Submitted 16 March 2016Accepted 9 May 2016Published 7 June 2016

Corresponding authorKaroliacutena Biacutelaacute kcernavolnycz

Academic editorStuart Pimm

Additional Information andDeclarations can be found onpage 16

DOI 107717peerj2094

Copyright2016 Biacutelaacute et al

Distributed underCreative Commons CC-BY 40

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Consequences for selected high-elevationbutterflies and moths from the spread ofPinus mugo into the alpine zone in theHigh Sudetes MountainsKaroliacutena Biacutelaacute12 Jan Šipoš3 Pavel Kindlmann24 and Tomaacuteš Kuras1

1Department of Ecology and Environmental Sciences Faculty of Science Palackyacute University OlomoucOlomouc Czech Republic

2Department of Biodiversity Research Global Change Research Institute CAS Brno Czech Republic3Department of Biology and Ecology Institute of Environmental Technologies Faculty of Science Universityof Ostrava Ostrava Czech Republic

4 Institute of Environmental Studies Charles University Prague Prague Czech Republic

ABSTRACTDue to changes in the global climate isolated alpine sites have become one of the mostvulnerable habitats worldwide The indigenous fauna in these habitats is threatened byan invasive species dwarf pine (Pinus mugo) which is highly competitive and could beimportant in determining the composition of the invertebrate community In this studythe association of species richness and abundance of butterflies with the extent of Pinusmugo cover at individual alpine sites was determined Butterflies at alpine sites in theHigh Sudetes Mountains (Mts) were sampled using Moericke yellow water traps Theresults of a Canonical Correspondence Analysis (CCA) indicated that at a local scale thearea of alpine habitats is the main limiting factor for native species of alpine butterfliesButterfly assemblages are associated with distance to the tree-line with the optimumsituated in the lower forest zone In addition the CCA revealed that biotic factors (iePinus mugo and alpine tundra vegetation) accounted for a significant amount of thevariability in species data Regionally the CCA identified that the species compositionof butterflies andmoths is associated with presence and origin of Pinus mugo Our studyprovides evidence that the structure of the Lepidopteran fauna that formed during thepostglacial period and also the present composition of species assemblages is associatedwith the presence of Pinus mugo With global warming Pinus mugo has the potentialto spread further into alpine areas and negatively affect the local species communities

Subjects Biodiversity Biogeography Conservation Biology Entomology Environmental SciencesKeywords Afforestation Alpine tundra Lepidoptera Dwarf pine Postglacial developmentCentral European mountains Biodiversity loss

INTRODUCTIONAlpine habitats are unique and highly vulnerable because their existence depends onabiotic conditions island phenomena and postglacial history (Taberlet et al 1998 Schmittamp Haubrich 2008) These habitats have been affected by changes in global climate andanthropogenic activities which favour the spread of non-indigenous species (Nagy ampGrabherr 2009) However there are few studies on the effect of biotic factors on the

How to cite this article Biacutelaacute et al (2016) Consequences for selected high-elevation butterflies and moths from the spread of Pinus mugointo the alpine zone in the High Sudetes Mountains PeerJ 4e2094 DOI 107717peerj2094

composition of the community in this habitat which developed during the postglacialperiod It is highly likely that competitive species determined the structure of the communityand survival of other species (Maron amp Vilagrave 2001)

Dwarf pine (Pinus mugo) is a typical species of Central European mountains where it isusually dominant at the lower margin of the subalpine altitudinal zone Dwarf pine limitsthe distribution of many other plants and animals within the alpine zone (Cavalli et al2011 Zeidler et al 2012) This successful colonist spreads not only at localities within itsindigenous distribution area but particularly on mountain summits where it was artificiallyplanted (Dullinger Dirnboumlck amp Grabherr 2003Treml et al 2010) The spread of dwarf pineis negatively affecting alpine zones by overgrowing the native treeless area fragmenting andincreasing its isolation decreasing the amount of food available for insects and changingthe microclimate (Svoboda 2001)

As far as we know only one study records the negative effects of Pinus mugo on theindigenous fauna in particular beetles (Kašaacutek et al 2015) Here we present recent findingsand hypothesize that Pinus mugo has a negative effect on Lepidoptera richness and speciescomposition at both local and regional levels Both these effects of Pinus mugo are discussedin terms of recent and postglacial development of invertebrate communities

At the local level we estimate the effects of (i) habitat quality (ii) area of the alpinesite and (iii) edge effect on species richness of butterflies at the regional level we estimatethe effects of (i) the extent of the area covered by Pinus mugo (naturally occurring andplanted) (ii) Pinus mugo origin and (iii) area of the alpine site on both butterflies andmoths

MATERIALS AND METHODSEthics statementOur field survey was approved by the Czech Ministry of Environment and we were allowedto collect species of Lepidoptera at all alpine sites studied which are part of the ProtectedLandscape area Jeseniacuteky The permission was issued for a group of scientists one of whichwas Dr Tomaacuteš Kuras Permission Nr MŽP1334104-620231904 from the 21st of July2004 valid untill the 30th of November 2007

Study areaThe High Sudetes Mts form a natural border between the Czech Republic and Poland(Jeniacutek 1961) and are one of the middle-high mountain systems in Central Europe Thereare sites in the alpine zone in the High Sudetes Mts with different degrees of isolationand different Pinus mugo origin ie these mountains are at the edge of the distribution ofPinus mugo with some sites occupied by this indigenous dwarf pine and others where thisshrub is absent or was artificially planted In the Czech Republic the High Sudetes Mtsconsist of three mountain ranges (Fig 1) the Krkonoše Mts (RiesengebirgeGiant Mts)Hrubyacute Jeseniacutek Mts (Altvatergebirge) and Kralickyacute Sněžniacutek Mts (Glatzer Schneegebirge)Pinus mugo occurs naturally in the Krkonoše Mts but not in the Kralickyacute Sněžniacutek andHrubyacute Jeseniacutek Mts

