Table 4A summary of HPLC analyses of orellanine in C. armillatus, C. rubellus, and a spiked cont

Mushroom species Sample Calibration range of orellanine, mg Linearity

C. armillatus 1 0.25e2.5 0.9962 1e10 0.999

C. rubellus 1 0.25e2.5 0.997

Negative control (L. edodes) 1 0.25e2.5 0.997

137 mg g�1 for the two separate analyses, respectively. The meanconcentration from the two analyses was 145 mg g�1. The calibra-tion curve for testing orellanine in C. rubellus gave a linearity of0.997 (R2) in the range of 0.25e2.5 mg, indicating reliable calibra-tion. The concentration of orellanine in C. rubellus was24,000 mg g�1 [2.4% (w/w)], two orders of magnitude higher thanthat in C. armillatus, consistent with the fact that it is a potent

rol sample.

(R2) Concentration, mg g�1 Average concentration, mg g�1 Spike recovery, %

153 145 e

1372.4 � 104

(2.4% (w/w))2.4 � 104

(2.4% (w/w))e

e e 78.3

D. Shao et al. / Toxicon 114 (2016) 65e74 71

mushroom. The spike recovery of the negative control specimenwas 78.3%. The lowest quantitation standard was 0.25 mg, equiva-lent to 17 mg g�1 orellanine based on this method. All othermushrooms tested negative for orellanine at the lowest quantifi-able standard of 17 mg g�1 of orellanine.

3.2. LC-MSMS

The chromatographs of orellanine in negative control Shittake(L. edodes) mushrooms, spiked L. edodes mushrooms, C. armillatusmushrooms, and C. rubellus mushrooms by LC-MSMS are shown inFig. 4. The slightly off retention time of the orellanine peak in theC. rubellusmushroommay be due to vast difference in the amountsof toxin being chromatographed. LC-MSMS analyses for orellaninein C. armillatus showed a concentration of 130 mg g�1. The differencebetween this and the HPLC results was only 10%. By HPLC, the meanorellanine concentration was 145 mg g�1. The concentration fororellanine in C. rubellus was 26,000 mg g�1[2.6% (w/w)]. This wasalso comparable to HPLC results, with only 7% difference notedbetween the two results. The spike recovery was 85.0% and verycomparable to that of HPLC (78.3%). A linearity with a good corre-lation coefficient of 0.989 (R2) was obtained in the range of0.1 mge0.2 mg for calibration curves. A summary of these results isshown in Table 5. Results of LC-MSMS provide definitive confir-mation of the identity of the toxin in C. armillatus as orellanine.

3.3. Genetic identification of Cortinarius armillatus

It was essential to have definitive genetic identification ofC. armillatus. The post trimmed ITS sequences for RH1535 (345 bp,Genbank accession KT750146) and RH1577 (346 bp, Genbankaccession KT750150) included ITS1 and 5.8s. The sequence forRH1593 (141 bp) was too short and therefore not included in

Fig. 4. Chromatographs of orellanine in (a) negative control Shiitake (Lentinula edodes) muscontrol) by LC-MSMS analysis.

further analyses. RH1535 and RH1577 sequences were highlysimilar to the sequence from the neotype specimen for C. armillatus(DQ114744) with one bp difference. Since we did not sequence theITS2 region, the length was 334 bp rather than the full 605 bp, sothere could be more differences. However, the nearly perfect matchof the 346 nucleotides in the ITS1 and 5.8S region helps tomolecularly identify this taxon as C. armillatus. Phylogenetic anal-ysis of our ITS sequences in an alignment with highly similar se-quences downloaded from a BLAST search against the nucleotidedatabase in GenBank, and other species delimited in the armillatisection of Cortinarius (Niskanen et al., 2011) inferred our sequencesas unambiguously consistent with that of the neotype specimensequence for C. armillatus (Supplementary Fig. 1). The 28s locus wassequenced for RH1577 (770 bp) and RH1535 (819 bp). This locus iswell conserved in Cortinarius, and though the 28S sequences of ourspecimens were a perfect match to C. armillatus, they were also 99%similar to Cortinarius luteo-ornatus and Cortinarius pinigaudis.

