A Single Genetic Lineage of Erebia Euryale (Nymphalidae) Along the Northern Iberian Mountains

Accepted on 20 October 2010 2010 Blackwell Verlag GmbH J Zool Syst Evol Res doi: 10.1111/j.1439-0469.2010.00587.x

1Department of Molecular and Cell Biology, Faculty of Sciences, University of Corun˜a, A Corun˜a, Spain; 2Department of Physiology, Faculty of Medicine, University of Santiago de Compostela, Santiago de Compostela, Spain; 3Department of Biogeography, Trier University, Trier, Germany

Some butterﬂies do not care much about topography: a single genetic lineage of Erebia euryale (Nymphalidae) along the northern Iberian mountains

Marta Vila1,Neus Marı´-Mena1,Ana Guerrero2 and Thomas Schmitt3

Abstract The phylogeography of montane species often reveals strong genetic differentiation among mountain ranges. Both classic morphological and genetic studies have indicated distinctiveness of Pyrenean populations of the butterfly Erebia euryale. This hypothesis remained inconclusive until data from the westernmost populations of the distribution area (Cantabrian Mountains) were analysed. In the present study, we set out to describe the population structure of Erebia euryale in western Cantabria, where the species occurs in scattered localities. For this goal, we estimate the genetic diversity and differentiation found in 218 individuals from six Cantabrian (North Spain) localities genotyped by 17 allozyme loci. We also sequence 816 bp of the cytochrome oxidase subunit I mitochondrial gene in 49 individuals from Cantabrian localities and 41 specimens from five other European sites. Mitochondrial data support the recognition of four major genetic groups previously suggested for the European populations based on allozyme polymorphisms. Both mitochondrial and nuclear markers reveal genetic distinctiveness of a single Pyrenean– Cantabrian lineage of E. euryale. The lack of geographical structure and the star-like topology displayed by the mitochondrial haplotypes indicate a pattern of demographic expansion in northern Iberia, probably related to Upper Pleistocene climatic oscillations. By contrast, within the Pyrenean–Cantabrian lineage, Cantabrian samples are genetically structured in nuclear datasets. In particular, San Isidro is significantly differentiated from the other five populations, which cluster into two groups. We recognize an evolutionary significant unit for Pyrenean– Cantabrian populations of Erebia euryale. Our results also illustrate that the evolutionary history of a species may be shaped by processes undetectable by using mtDNA alone.

Key words: allozymes – phylogeography – population genetics – Lepidoptera – evolutionarily signiﬁcant unit – mtDNA – cytochrome oxidase I (COI)

Introduction et al. 2006). However, some other surveys have revealed a The evolutionary history of many Palearctic taxa is undoubt- common lineage present all along the northern part of Spain edly linked to the environmental changes that occurred during and south-eastern France, e.g. the bank vole Myodes glareolus Quaternary climatic oscillations (Hewitt 1996). The so-called (Schreber, 1780) (Deffontaine et al. 2009). In addition, both Iberian refuge hosted several refugial areas that likely varied in the Pyrenean and Cantabrian Mountains currently show area and location during the different glacial stages (Go´mez contact zones for some previously separated lineages, such as and Lunt 2007). Some of them were used by populations that the capercaillie, Tetrao urogallus Linnaeus, 1758, in the surmounted the Pyrenees and expanded northwards when Pyrenees (Rodrı´guez-Mun˜ oz et al. 2007) and the fire salaman- climatic conditions were more suitable, such as those of the der, Salamandra salamandra (Linnaeus, 1758), in the Canta- zygaenid moth Aglaope infausta (Linnaeus, 1767) (Schmitt and brian Mountains (Garcıá-Parı´s et al. 2003). These studies Seitz 2004) or the southern water vole Arvicola sapidus Miller, comprise taxonomically and ecologically diverse species. The 1908 (Centeno-Cuadros et al. 2009). In contrast, other areas lack of a common pattern in their genetic structure may reflect did not contribute to the current genetic pattern of populations not only different evolutionary histories but also differences in outside the Iberian Peninsula, e.g. the grasshopper Chorthippus life history traits (e.g. dispersal ability, susceptibility to parallelus (Zetterstedt, 1821) (Cooper et al. 1995) or the Scots extreme temperatures) that may have shaped their phylogeo- pine Pinus sylvestris L. (Naydenov et al. 2007; Pyha¨ ja¨ rvi et al. graphical pattern. The study of closely related and recently 2008) and so the areas in question might therefore be diverged species has proved extremely useful for incorporating considered as ÔrelictsÕ rather than refuges (Petit et al. 2005), the role of the evolutionary constraints upon which intraspe- reflecting a model with lack of range movement. cific processes act in shaping the genetic structure of popula- To the best of our knowledge, the phylogeography of tions. In fact, several investigations have already showed that terrestrial taxa currently occurring along the northern Iberian closely related taxa may also show differing population mountain ranges (Cantabrian Mountains and Pyrenees) does histories (Pauls et al. 2009 and references therein). not show such a general genetic pattern. Some investigations One suitable model group for such a comparative approach have indicated that the mountains of northern Spain currently is the genus Erebia Dalman, 1816 (Nymphalidae: Satyrinae: host glacial relicts, e.g. the Convallariaceae plant Polygonatum Satyrini), which underwent a rapid diversification, like many verticillatum (L.) (Kramp et al. 2009). This is also the case for of its tribe (Pen˜ a et al. in press), possibly because of the spatial the chamois Rupicapra pyrenaica (Linnaeus, 1758) (Pe´rez et al. and temporal restriction of its species, which has resulted in a 2002) and the caddisfly Drusus discolor (Rambur, 1842) (Pauls large number of endemic taxa (Martin et al. 2000). This genus is considered to be composed of numerous glacial relicts Corresponding author: Marta Vila ([email protected]) because of the typical narrow elevational distribution of its Contributing authors: Neus Marı´-Mena ([email protected]), Ana Guer- many species in central and southern Europe (revised by rero ([email protected]), Thomas Schmitt ([email protected]) Tennent 2008). Some species of Erebia have already provided