Biacutelaacute et al (2016) PeerJ DOI 107717peerj2094 220

Figure 1 Map of the High Sudetes Mts which consist of three mountain ranges (Krkonoše MtsKralickyacute SněžniacutekMts and Hrubyacute Jeseniacutek Mts) showing the location of the alpine sites studied(abbreviations used in Fig 6 in brackets) 1 Kralickyacute Sněžniacutek (KraSneznik) 2 Šeraacutek (Serak) 3 Keprniacutek(Keprnik) 4 Červenaacute hora (Cerhora) 5 Malyacute Děd (MalDed) 6 Mravenečniacutek-Vřesniacutek (MrVresnik)7 Praděd (Praded) 8 Vysokaacute hole (Vyshole) 9 Krkonoše-West (KrkoW) and 10 Krkonoše-East(KrkoE)

We also studied sites at a wider regional scale by including the two geographically nearestalpine habitats of different origins with Pinus mungo present or absent ie Babia GoraSlovakiaPoland with Pinus mugo and Harz Germany without Pinus mugo (Fig 2)

Historical contextSummits in the High Sudetes usually reach an altitude of not more than 1600 m and havea narrow belt of treeless arctic-alpine tundra at the summits Although the insect faunaof middle-high mountains is typically poor compared to that at lower altitudes (DennisShreeve amp Williams 1995 Fleishman Austin amp Weiss 1998Nagy et al 2003) alpine sites inthe High Sudetes Mts host a unique fauna comprised of arcto-alpine and boreo-montanespecies plus endemic species and glacial relicts (Mazalovaacute Kašaacutek amp Kuras 2012) A similarfauna does not occur anywhere else in Central Europe and the sites studied in the HighSudetes Mts strongly differ in species composition Therefore the High Sudetes have ahigh conservation value The exceptional nature of these mountains is associated with theirlocation near the northern-most border of Pinus mugo distribution (Hamerniacutek amp Musil2007) Dwarf pine is indigenous in most Central European mountains (incl WesternCarpathians Beskydy Mts Krkonoše MtsRiesengebirge) and artificially planted in theHrubyacute Jeseniacutek MtsAltvatergebirge and the Kralickyacute Sněžniacutek MtsGlatzer Schneegebirge(Klimeš amp Klimešovaacute 1991 Rybniacuteček amp Rybniacutečkovaacute 2004) Afforestation of most of the

Biacutelaacute et al (2016) PeerJ DOI 107717peerj2094 320

Figure 2 Map showing the areas studied at the Central European scale

summits in Hrubyacute Jeseniacutek Mts and Kralickyacute Sněžniacutek with Pinus mugo started at the endof the 19th century (Jeniacutek amp Hampel 1992) and resulted in it colonizing the area whereit currently covers about 63 of what was previously alpine habitat (Treml et al 2010)On the other hand this latter afforestation allows us to test recent vs postglacial effects ofPinus mugo

Insect groups studied and sampling methodA survey of butterflies was carried out in the Hrubyacute Jeseniacutek and the Kralickyacute Sněžniacutek Mtsa group of invertebrates with high conservation and bio-indication importance Butterflieswere captured using yellow plastic traps (lsquolsquoMoericke water trapsrsquorsquo) which are widely usedfor monitoring nectar feeding insects and thus also suitable for butterflies (Bartaacutek 1997Rohaacuteček Bartaacutek amp Kubiacutek 1998 Beneš Kuras amp Konvička 2000) The traps were made ofcircular plastic pans (upper diameter 13 cm depth 6 cm) painted yellow inside (Industrol6200 Rcopy) At the study sites the pans were filled to a depth of about 2 cm with a solution ofdetergent and water (1 ml of detergent per 1 l of water) We placed the traps approximately20 m apart along transects at each study site with the number of traps set dependent on thearea of the site The traps were set during the flight period of the butterflies (Kuras et al2001b Kuras et al 2003 Konvička Beneš amp Kuras 2002) ie from July 15 to September 3

Biacutelaacute et al (2016) PeerJ DOI 107717peerj2094 420

Table 1 Trapping sites number of traps and dates on which they were checked

Site Nr of traps Dates checked

Kralickyacute Sněžniacuteka 10 197 237 18 98 168 268 29 2005Hrubyacute Jeseniacutek - Šeraacuteka 7 197 237 18 98 168 268 29 2005Hrubyacute Jeseniacutek - Keprniacuteka 10 197 237 18 98 168 268 29 2005Hrubyacute Jeseniacutek - Červenaacute horaa 10 197 237 18 98 168 268 29 2005Hrubyacute Jeseniacutek ndash Malyacute Dědb 7 207 247 28 108 178 278 39 2005Hrubyacute Jeseniacutek - Pradědb 13 207 247 28 108 178 278 39 2005Hrubyacute Jeseniacutek - Vřesniacutekb 10 207 237 28 98 168 268 29 2005Hrubyacute Jeseniacutek - Vysokaacute holeb 17 207 247 28 108 178 278 39 2005Total 84 7

NotesaTraps set on July 15 2005bTraps set on July 16 2005

in 2005 and checked every week (Table 1)We alsomeasured the distance of each trap fromthe tree-line (in m) using software ArcGIS 9 (ESRI 2004) and the tree-line specificationof Treml amp Banaš (2000)

The yellow plastic traps provide a simpler and quicker way of acquiring large quantitativedata sets than monitoring transects or capture-recapture Using yellow water traps avoidshuman errors as the same principle determines the catch at all sites which are all caughtat the same time This method is also very effective for faunal surveys of alpine habitats(including butterflies) as very few plants are in flower above the tree-line during thevegetation season (Beneš Kuras amp Konvička 2000)