Morphologically, these specimens fit the description forC. armillatus. Spore size was 9.6-10-10.4 � 6e6.4 mm, they weredextrinoid in Melzer's reagent, and the spore wall was relativelythick, ~0.4 mm. Color of annulus was initially whitish (Fig. 1 A) butbecame rust-colored due to spore deposit.

Both morphology and molecular analyses confirmed the iden-tity of the Cortinarius samples used in this study as C. armillatus. Itappears that this species has a widespread distribution, beingfound in both the North Eastern US and in Europe, where it isknown to associate with birch trees (Niskanen et al., 2011). Wesequenced C. armillatus from birch roots at the site of the collec-tions of fruit bodies in Harvard Forest.

4. Discussion

Orellanine-containingmushrooms are present in North America

hrooms, (b) spiked L. edodes mushrooms, (c) C. armillatus, and (d) C. rubellus (positive

Table 5A summary of LC-MSMS analyses of orellanine in C. armillatus, C. rubellus, and a spiked control sample.

Mushroom species Calibration range of orellanine, mg Linearity (R2) Concentration, mg g�1 Spike recovery, %

C. armillatus 0.1e0.2 0.989 130 e

C. rubellus 0.1e0.2 0.989 2.6 � 104

(2.6% (w/w))e

Negative control (L. edodes) 0.1e0.2 0.989 e 85.0

D. Shao et al. / Toxicon 114 (2016) 65e7472

and Europe. Some of these mushrooms are edible, while othersespecially those belonging to the subgenus Cortinarius sectionOrellani are toxic (Bresinsky and Besl, 1990; as subgenus Leprocybe).In North America, C. rubellus is found in Northern areas of the eastand west coasts, while C. orellanosus has been reported in Michigan.These toxic Cortinarius mushroom species contain orellanine, abipyridyl compound similar to herbicides Paraquat and Diquat.Both people and animals are susceptible to orellanine, a toxin withpotentially multiple toxic outcomes, of which nephrotoxicity is themost widely recognized. All previously known orellanine-containing mushroom species belonging to the subgenus Corti-narius section Orellani are toxic. Toxicosis is notably characterizedby a gradually developing acute renal failure over a period of daysto up to 2 weeks following a onetime toxic exposure.

Orellanine poisoning is well recognized in Europe (Denal et al.,2001; Prast et al., 1988; Schumacher and Høiland, 1983). There isone reported case of human poisoning in North America involvingC. orellasosus, in which a definitive identification of Cortinariusmushrooms was made (Judge et al., 2010). Since neither measure-ment of orellanine in mushrooms nor definitive genetic identifi-cation of mushrooms were done, there is doubt that otherpreviously reported cases of orellanine poisoning in North Americawere actually caused by Cortinarius mushrooms (Bronstein et al.,2008, 2010; Denal et al., 2001; Judge et al., 2010; Karlson-Stiberand Persson, 2003; Litovitz et al., 1996; Moore et al., 1991; Raffet al., 1992). Lack of awareness of species complexes and use ofoutdated mushroom field guides have also contributed to erro-neous identifications. Outdated mushroom field guides use a singlename to erroneously label multiple species, and yet these speciesmay not share the same chemical or ecological properties(Harrington and Wingfield, 1995; Jaklitsch et al., 2013; Zhang et al.,2015). Mycologists are now more aware of these potential prob-lems, and as a result, many groups of mushrooms are currentlyundergoing revision, with new species being discovered (Folz et al.,2013; Justo et al., 2014; Liimatainen et al., 2014). Establishment ofselective, sensitive analytical tests for testing mushrooms as thosedescribed here will add value by leading to accurate diagnosis ofCortinarius mushroom poisoning.

Although our taxon is unambiguously C. armillatus as delimitedby Niskanen et al. (2011), it is possible that there are variationswithin populations of this species that could be parsed out usingmethods of genotyping more refined than the ITS barcoding region.In this regards, it would be of interest to test the orellanine contentof European C. armillatus to compare with the North AmericanC. armillatus, as well as testing other species in the armillati section.Considering that some of the Cortinarius mushrooms are edible, itmay be of interest to assess the potential toxicity of other Corti-narius mushrooms as well. Assessing the entire genus of an esti-mated 2000 species is not necessary, however. It is known thatorellanine-containing tissue will fluoresce under UV light, sochoosing species that fluoresce would filter out the species that donot have the toxin. Fluorescence alone, however, does not defini-tively identify orellanine (Keller-Dilitz et al., 1985).