J Zool Syst Evol Res (2011) 49(2), 119–132 120 Vila, Marı´-Mena, Guerrero and Schmitt interesting scenarios for studying incipient speciation in for one Pyrenean, one eastern European and three Alpine northern Iberia (e.g. Vila et al. 2006). localities. Derived from the above-mentioned two main For the present study, as a starting point for a comparative objectives, we also addressed the following questions: phylogeographical study on northern Iberian Erebia species, (3) Did the ancestors of the Cantabrian and Pyrenean we selected one of the Erebia species present in both the populations suffer drastic demographic changes during the Cantabrian and the Pyrenean Mountains: the large ringlet last glacial stages? Erebia euryale (Esper, 1805). As many as 19 of the approx- (4) Do the mitochondrial and nuclear data provide a concor- imately hundred recognized species of Erebia occur in dant pattern? the Iberian Peninsula. Most of them (17) are present in the Pyrenees, but eight Erebia species occur only along the northern mountains of Spain (Pyrenean and Cantabrian Material and Methods mountain ranges) (Garcıá-Barros et al. 2004). The Cantabrian Study species Mountains pose an interesting biogeographical framework for The large ringlet Erebia euryale occurs in several European mountain this group, as one of the two Iberian species of Erebia absent systems, from the Cantabrian Mountains to the Balkans, Urals and from the Pyrenees inhabits the Cantabrian Mountains. Kanin Peninsula. Alpine populations are biennial (Lafranchis 2000; The survival of Erebia euryale in mountain areas of northern Sonderegger 2005), but Spanish populations likely follow an annual Spain during glacial stages was postulated by Schmitt and cycle (H. Mortera and R. Vila, pers. comm.). According to Tolman and Lewington (1997), the speciesÕ habitats are grassy, flowery places Haubrich (2008), although their work proposed the existence in pinewood and spruce forest clearings and grassy slopes above the of a refugial area in the northern foot hills of the Pyrenees. treeline (elevational range = 750–2500 m a.s.l.). Larval host plants Another interesting issue raised by their study was the include Poaceae species (genera Sesleria, Festuca, Poa and Calama- suggestion that the most ancient split within the European grostis) as well as several species from the family Cyperaceae, i.e. Erebia euryale was one between the western Alpine lineage Carex flacca, Carex ferruginea. It shows marked local and regional (E. e. adyte) and all the other surveyed populations. The same morphological differences (Cupedo 2010). This has led to the definition of several subspecies, forms and variants, and even an Erebia euryale work suggested that the Pyrenean populations might represent species complex (as revised by Korshunov et al. http://www.jugan. a unique genetic entity within Erebia euryale; however, that narod.ru/satyridae1_eng.htm, accessed October 19, 2010). This taxon study included only one population from the eastern Pyrenees, is classified as of Least Concern by the European Red List of which limited the possible interpretations. Butterflies (van Swaay et al. 2010). Schmitt and Haubrich (2008) The hypothesis of a single and distinct genetic lineage for the reported the following four major phenotypic groups, called subspecies Cantabrian and Pyrenean populations of Erebia euryale is E. e. isarica Heyne, 1895 (widespread in western Europe as far East as supported by classical intraspecific taxonomy (Appendix S1). the northern Carpathians), E. e. adyte (Hu¨ bner, 1822) (restricted to the western and south-western Alps), E. e. ocellaris Staudinger, 1861 However, in the light of the scattered distribution of the species (endemic to a particular area of the south-eastern Alps), and in northern Spain and the intraspecific taxonomy, we also E. e. syrmia Fruhstorfer, 1910 (widespread in the southern and eastern expected a low but significant degree of genetic differentiation Carpathians as well as in the Balkan Peninsula). within that lineage. In the present study, we aimed to: (1) describe the population structure of the westernmost part of Sampling the distribution area of Erebia euryale (i.e. the Cantabrian A total of 218 individuals of E. euryale were collected at six localities Mountains), where the species occurs in scattered localities; and from northern Spain (Fig. 1). For conservation purposes, only males were captured. Between 33 and 40 specimens per locality were caught (2) determine whether the Cantabrian and Pyrenean localities at these Cantabrian sites (Fig. 1, Tables 1 and 2, Table S11). The should be considered as a single evolutionarily significant unit imagos were hand-netted between July 6 and 23, 2006, and stored in or not (following Fraser and Bernatchez 2001). liquid nitrogen until analysis. We studied the allozyme polymorphisms of six populations from the Cantabrian Mountains and compared the results against populations from the Pyrenees, Alps and eastern Laboratory procedures European Mountains. Furthermore, we sequenced 816 bp of We genotyped the samples for 17 allozyme loci as described in Schmitt the mtDNA COI gene for these Cantabrian populations and and Haubrich (2008). The obtained dataset was compared against their