This method is non-selective as different taxa of nectar feeding insects were caught Thecontents of the traps were separated into different taxa with the species and sex of thebutterflies recorded (Table 2) and other insect groups conserved for future use Duringthe trapping period only 15 species were caught Aglais urticae Boloria dia Coenonymphapamphilus Erebia epiphron silesiana Erebia euryale Erebia ligea Erebia sudetica sudeticaGonepteryx rhamni Inachis io Issoria lathonia Lycaena hippothoe Pieris brassicae Pierisnapi Pieris rapae and Polygonia c-album The species richness recorded is rather lowconsisting mostly of species that migrate into alpine zones from lower altitudes A totalof 7651 individuals were caught during the summer of 2005 Erebia spp were the mostabundant (942 of the individuals sampled) mainly because of their exclusive associationwith alpine and montane habitats and low probability of occurring at lower altitudes thanthe other species captured which are generalists and good migrants Therefore we usedonly four species of Erebia (ie E epiphron silesiana E euryale E ligea E s sudetica) inthe analysis and testing of the effects of environmental factors on autochthonous alpinebutterfly assemblages

Classification of the vegetationVegetation at the alpine sites in the Hrubyacute Jeseniacutek Mts was classified according to theHabitat Catalogue of the Czech Republic (Chytryacute et al 2010) where habitats are specifiedin terms of the dominant diagnostic and other plant species present There are five types

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Table 2 Number of butterflies caught by theMoericke water traps in the High Sudetes Mts during the summer of 2005

Alpine site Species KralickyacuteSněžniacutek

Šeraacutek Keprniacutek Červenaacutehora

MalyacuteDěd

Praděd Mravenečniacutek-Vřesniacutek

Vysokaacutehole

Aglais urticae 35 2 40 20 3 26 18 25Aglais urticae 32 0 53 26 9 37 22 39Boloria dia 0 0 0 0 1 0 0 0Coenonympha pamphilus 0 1 0 0 0 0 0 0Erebia epiphron silesiana 0 1 0 0 195 156 257 509Erebia epiphron silesiana 0 0 0 0 96 70 76 149Erebia euryale 893 264 76 591 379 534 594 287Erebia euryale 405 109 20 391 222 307 289 182Erebia ligea 0 1 0 14 1 0 27 0Erebia ligea 0 0 0 7 1 1 26 0Erebia sudetica sudetica 0 5 0 0 24 9 1 0Erebia sudetica sudetica 0 0 0 0 30 12 0 0Gonepteryx rhamni 0 0 0 0 0 1 0 1Gonepteryx rhamni 0 0 0 1 0 0 0 0Inachis io 0 0 1 0 1 0 0 1Inachis io 0 0 0 1 0 0 0 0Issoria lathonia 0 0 1 1 0 0 0 0Lycaena hippothoe 0 0 0 0 0 0 0 1Pieris brassicae 1 0 0 0 0 0 0 0Pieris brassicae 0 0 0 0 0 0 1 1Pieris napi 0 0 1 1 0 0 0 0Pieris napi 0 0 0 1 1 0 0 1Pieris rapae 0 2 1 0 2 1 2 3Pieris rapae 1 3 4 2 3 1 2 3Polygonia c-album 0 0 1 0 0 0 0 0Polygonia c-album 0 0 1 0 0 1 0 0

of habitat at the alpine sites studied (1) Alpine heathland dominated by Calluna vulgaris(2) Subalpine Vaccinium vegetation dominated by Vaccinium myrtillus and Vacciniumvitis-idaea (3) Alpine grassland dominated by Avenella flexuosa Nardus stricta and Festucasupina (4) Subalpine tall-herbaceous vegetation dominated by Molinia coerullea and (5)Pinus mugo scrub dominated by dwarf pine These habitats occurred at all the sites studiedWe recorded the percentage vegetation cover made up of each type of habitat within anarea of 10 m radius around each trap The percentage cover of Pinus mugo was obtainedfrom aerial photographs taken in 2005 with a grid of 03 m pixel resolution (Wild 2005)(Table 3)

DATA ANALYSISLocal effect of Pinus mugoWe used a unimodal direct gradient analysis ie a CCA (Canonical CorrespondenceAnalysis) to test the association of the abundances of the different species of Erebia

Biacutelaacute et al (2016) PeerJ DOI 107717peerj2094 620

Table 3 Altitude area extent of cover and origin of Pinus mugo in the alpine sites studied

Alpine site Altitude (m asl) Area (ha) Pinus mugo () P mugo origina

Šeraacutek 1351 219 360 0Mravenečniacutek-Vřesniacutek 1342 467 0 0Malyacute Děd 1368 550 247 0Červenaacute hora 1333 657 357 0Keprniacutek 1423 801 373 0Kralickyacute Sněžniacutek 1424 896 125 0Praděd 1492 1425 221 0Vysokaacute hole 1464 6785 143 0Krkonoše-East 1602 32126 460 1Krkonoše-West 1508 21161 570 1Harz 1142 1310 50 0Babia Gora 1725 1940 750 1

NotesaPinus mugo origin occurs naturally in the area= 1 planted or absent= 0

with the environmental variables measured (Ter Braak amp Šmilauer 1998) Vegetationtypes (Calluna Vaccinium Avenella Molinia and Pinus) area of the alpine sites (ha) anddistance to the tree-line (m) were considered as explanatory variables There was littledifference in the altitudes of the sites and as it had an insignificant effect it was not includedin the model The covariates of the model were selected by forward selection and weretime of year (ie number of days from 1st July) and degree of isolation of each alpine site(ie distance to the nearest alpine site in km) We configured the CCA model with anaxis scaled in terms of inter-species distances Species abundances were log transformedand results for males and females were identified in the analysis Significance of canonicalaxis and explanatory variables was tested using a Monte-Carlo permutation test (5000restricted permutations) The permutation test was restricted by the split-plot design (timeseries and linear transect as a split-plot and freely exchangeable whole plot) Relationshipbetween species abundance and cover of Pinus mugo was tested using a Generalized LinearModel (GLM) and a Poisson distribution We tested both sexes of butterflies for possibledifferences in their behaviour (occupancy and usage of the habitat) especially for Erebiaspp (Kuras Beneš amp Konvička 2001a Konvička Beneš amp Kuras 2002)