Besides being hazardous to people, toxic Cortinariusmushroomsare also a potential danger to pets, especially dogs which are knownto have indiscriminate eating habits. Toxic Cortinarius mushrooms

are also a danger to other animal species which can consumemushrooms. No single diagnosis of Cortinarius mushroompoisoning in animals including pets has ever been reported inNorth America, yet toxic Cortinarius mushrooms includingC. rubellus and C. orellanosus are reported to exist in North America.C. rubellus is found in northern areas of both the east and westcoasts. C. orellanosus has only recently been identified reported inMichigan but its entire geographic distribution is currently un-known. Veterinarians generally are not aware of dangers posed bythese mushrooms. It is possible that a lack of awareness amongveterinarians, coupled with a lack of suitable diagnostic tests mayaccount for a lack of recognition of Cortinariusmushroom poisoningin animals in North America. Cortinarius mushroom intoxicationshould be considered as part of the differentials of causes of acuterenal failure in pets. Cortinarius mushroom poisoning in small ru-minants was reported in Norway (Overås et al., 1979). In that case 4sheep died after grazing pastures containing Cortinarius mush-rooms. The diseasewas experimentally reproduced confirming thatCortinarius mushrooms were responsible for the deaths (Overåset al., 1979).

In this collaborative study, we developed highly sensitive andspecific analytical test methods for detection and quantitation oforellanine in mushrooms. We used these analytical test methods toanalyze several Cortinarius mushroom species collected from theEast Coast to the Midwest USA. Our results indicated that amongseveral North American Cortinarius mushrooms analyzed forpresence of orellanine, only C. armillatus was found to containorellanine. This is an important finding. Danel et al. indicated that itis impossible to establish an extensive list of toxic Cortinariusmushroom species and recommended avoiding all Cortinariusspecies (Denal et al., 2001). This study suggests that most of themushrooms studied did not contain orellanine. Although the con-centration of orellanine in C. armillatuswas low compared to that ofC. rubellus, more work is needed to determine the concentrationrange of this toxin in this mushroom species. The content of naturaltoxins in plants and mushrooms varies depending on environ-mental conditions. It is possible that the concentration of orellaninein C. armillatus is variable due to genetic differences, but may alsobe affected by environmental factors such as soils, and weather.Thus more studies are needed to establish the concentration oforellanine in C. armillatus. It is important that clinicians are awarethat C. armillatus contains orellanine because there are many fac-tors which influence a toxic outcome. Whereas ingesting lowamounts of orellanine may not cause renal failure in young in-dividuals or animals at the prime of their health, low amounts oforellanine in C. armillatus may push patients with pre-existingrenal disease over the edge to cause renal failure. Besides, mush-room hunters should be aware of this information so that theymake personal decisions whether or not to consume thesemushrooms.

The other significance of this study is that the analytical testmethods for orellanine we have established in our laboratory canbe extended and used for diagnostic purposes. Toxic-induced acuterenal failure is reported in pets and there are multiple causes(Rumbeiha, 2000). There are no pathognomonic signs for orellaninepoisoning. Orellanine poisoning causes a severe renal disease.

D. Shao et al. / Toxicon 114 (2016) 65e74 73

Testing suspect mushrooms for orellanine as early as possiblebefore clinical signs set in gives patients the best chances of survivaland also minimizes cost of treatment. This test is now available foranalyzing suspect ingested mushrooms to determine if theycontain orellanine. Orellanine causes irreversible renal failure andso it is important that an early diagnosis is made. The test methodswe have developed will be helpful in confirming or ruling outorellanine poisoning, among other toxin-induced causes of acuterenal failure. Both HPLC and LC-MSMS analytical tests are availableand can be used to detect and quantify orellanine in mushrooms.Both methods are specific for orellanine. Taking into account thevariations between instrument and detection techniques, the twomethods are complimentary for assessment of orellanine inmushrooms.