Regional effect of Pinus mugoFaunal structure and its historical development in the presence and absence of Pinusmugo was tested using methods based on diagnostic species of both butterflies and mothsDiagnostic species are alpine and boreal species with an exclusive association with alpinehabitats or generally unlikely to be found below the tree-line (Patočka amp Kulfan 2009)From an eco-zoogeographical point of view (sensu Krampl 1992) it means species witheuboreal boreo-alpine arctic-alpine and subalpine distributions with disjunct and verynarrow dispersal ranges within Central European mountains All literature sourcesrelated to these mountain ranges were critically reviewed with regard to recent findings(Soffner 1960 Krampl 1992 Jahn Kozlowski amp Pulina 1997 Liška 1997 Liška amp Skyva

Biacutelaacute et al (2016) PeerJ DOI 107717peerj2094 720

1997 Liška 2000 Kuras et al 2009) In these publications the following species wereconsistently claimed to be exclusively associated with the alpine environment and aretherefore referred to as lsquolsquodiagnosticrsquorsquo in this paper (Table 4) Argyresthia amiantellaBlastesthia mughiana Callisto coffeella Catoptria maculalis Catoptria petrificella Chio-niodes viduellus Clepsis rogana Clepsis steineriana Elachista dimicatella Elachistakilmunella Elophos operarius Epichnopterix ardua Erebia epiphron Erebia sudetica Glaciesalpinatus Incurvaria vetulella Kessleria zimmermanni Lampronia rupella Olethreutesobsoletanus Psodos quadrifarius Rhigognostis senilella Sparganothis rubicundana andXestia alpicola Pinus mugo cover nearest distances and areas of the alpine sites wereestimated using aerial photographs and GIS analysis

We used CCA to determine the effect of area of the alpine site Pinus mugo cover andits origin (naturally occurringplanted) for all summits in the High Sudetes Mts plus twogeographically nearest and isolated alpine sites ie Babia Gora Mts (Slovakia) and HarzMts (Germany) Significance of the canonical axis and explanatory variables was testedusing a Monte-Carlo permutation test (5000 unrestricted permutations)

Next we tested the similarity in the species composition at all the sites Diagnosticspecies were allocated values 10 (presentabsent) and tested using the UPGMA (un-weighted pair group using arithmetical average) clustering method (Everitt amp Hothorn2006) Significance of each cluster was estimated and possible subjectivity of the diagnosticspecies list decreased using bootstrap methods so that a dissimilarity matrix of the alpinesites was generated for each cluster and bootstrap probabilities calculated using multi-scalebootstrap re-sampling in order to acquire AU (Approximately Unbiased) p-values

RESULTSLocal effect of Pinus mugoAfter filtering out the effects of covariates the CCA model explained 150 (adjustedexplained variation 135) of the variance of the results for this species As shown in theordination graph (Table 5 and Fig 3) most of the variability is accounted for in terms ofthe distance to the tree-line area of the alpine site and vegetation cover of Avenella andPinus With increase in the area of the site we recorded an increase in the abundance ofErebia epiphron a typical alpine species Other Erebia spp are negatively correlated withthe area of the site

Using the CCA model we also tested each parameter separately Each environmentalvariable was included in the model individually and remaining parameters acted ascovariates We recorded that individual environmental parameters (after subtractingsummarized variability of the covariates) accounted for the variability as follows time ofyear 119 distance from the tree-line 71 distance to the next nearest alpine site 27area of alpine site 31 vegetation cover of Calluna 05 Avenella 09 Pinus 09Molinia 04 and Vaccinium 04

The association between species abundance and area covered by Pinus mugo was furthertested using a Generalized LinearModel Themountain specialists (Erebia spp) are affectedmore than the generalists (Aglais urticae and Pieris rapae) by Pinus mugo (Table 6) The

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Table 4 Diagnostic species in the High Sudetes Mts the Kralickyacute SněžniacutekMt the Hrubyacute Jeseniacutek Mts and the Krkonoše Mts (Czech Republic) and the two geo-graphically nearest alpine habitats Harz (Germany) and Babia Gora (SlovakiaPoland)

Alpine site Species KralickyacuteSněžniacutek

Hrubyacute Jeseniacutek Mts Krkonoše Mts Harz BabiaGora

Šeraacutek Keprniacutek Červenaacutehora

MalyacuteDěd

MravenečniacutekVřesniacutek

Praděd Vysokaacutehole

Krkonoše-West

Krkonoše-East

Incurvaria vetulella + + + + +

Lampronia rupella + + + + +

Argyrhestia amianthella + +

Rhigognostis senilella + + + +

Elachista kilmunella + + + +

Elachista dimicatella +

Chioniodes viduellus + + + +

Sparganothis rubicundana + + + +

Clepsis steineriana + + +

Clepsis rogana + + + + + + + + +

Blastesthia mughiana + +

Olethreutes obsoletanus +

Catoptria maculalis + +

Catoptria petrificella + + + + + +

Erebia epiphrona + + + + +

Erebia sudetica + + + + + + +

Psodos quadrifarius + +

Glacies alpinatus + + + + +

Epichnopterix arduab + + + + + + +

Xestia alpicolla + +

Elophos operarius + +

Callisto coffeella +

Kessleria zimmermanni +

NotesaErebia epiphron was introduced into the Krkonoše Mts in 1932 (Kuras et al 2001b Schmitt Čiacutežek amp Konvička 2005) thus this species is not included in the analysis of the results for the Krkonoše MtsbSeveral authors mention this species as E sieboldi (cf Laštůvka amp Liška 2011)

Biacutelaacute

etal(2016)PeerJDOI107717peerj2094

920

Table 5 Summary of Monte Carlo test of the CCAmodel lsquolsquospeciessim env variables area of alpine site(ha) distance to the tree-line (m) cover of the different types of vegetation covariates time of yeardistance to the nearest alpine site (km)rsquorsquo used to determine the local effect

Axes I II III IV

Eigenvalues 0117 0039 0011 0002Species-environment correlations 0620 0439 0222 0139Cumulative percentage variance

of species data 103 137 147 149of species-environment relation 689 917 980 994

Sum of all eigenvalues 1134Sum of all canonical eigenvalues 0150Test of significance of first canonical axis F-ratio= 47419 P-value= 00012Test of significance of all canonical axes F-ratio= 10376 P-value= 00012

Figure 3 CCA ordination graph with the environmental variables and butterflies that occurred at thealpine sites in the Kralickyacute Sněžniacutek and Hrubyacute Jeseniacutek Mts Length of the arrows indicate the amount ofvariance accounted for by a particular variable Species positions are indicated by black dots in the reducedordination area of the first and second canonical axes Acronyms for the species ErEpi Erebia epiphronsilesiana ErEur Erebia euryale ErLig Erebia ligea ErSud Erebia sudetica sudetica+ femaleminus male

Biacutelaacute et al (2016) PeerJ DOI 107717peerj2094 1020

Table 6 Results of GLM regressions of the abundances of the different butterflies and percentagecover of Pinus mugo

Species Beta SE T -value F p AIC

Aglais urticae ndash0014 0004 ndash375 385 gt005 1077618Aglais urticae ndash0012 0003 ndash381 396 lt005 1297152Erebia ligea ndash0005 0006 ndash088 017 gt005 530437Erebia ligea ndash0062 0020 ndash316 727 lt001 389823Er epiph silesiana ndash0027 0002 ndash1507 1245 lt001 7035796Er epiph silesiana ndash0019 0003 ndash735 794 lt001 2445229Erebia euryale ndash0010 0001 ndash1570 617 lt005 151e+004Erebia euryale ndash0015 0001 ndash1526 1868 lt001 5853988Er sudet sudetica 0000 0005 009 000 lt005 620972Er sudet sudetica ndash0119 0034 ndash354 847 lt001 614618Pieris rapae 0020 0008 267 353 gt005 165006Pieris rapae 0003 0009 034 005 gt005 217394

NotesKey Model resid DF (all models)= 442 Link function Log Poissonlsquos distribution lsquolsquoBetarsquorsquo indicates the slope of regressionline

Table 7 Summary of Monte Carlo test of CCAmodel lsquolsquospeciessim env variables area of alpine site cover of Pinus mugo and its originrsquorsquo for determining the regional effect

Axes I II III IV

Eigenvalues 0610 0398 0079 0224Species-environment correlations 0950 0883 0784 0000Cumulative percentage variance

of species data 313 518 559 674of species-environment relation 561 927 1000 00

Sum of all eigenvalues 1947Sum of all canonical eigenvalues 1087Test of significance of first canonical axis F-ratio= 3646 P-value= 00120Test of significance of all canonical axes F-ratio= 3374 P-value= 00024

abundance of the two most abundant specialists (Erebia epiphron and Erebia euryale) isconsiderably reduced in areas where there is a relatively low cover of Pinus mugo (Fig 4)We also tested the association of both sexes of these butterflies with the area covered byPinus mugo which revealed no significant difference between males and females in spite ofthe higher numbers of males caught

Regional effect of Pinus mugoCCA accounted for 61 of the variance in the speciesrsquo environment (ie area of alpinesite percentage cover and origin of Pinus mugo) (Table 7 Fig 5) The effect of Pinus mugoorigin and area of alpine site were significant but not the percentage cover of Pinus mugo

Relationship between species and environmental variables identified by CCA wasfurther tested using a similarity analysis (Fig 6) and three different groups of alpine sites

Biacutelaacute et al (2016) PeerJ DOI 107717peerj2094 1120

Figure 4 GLM regressions of the relationships between the twomost abundant species of butterfly andPinus mugo cover [] Species names are abbreviated and differentiated according to gender ErEpi Ere-bia epiphron silesiana ErEur Erebia euryale+ female ndash male

were identified The first cluster is of alpine habitats with naturally occurring Pinus mugo(Krkonoše-West and Krkonoše-East) which are clearly distinguished from other sites(Multi-scale Bootstrap Re-sampling AU = 98 p = 001) The second cluster includesalpine summits in the Hrubyacute Jeseniacutek Mts ie Vysokaacute hole Praděd and Malyacute Dědwhich is presumably associated with the presence of planted Pinus mugo and their largeareas Furthermore we can also consider the cluster that includes Kralickyacute Sněžniacutek HarzKeprniacutek Červenaacute hora Šeraacutek and Mravenečniacutek-Vřesniacutek (last four are in the Hrubyacute JeseniacutekMts) which are small areas with a dense cover of planted Pinus mugo These two clusterswere separated at a level of significance AU= 92 p= 007 The position of the Babia GoraMt in the dendrogram seems to be ambiguous (AU = 86 p = 011) due to a combinationof small species pool small alpine area and the presence of naturally occurring Pinus mugo

Biacutelaacute et al (2016) PeerJ DOI 107717peerj2094 1220

Figure 5 CCA ordination graph with the environmental variables and Lepidopteran species thatoccurred at the alpine sites in the High Sudetes Mts (CZ) Babia Gora Mts (SK) and Harz Mts(D) Species positions are indicated by black dots in the reduced ordination area of the first andsecond canonical axes Acronyms for species ArgyAmi Argyresthia amiantella BlastMug Blastesthiamughiana CallCof Callisto coffeella CatoMac Catoptria maculalis CatoPet Catoptria petrificella ChioVidChioniodes viduellus ClepRog Clepsis rogana ClepSte Clepsis steineriana ElacDim Elachista dimicatellaElacKil Elachista kilmunella ElopOpe Elophos operarius EpicArd Epichnopterix ardua ErebEpi Erebiaepiphron ErebSud Erebia sudetica GlacAlp Glacies alpinatus IncuVet Incurvaria vetulella KessZimKessleria zimmermanni LampRup Lampronia rupella OletAbs Olethreutes obsoletanus PsodQua Psodosquadrifarius RhigSen Rhigognostis senilella SparRub Sparganothis rubicundana XestAlp Xestia alpicola