As expected, the LC-MSMS method is more sensitive than theHPLC method. The LOQ for the two methods was 30 ng g�1 and17 mg g�1 respectively. For routine diagnosis, we recommend theHPLC method first and the LC-MSMS for confirmation. The LC-MSMS is specific for orellanine because this method looks at thespecific product ions from the parent orellanine ions and is rec-ommended as a confirmatory method. Other methods, includingthin layer chromatography, have limitations of causing false posi-tives, lack of sensitivity, and are difficult to perform (Danel, 2001).Ours is the only veterinary diagnostic laboratory which has estab-lished a diagnostic test for orellanine in the US. This method can beutilized both for veterinary diagnostic purposes and for research totest for presence of orellanine in suspect mushrooms. For best re-sults we recommend that suspect mushrooms be submitted to thelab in paper bags, shipped overnight to prevent decomposition ofspecimens.

In conclusion, we have established two tests for detection andquantitation of orellanine in mushrooms using HPLC and LC-MSMSmethods. Using these tests, we have identified C. armillatus as anovel orellanine containing mushroom. The concentration of orel-lanine was low compared to that found in the more potent mush-rooms such as C. rubellus. However, more research is recommendedon C. armillatus to determine the range of toxin concentration.Although the orellanine concentration in C. armillatus is low andunlikely to poison young healthy individuals or animals at theprime of their health, it is likely to be toxicologically relevant inpatients with predisposing conditions such as pre-existing renaldiseases. This knowledge is also important because our knowledgeof orellanine toxicity in animals is still rudimentary and the mini-mum toxic dose of this toxin is not currently known for all animalsunder care of veterinarians. Other Cortinarius mushroom speciesanalyzed in this study including C. distans, C. alboviolaceus, C.glaucopus, C. cinnabarinus, C. castaneus, C. alboviolaceus, C. corru-gatus, and C. coalescens, tested negative for orellanine. Becausethere is limited research done on Cortinarius mushroom poisoningin North America more research is recommended, particularly ondifferent geographic isolates of C. armillatus and other species insection armillati, to definitively identify orellanine-containinghazardous species. Also, because of the lack of data on thetoxicity of orellanine in pets, livestock andwildlife, more research isrecommended in this area as well. Finally, the analytical tests wehave established in our laboratory are available to assist veterinaryand medical practitioners confirm cases of intoxications caused byorellanine containing mushrooms and also for research purposes.

Conflicting interest

The authors declare that there are no conflicts of interest.

Ethical statements

This study did not involve any human or animal studies. Themushroom species studied were obtained frommuseums or from aforest where permission for collecting was granted to RH.

Acknowledgments

The authors thank an anonymous reviewer for helpful com-ments that improved this manuscript. The authors are grateful toDeborah A. Lewis of Ada Hayden Herbarium, Iowa State Universityfor her generous assistance in providing mushroom samples.Healy's work was funded through a Sargent Award from the Har-vard Arnold Arboretum with matching support from Harvard For-est. This work was also partially funded by Iowa State UniversityStartup funds toWilson K Rumbeiha. The authors would also like toacknowledge Dr. Otto Miettinen of Finnish Museum of NaturalHistory, Botany Unit, University of Helsinki for providing C. rubellusmushroom specimen. They also express their gratitude to Dr. TobiasGuldberg Frøslev of Natural History Museum of Denmark and Dr.Matthew E. Smith of the University of Florida for enlighteningdiscussion about identification and classification of mushroomspecies.

Transparency document

Transparency document related to this article can be foundonline at http://dx.doi.org/10.1016/j.toxicon.2016.02.010

Appendix A. Supplementary data

Supplementary data related to this article can be found at http://dx.doi.org/10.1016/j.toxicon.2016.02.010

References

Bertini, L., Amicucci, A., Agostini, D., Polidori, E., Guidi, C., Stocci, V., 1999. A new pairof primers designed for amplification of the ITS region in Tuber species. FEMSMicrobiol. Lett. 173, 239e245.

Bonito, G., Smith, M.E., Nowak, M., Healy, R.A., Guevara, G., C�azares, E., Kinoshita, A.,Nouhra, E.R., Domínguez, L.S., Tedersoo, L., Murat, C., Wang, Y., Moreno, B.A.,Pfister, D.H., Nara, K., Zambonelli, A., Trappe, J.M., Vilgalys, R., 2013. Historicalbiogeography and diversification of truffles in the Tuberaceae and their newlyidentified southern hemisphere sister lineage. PLOS One 8, 1e15 e52765.