DISCUSSIONLocal effect of Pinus mugoIn a local context we tested for the recent effect of Pinus mugo on the butterfly assemblagenamely itrsquos overgrowing of alpine sites and competing with native vegetation We testedthe response of males and females separately to the vegetation cover because previousresearch showed that males fly more frequently in search of food than females whichmainly bask and feed on nectar (Kuras Beneš amp Konvička 2001a Konvička Beneš amp Kuras2002) However our analyses did not indicate any significant differences between the sexesof Erebia spp except for the greater probability of catching males

The presence of Erebia spp was most significantly associated with the area of an alpinesite and its distance to the tree-line followed by vegetation dominated by Avenella(a characteristic species of alpine tundra) and Pinus mugo Correlation of E epiphron

Biacutelaacute et al (2016) PeerJ DOI 107717peerj2094 1320

Figure 6 Cluster analysis (dendrogram) indicating the differences between alpine sites in the Cen-tral Europeanmiddle high mountains calculated on the basis of the occurrence of diagnostic speciesof Lepidoptera (see chapter MethodsndashData Analysis) and a clustering method UPGMA (unweightedpair group using arithmetical average) AUBP values AU (Approximately Unbiased) p-value is moreaccurate than the BP value as an unbiased p-value and is computed by multiscale bootstrap resamplingBP (Bootstrap Probability) value is a simple statistic computed by bootstrap resampling and tends to havea biased p-value when the absolute value of c (ie the value related to geometric aspects of hypotheses)is large Site codes KrkoE Krkonoše-East KrkoW Krkonoše-West BabGora Babia Gora MalDedMalyacute Děd Praded Praděd Vyshole Vysokaacute hole KraSneznik Kralickyacute Sněžniacutek Harz Harz KeprnikKeprniacutek MrVresnik Mravenečniacutek-Vřesniacutek Serak Šeraacutek Cerhora Červenaacute hora)

abundance with the area of the sites supports an alpine origin for this species as does itsassociation with alpine grassland where vegetation dominated by Avenella and Callunaprevail At small sites there are fewer E epiphron and E sudetica (Kuras et al 2003) specieswith a high association with treeless sites or these species do not occur there (Kuras et al2001b) Thus the area of an alpine site alone seems to be of greater importance for thebutterfly assemblage than its heterogenity in terms of different types of alpine vegetationThe congeneric species E Euryale and E Ligea also occur at alpine sites but mainly atlower altitudes ie in mountain taiga (Kuras Beneš amp Konvička 2000) or like Es sudeticanear springs close to the upper tree-line (Kuras et al 2001b) This fact accounts for thesignificant effect of distance to the tree-line on the species studied The relatively small

Biacutelaacute et al (2016) PeerJ DOI 107717peerj2094 1420

effect of site heterogenity can be due to the similarity of the different types of vegetationand high mobility of the butterflies (Chytryacute et al 2010)

Even though adult butterflies are highly mobile Pinus mugo serves as a barrier to theirdispersal (Kuras Beneš amp Konvička 2001a Konvička Beneš amp Kuras 2002) This is alsosupported by Fig 4 Dwarf pine overgrows alpine vegetation and in this way is changingthe characteristics of this habitat (food available for adults and quantity of suitable plantsfor caterpillarsmdashsee Zeidler et al 2012) and decreasing the area of native grass-herbaceousvegetation at alpine sites Over the last 30 years the extent of the cover of planted dwarfpine has increased by 63 and the percentage cover at small alpine sites in the HrubyacuteJeseniacutek Mts is currently more than 30 and is continuing to increase (Treml et al 2010)

Regional effect of Pinus mugoPinus mugo significantly affected the fauna and flora of alpine sites in the Central EuropeanMountains during their postglacial development (Jeniacutek 1961 Gutierrez 1997 Čiacutežek etal 2003) We found a significant effect of the presence of Pinus mugo but not the areacovered by this species at alpine sites We suppose this is a consequence of the postglacialdevelopment at the sites studied The tree-line shifted during the postglacial era and the areaof treeless sites obviously changed (Treml amp Banaš 2008) During the Holocene climateoptimum the conditions for alpine species were even less favourable than now because themountain summits were overed with dwarf pine or spruce (Rybniacuteček amp Rybniacutečkovaacute 2004)Therefore species confined to treeless habitats could survive only at very few localitieseg glacial cirques stone debris fields rocks or wind swept summits

We also recorded significant differences in the Lepidopteran fauna at the alpine sitesstudied in the High Sudetes Mts which were associated with differences in the historicaloccurrence of Pinus mugo Our results (Fig 6) are clearly similar to the effect that Pinusmugo had on the fauna of alpine sites during the Holocene Alpine sites with naturallyoccurring Pinus mugo (Krkonoše Mts Babia Gora Mt) which most probably completelycovered or at least a large part of the summits of these mountains in the past hostpredominantly species of Lepidoptera that are able to feed on Pinus or those that survivedin glacial cirques or stone debris fields Alpine sites where dwarf pine was not present (allsummits of the Hrubyacute Jeseniacutek Mts Kralickyacute Sněžniacutek Mt and Harz Mt) were covered withdwarf spruce which does not form such dense formations as dwarf pine Thus speciesof Lepidoptera associated with grassy tundra could survive there until colder conditionsresulted in the tree line occurring at lower altitude

These changes selected the species assemblages currently recorded at the different alpinesites and the most critical time was when the summits were either completely or nearlycovered by trees during the Holocene Recent climate changes favoured pine which becameamore prominent element in the landscape again and so reversed a natural multicentennialor evenmillennial trend in tree line decline and recession (Kullman 2007) Global warmingis now favouring a similar spread of planted Pinus mugo into alpine sites where it previouslydid not occur Even though its current coverage at these sites is not very extensive Pinusmugo has the potential to change these treeless sites and increase their degree of isolation(Biacutelaacute et al 2012)

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CONCLUSIONSThe spread of Pinus mugo into alpine treeless sites is rapid as it covers approximatelyanother 2 of the alpine area in the High Sudetes each year (Zeidler et al 2012) Dwarfpine together with spruce spread into grassy tundra and out compete the native fauna andflora In addition dwarf pine can also colonize glacial cirques Because of its competitiveability and fast growth Pinus mugo (Wild amp Wildova 2002 Wild 2005) is likely to rapidlycolonize the alpine zone particularly sites in areas where Pinus mugowas planted Themostthreatened are small alpine sites in theHrubyacute JeseniacutekMts where the authorities responsiblefor this Protected Landscape Area have already started to remove dwarf pine This studyalso helped to identify sites with the highest priority and the process of eradicating dwarfpine at these sites is ongoing Other effects of climate change on Lepidoptera such aschanges in species and sources of food or population dynamics would be interestingtopics for further research

ACKNOWLEDGEMENTSThe authors thank Vaacuteclav Treml for providing the data on position of the tree-line andlocality of alpine sites in the High Sudetes Mts and for his permission to print maps ofthe study area (Figs 1 and 2) We also thank Tony Dixon for improving the English of thispaper

ADDITIONAL INFORMATION AND DECLARATIONS

FundingThis research was funded by the Ministry of Environment of the Czech Republic (projectVaV SM67005) grant No IG UP 91310404131 and by the MŠMT grants LC06073 andLO1415 The funders had no role in study design data collection and analysis decision topublish or preparation of the manuscript

Grant DisclosuresThe following grant information was disclosed by the authorsMinistry of Environment of the Czech Republic IG UP 91310404131MŠMT LC06073 LO1415

Competing InterestsThe authors declare there are no competing interests

Author Contributionsbull Karoliacutena Biacutelaacute conceived and designed the experiments performed the experimentsanalyzed the data contributed reagentsmaterialsanalysis tools wrote the paperprepared figures andor tables reviewed drafts of the paperbull Jan Šipoš analyzed the data contributed reagentsmaterialsanalysis tools preparedfigures andor tables reviewed drafts of the paperbull Pavel Kindlmann wrote the paper reviewed drafts of the paper

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bull Tomaacuteš Kuras conceived and designed the experiments analyzed the data contributedreagentsmaterialsanalysis tools wrote the paper prepared figures andor tablesreviewed drafts of the paper

Field Study PermissionsThe following information was supplied relating to field study approvals (ie approvingbody and any reference numbers)

This field survey was approved by the Czech Ministry of Environment and we wereallowed to collect Lepidoptera species on all studied alpine sites which are part of theProtected Landscape area Jeseniacuteky The permission was issued for a group of scientistsone of which was Dr Tomaacuteš Kuras Permission Nr MŽP1334104-620231904 from the21st of July 2004 valid until the 30st of November 2007

Data AvailabilityThe following information was supplied regarding data availability

The research in this article did not generate any raw data

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Dennis RLH Shreeve TGWilliamsWR 1995 Taxonomic differentiation in speciesrichness gradients among european butterflies (Papilionoidea Hesperioidea)contribution of macroevolutionary dynamics Ecography 1827ndash40DOI 101111j1600-05871995tb00116x

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Gutierrez D 1997 Importance of historical factors on species richness and compositionof butterfly assemblages (Lepidoptera Rhopalocera) in northern Iberian mountainrange Journal of Biogeography 2477ndash88 DOI 101111j1365-26991997tb00052x

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Kašaacutek J MazalovaacuteM Šipoš J Kuras T 2015 Dwarf pine invasive plant threatensbiodiversity of alpine beetles Biodiversity and Conservation 24(10)2399ndash2415DOI 101007s10531-015-0929-1

Klimeš L Klimešovaacute J 1991 Alpine tundra in the Hrubyacute Jeseniacutek Mts the Sudeten andits tentative development in the 20th century Preslia 63245ndash268

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Krampl F 1992 Boreal macro-moths in Central Europe (Czechoslovakia) and their eco-geographical characteristics (Lepidoptera Geometridae Noctuidae Notodontidae)Acta Entomologica Bohemoslovaca 89237ndash262

Kullman L 2007 Tree line population monitoring of Pinus sylvestris in the swedishscandes 1973ndash2005 implications for tree line theory and climate change ecologyJournal of Ecology 9541ndash52 DOI 101111j1365-2745200601190x

Kuras T Beneš J Fric Z KonvičkaM 2003 Dispersal patterns of endemic alpinebutterflies with contrasting population structures Erebia epiphron and E sudeticaPopulation Ecology 45115ndash123 DOI 101007s10144-003-0144-x

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Kuras T Beneš J KonvičkaM 2001a Behaviour and within-habitat distribution ofadult Erebia sudetica sudetica endemic of the Hrubyacute Jeseniacutek Mts Czech Republic(Nymphalidae Satyrinae) Nota Lepidopterologica 2469ndash83

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Kuras T KonvičkaM Beneš J čiacutežek O 2001b Erebia sudetica and Erebia epiphron(Lepidoptera Nymphalidae Satyrinae) in the Czech Republic review of presentand past distribution conservation implication Časopis Slezskeacuteho Muzea Opava (A)5057ndash81