Bresinsky, A., Besl, H., 1990. A Colour Atlas of Poisonous Fungi: a Handbook forPharmacists, Doctors, and Biologists, first ed. Wolfe Publishing Ltd., London,pp. 50e61.

Bronstein, A.C., Spyker, D.A., Cantilena Jr., L.R., Greenn, J.L., Rumack, B.H., Heard, S.E.,2008. 2007 annual report of the american association of poison control centers'national poison data system (NPDS): 25th annual report. Clin. Toxicol. (Phila) 46(10), 927e1057.

Bronstein, A.C., Spyker, D.A., Cantilena Jr., L.R., Greenn, J.L., Rumack, B.H., Giffin, S.L.,2010. 2009 annual report of the american association of poison control centers'national poison data system (NPDS): 27th annual report. Clin. Toxicol. (Phila) 47(10), 911e1084.

Cantin, D., Richard, J.-M., Alary, J., 1989. Chromatographic behaviour and determi-nation of orellanine, a toxin from the mushroom Cortinarius orellanus.J. Chromatogr. 478, 231e237.

Denal, V.C., Saviuc, P.F., Garon, D., 2001. Main features of Cortinarius spp. poisoning:a literature review. Toxicon 39 (7), 1053e1060.

Drummond, A.J., Ashton, B., Buxton, S., Cheung, M., Cooper, A., Duran, C., Heled, J.,Kearse, M., Markowitz, S., Moir, R., Stones-Havas, S., Sturrock, S., Swidan, F.,Thierer, T., Wilson, A., 2012. Geneious v5.6. http://www.geneious.com/.

Folz, M.J., Perez, K.E., Volk, T.J., 2013. Molecular phylogeny and morphology revealthree new species of Cantharellus within 20 m of one another in westernWisconsin, USA. Mycologia 105, 447e461.

Harrington, T.C., Wingfield, B.D., 1995. A PCR-based identification method for spe-cies of Armillaria. Mycologia 87, 280e288.

Herrmann, A., Hedman, H., Ros�en, J., Janssen, D., Haraldsson, B., Hellen€as, K.E., 2012.Analysis of the mushroom nephrotoxin orellanine and its glucosides. J. Nat.Prod. 75, 1690e1696.

Jaklitsch, W.M., Samuels, G.J., Ismaiel, A., Voglmayr, H., 2013. Disentangling theTrichoderma viridescens complex. Persoonia-Mol. Phylogeny Evol. Fungi 31,112e146.

D. Shao et al. / Toxicon 114 (2016) 65e7474

Judge, B.S., Ammirati, J.F., Lincoff, G.H., Trestrail III, J.H., Matheny, P.B., 2010. Inges-tion of a newly described North American mushroom species from Michiganresulting in chronic renal failure: Cortinarius orellanosus. Clin. Toxicol. 48,545e549.

Justo, A., Malysheva, E., Bulyonkova, T., Vellinga, E.C., Cobian, G., Nguyen, N.,Minnis, A.M., Hibbett, D.S., 2014. Molecular phylogeny and phylogeography ofHolarctic species of Pluteus section Pluteus (Agaricales: Pluteaceae), withdescription of twelve new species. Phytotaxa 180, 1e85.

Karlson-Stiber, C., Persson, H., 2003. Cytotoxic fungi: an overview. Toxicon 42 (4),339e349.

Keller-Dilitz, H., Moser, M., Ammirati, J.F., 1985. Orellanine and other fluorescentcompounds in the genus Cortinarius, section Orellani. Mycologia 77 (5),667e673.

Kirk, P., 2016. Index Fungorum Partnership. CABI. http://www.indexfungorum.org.Koller, G.E.B., Høiland, K., Janak, K., Størmer, F., 2002. The presence of orellanine in

spores and basidiocarp from Cortinarius orellanus and Cortinarius rubellus.Mycologia 94, 752e756.

Liimatainen, K., Niskanen, T., Dima, B., Kytovuori, I., Ammirati, J.F., Frøslev, T.G., 2014.The largest type study of Agaricales species to date: bringing identification andnomenclature of Phlegmacium (Cortinarius) into the DNA era. Persoonia-Mol.Phylogeny Evol. Fungi 33, 98e140.

Lincoff, G., 2011. The Complete Mushroom Hunter: an Illustrated Guide to Finding,Harvesting, and Enjoying Wild Mushrooms, Quarry Books. USA, Beverly, MA.

Litovitz, T.L., Felberg, L., White, S., Klein-Schwartz, W., 1996. 1995 annual report ofthe american association of poison control centers toxic exposure surveillancesystem. Am. J. Emerg. Med. 14 (5), 487e537.

Matheny, P.B., Aime, M.C., Bougher, N.I., Buyck, B., Desjardin, D.E., Horak, E.,Kropp, B.R., Lodge, D.J., Soytong, K., Trappe, J.M., Hibbett, D.S., 2009. Out of thePalaeotropics? Historical biogeography and diversification of the cosmopolitanectomycorrhizal mushroom family. Inocybaceae J. Biogeogr. 36, 577e592.

Miller, M.A., Pfeiffer, W., Schwartz, T., 2010, November. Creating the CIPRES sciencegateway for inference of large phylogenetic trees. In: Gateway Computing En-vironments Workshop (GCE), 2010. IEEE, pp. 1e8.

Moore, B., Burton, B.T., Lindgren, J., Rieders, F., Kuehnel, E., Fisher, P., 1991. Corti-narius mushroom poisoning resulting in anuric renal failure. Vet. Hum. Toxicol.33, 369.

Niskanen, T., Kyt€ovuori, I., Liimatainen, K., 2009. Cortinarius sect. Brunnei (Basidio-mycota, Agaricales) in North Europe. Myco. Res. 113, 182e206.

Niskanen, T., Kyt€ovuori, I., Liimatainen, K., 2011. Cortinarius sect. Armillati innorthern Europe. Mycologia 103 (5), 1080e1101.

O'Donnell, K., Rooney, A.P., Mills, G.L., Kuo, M., Weber, N.S., Rehner, S.A., 2011.Phylogeny and historical biogeography of true morels (Morchella) reveals anearly Cretaceous origin and high continental endemism and provincialism inthe Holarctic. Fungal Genet.. Biol. 48, 252e265.

Oubrahim, H., Richard, J.-M., Cantin-Esnault, D., Seigle-Murandi, F., Tr�ecourt, F.,1997. Novel methods for identification and quantification of the mushroomnephrotoxin orellanine. Thin-layer chromatography and electrophoresisscreening of mushrooms with electron spin resonance determination of thetoxin. J. Chromatogr. A 758, 145e157.

Overås, J., Ulvund, M.J., Bakkevig, S., Eiken, R., 1979. Poisoning in sheep induced bythe mushroom Cortinarius speciosissimus. Acta. Vet. Scan 20 (1), 148e150.

Peintner, U., Moncalvo, J.M., Vilgalys, R., 2004. Toward a better understanding of theinfrageneric relationships in Cortinarius (Agaricales, Basidiomycota). Mycologia96, 1042e1058.

Phillips, R., 2006. Mushrooms: a Comprehensive Guide to Mushroom Identification.

Pan McMillan, London.Prast, H., Werner, E.R., Pfaller, W., Moser, M., 1988. Toxic properties of the mush-

room Cortinarius orellanus. I. Chemical characterization of the main toxin ofCortinarius orellanus (Fries) and Cortinarius speciosissimus (Kühn & Romagn)and acute toxicity in mice. Arch. Toxicol. 62, 81e88.

Puschner, B., Rose, H.H., Filigenzi, M.S., 2007. Diagnosis of Amanita toxicosis in a dogwith acute hepatic necrosis. J. Vet. Diagn. Investig. 19 (3), 312e317.

Puschner, B., Wegenast, C., 2012. Mushroom poisoning cases in dogs and cats:diagnosis and treatment of hepatotoxic, neurotoxic, gastroenterotoxic, neph-rotoxic, and muscarinic mushrooms. Vet. Clin. N. Am. Small Anim. Pract. 42 (2),375e387.