Kuras T Sitek J Liška J MazalovaacuteM černaacute K 2009Motyacuteli (Lepidoptera) naacuterodniacutepřiacuterodniacute rezervace Praděd (CHKO Jeseniacuteky) implikace poznatku v ochraně uacutezemiacute[Lepidoptera of the Praděd National Nature Reserve (Jeseniacuteky Protected LandscapeArea) conservation implications] Časopis Slezskeacuteho Muzea Opava (A) 58250ndash288

Liška J 1997 Motyacuteliacute fauna Uacutepskeacuteho a Černohorskeacuteho rašeliniště v Krkonošiacutech [Lepi-doptera of the Uacutepskeacute and Černohorskeacute rašeliniště bogs in the Krkonoše Mts] InGeoekologiczne problemy Karkonoszy Materialy z sesji naukowej w Przesiece 15-18 X Poznan Wydawnictwo Acarus

Liška J 2000 Pokus o srovnaacuteniacute motyacuteliacute fauny subalpinskyacutech poloh Vysokyacutech Sudet[Attempt at comparing lepidopteran fauna of subalpine areas of the High Sudetes]Opera Corcontica 37286ndash290

Liška J Skyva J 1997Historical and recent occurrence of Lepidoptera in mountains sitesof the Giant Mountains (Czech Republic) Biologia Bratislava 52163ndash165

Laštůvka Z Liška J 2011 Komentovanyacute seznam motyacutelů Českeacute republiky (Annotatedchecklist of moths and butterflies of the Czech Republic (Insecta Lepidoptera) BrnoBiocont Laboratory

Maron JL Vilagrave M 2001When do herbivores affect plant invasion evidencefor the natural enemies and biotic resistance hypotheses Oikos 95361ndash373DOI 101034j1600-07062001950301x

MazalovaacuteM Kašaacutek J Kuras T 2012 Annotated entomological bibligraphy of the PradědNational Nature Reserve (Hrubyacute Jeseniacutek Mts) Casopis Slezskeacuteho Muzea Opava (A)6123ndash42

Nagy L Grabherr G 2009 The biology of alpine habitats Oxford Oxford UniversityPress

Nagy L Grabherr G Koumlrner C Thompson D 2003 Alpine Biodiversity in Europe(Ecological Studies) New York Springer Berlin Heidelberg

Patočka J Kulfan J 2009Motyacutele Slovenska (bionomia a ekologia) [Lepidoptera of Slovakia(bionomics and ecology)] Slovakia Vydavatelstvo VEDA

Rohaacuteček J BartaacutekM Kubiacutek S 1998 Diptera Acalyptrata of the Hraničniacute (Luzenskaacute) slatpeat-bog in the Šumava Mts (Czech Republic) Časopis Slezskeacuteho Muzea Opava (A)471ndash12

Rybniacuteček K Rybniacutečkovaacute E 2004 Pollen analyses of sediments from the summit of thePraděd range in the Hrubyacute Jeseniacutek Mts (Eastern Sudetes) Preslia 76331ndash347

Schmitt T Čiacutežek O KonvičkaM 2005 Genetics of a butterfly relocation large smalland introduced populations of the mountain endemic Erebia epiphron silesianaBiological Conservation 12311ndash18 DOI 101016jbiocon200409018

Schmitt T Haubrich K 2008 The genetic structure of the mountain forest but-terfly Erebia euryale unravels the late Pleistocene and postglacial history of the

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mountain coniferous forest biome in EuropeMolecular Ecology 172194ndash2207DOI 101111j1365-294X200703687x

Soffner J 1960 Schmetterlinge aus dem Riesengebirge [Lepidoptera of the Giant Mts]Zeitschrift der Wiener Entomologischen Gesellschaft 4570ndash91

SvobodaM 2001 The effects of Pinus mugo (Turra) plantations on alpine-tundramicroclimate vegetation distribution and soils in Krkonoše National Park CzechRepublic Opera Corcontica 38189ndash206

Taberlet P Fumagalli L Wust-Saucy A-G Cosson JF 1998 Comparative phylogeog-raphy and postglacial colonization routes in EuropeMolecular Ecology 7453ndash464DOI 101046j1365-294x199800289x

Ter Braak CJF Šmilauer P 1998 Canoco Reference Manual and Userrsquos Guide to Canocofor Windows Software for Canonical Community Ordination (version 4) IthacaCentre for Biometry Wageningen (NL) and Microcomputer Power

Treml V Banaš M 2000 Alpine timberline in the high sudetes Acta UniversitatisCarolinae Geographica 3583ndash99

Treml V Banaš M 2008 The effect of exposure on alpine treeline position a casestudy from the High Sudetes Czeh Republic Arctic Antarctic and Alpine Research40751ndash760 DOI 1016571523-0430(07-060)[TREML]20CO2

Treml VWild J Chuman T PotůčkovaacuteM 2010 Assessing the change in coverof non-indigenous dwarfpine using aerial photographs a case study fromthe Hrubyacute Jeseniacutek Mts the Sudetes Journal of Landscape Ecology 490ndash104DOI 102478v10285-012-0029-9

Wild J 2005 Vyacutevoj plošneacuteho rozšiacuteřeniacute klečovyacutech porostů [Extent development of Pinusmugo cover] In Hošek J et al eds Vliv vyacutesadeb borovice kleče (Pinus mugo) nabiotopovou a druhovou diverzitu arkto-alpinskeacute tundry ve Vyacutechodniacutech Sudetech (CHKO Jeseniacuteky NPR Kralickyacute Sněžniacutek) Naacutevrh managementu těchto porostů Diacutelčiacute zpraacutevaprojektu VaV SM67005 40-46

Wild J Wildova R 2002 Interactions between dwarf pine shrubs and grassland vegeta-tion under different management Opera Corcontica 3917ndash33

Zeidler M Duchoslav M Banaš M LeškovaacuteM 2012 Impacts of introduced dwarf pine(Pinus mugo) on the diversity and composition of alpine vegetation CommunityEcology 13(2)213ndash220 DOI 101556ComEc132012211

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