Raff, E., Halloran, P.F., Kjellstrand, C.M., 1992. Renal failure after eating “magic”mushrooms. Can. Med. Assoc. J. 147 (9), 1339e1341.

Rambaut, A., 2007. Se-al: Sequence Alignment Editor. http://tree.bio.ed.ac.uk/software/seal/.

Richard, J.-M., Louis, J., Cantin, D., 1988. Nephrotoxicity of orellanine, a toxin fromthe mushroom Cortinarius orellanus. Arch. Toxicol. 62, 242e245.

Robertson, C.P., Wright, L., Gamiet, S., Machnicki, N., Ammirati, J., Birkebak, J.,Meyer, C., Allen, A., 2006. Cortinarius rubellus cooke from British Columbia,Canada and western Washington, USA. Pac. Northwest Fungi 1 (6), 1e7.

Rumbeiha, W.K., 2000. Nephrotoxins. In: Bonagura, J.D. (Ed.), Kirk's Current Vet-erinary Therapy XIII. WB Saunders, Philadelphia, PA, pp. 212e216.

Schoch, C.L., Sung, G.-H., Lopez-Giraldez, F., Townsend, J.P., Miadlikowska, J.,Hofstetter, V., Robbertse, B., Matheny, P.B., Kauff, F., Wang, Z., Gueidan, C.,Andrie, R.M., Trippe, K., Ciufetti, L.M., Wynns, A., Fraker, E., Hodkinson, B.P.,Bonito, G., Groenewald, J.Z., Arzanlou, M., Hoog, G.S.D., Crous, P.W., Hewitt, D.,Pfister, D.H., Peterson, K., Gryzenhout, M., Wingfield, M.J., Aptroot, A., Suh, S.-O.,Blackwell, M., Hillis, D.M., Griffith, G.W., Castlebury, L.A., Rossman, A.Y.,Lumbsch, H.T., Lücking, B.B., Rauhut, A., Dierderich, P., Ertz, D., Geiser, D.M.,Hosaka, K., Inderbitzin, P., Kohlmeyer, J., Volkmann-Kohlmeyer, B., Mostert, L.,O'Donnell, K., Sipman, H., Rogers, J.D., Shoemaker, R.A., Sugiyama, J.,Summerbell, R.C., Untereiner, W., Johnson, P.R., Stenroos, S., Zuccaro, A.,Dyer, P.S., Crittenden, P.D., Cole, M.S., Hansen, K., Trappe, J.M., Yahr, R.,Lutzoni, F., Spatafora, J., 2009. The Ascomycota tree of life: a phylum-widephylogeny clarifies the origin and evolution of fundamental reproductive andecological traits. Syst. Biol. 58, 224e239.

Schumacher, T., Høiland, K., 1983. Mushroom poisoning caused by species of thegenus. Cortinarius Fries. Arch. Toxicol. 53 (2), 87e106.

Stamatakis, A., 2014. RAxML version 8: a tool for phylogenetic analysis and post-analysis of large phylogenies. Bioinformatics. http://dx.doi.org/10.1093/bioin-formatics/btu033. http://bioinformatics.oxfordjournals.org/content/early/2014/01/21/bioinformatics.btu033.abstract.

Vilgalys, R., Hester, M., 1990. Rapid genetic identification and mapping of enzy-matically amplified ribosomal DNA from several Cryptococcus species.J. Bacteriol. 172, 4238e4246.

Waring, R.H., 2007. Mushroom toxins. In: Waring, R.H., Steventon, G.B., Mitchell, S.C.(Eds.), Molecules of Death, second ed. Imperial College Press, London,pp. 187e208.

Zhang, Y., Hagen, F., Stielow, B., Rodrigues, A.M., Samerpitak, K., Zhou, X., Feng, P.,Yang, L., Chen, M., Deng, S., Li, S., Liao, W., Li, R., Li, F., Meis, J.F., Guarro, J.,Teixeira, M., Al-Zahrani, H.S., Pire de Camargo, Z., Zhang, L., de Hoog, G.S., 2015.Phylogeography and evolutionary patterns in Sporothrix spanning more than 14000 human and animal case reports. Persoonia-Mol. Phylogeny Evol. Fungi 35,1e20.