Fig. 1. Iberian distribution area of Erebia euryale (yellow circles, adapted from Garcı´a-Barros et al. 2004) and sampling locations surveyed for the present genetic (allozymes + mtDNA) study. Allozyme data from La Gle` be were published in Schmitt and Haubrich (2008). Readers are referred to Fig. S10 for a map displaying the other European localities from where mitochondrial results are published in the present study J Zool Syst Evol Res (2011) 49(2), 119–132 2010 Blackwell Verlag GmbH Phylogeography of Erebia euryale 121

results for 11 E. euryale populations sampled in the Pyrenees (one locality), the Alps (seven sites), the southern Carpathians (two areas) and the Rila Mountains (one population). Data from the Alpine number accession Genbank locality of Scho¨ neck (Austria) was not included in the analyses because HQ176010 HQ176011 HQ176012 HQ176013 HQ176014 of small sample size (n = 7). We also sequenced the second half of the mitochondrial cytochrome E N

¢ c oxidase subunit I (COI) gene in 5–10 individuals per Cantabrian ¢ 811 HQ176017 HQ176018 HQ176019 31 21 locality. That part of the COI gene is the most variable, according to

25 prior literature (Lunt et al. 1996) and prior experience on species of 45 Erebia (Vila et al. 2005). Genomic DNA was extracted from adult thoracic tissue using the Genelute Mammalian Genomic DNA Miniprep kit (Sigma-Aldrich, Saint Louis, MO, USA). Primers and E N ¢ ¢ PCR conditions are described in Vila and Bjo¨ rklund (2004). To obtain 51 HQ176020 HQ176021 34 15 a wider pattern of the genetic diﬀerentiation of these samples, we also 13 47 sequenced this marker in 5–10 individuals from some of the populations analysed by Schmitt and Haubrich (2008) (Table 1). The total number of sequenced individuals was 90. The 816-bp sequences used for this survey start at position 706 of the COI gene E N ¢ ¢ (reference sequences for this alignment were GenBank accession 5 HQ176016 19 48 numbers DQ102703 and EF621724) and correspond to positions 12 46 2182–2997 of the mitochondrial genome of Drosophila yakuba Burla, 1954 (accession no. X03240). New haplotypes were deposited in

(rows) from each sampled location (columns). Geographical GenBank (Table 1). re Kalkstein Obertauern Sinaia ` N E ¢ ¢ Ise Õ 27 10 HQ176015 59 Statistical analyses: allozyme dataset 6 45 Allozyme alleles were labelled according to their relative mobility,

Erebia euryale starting with Ô1Õ for the slowest. Allele labels were the same as those used by Schmitt and Haubrich (2008). Consequently, some alleles are be Val d N ` E ¢ ¢ absent from the present dataset (e.g. alleles 1 and 2 at Apk locus). 40 13

Allele frequencies and parameters of genetic diversity [i.e. mean 2 42 number of alleles per locus (A), expected and observed heterozygosity (He, Ho), total percentage of polymorphic loci (Ptot) and percentage of polymorphic loci with the most common allele not exceeding 95% (P95)] were computed with G-Stat (Siegismund 1993). Analyses of N, W ¢ ¢ Hardy–Weinberg (HW) segregations were performed for all loci and 1 06 32

populations. We used the score test (U-test) to evaluate whether 4 43 observed deviations were due to heterozygote deficit both at population and at locus level (tests based on 1560 randomizations). U-tests calculated with statistica were also used to test for differences in genetic diversity values from different populations. We performed N, W ¢ ¢ exact probability tests to assess the adjustment to HW expectations for 59 24

each locus in each population. An unbiased estimate of the exact p- 6 42 value for those 45 tests was computed using the Markov Chain Monte Carlo Method as implemented in the software genepop 4.0.10 (Rousset 2008). Weighted F-statistics were calculated using the estimators described by Weir and Cockerham (1984). Their values and signiﬁ- N, W ¢ ¢ cance were estimated in fstat 2.9.3 (Goudet 1995) after 10 000 01 13 randomizations. We also reported estimates of the pairwise estimator 6 43 of diﬀerentiation Dest (Jost 2008) across loci calculated using smogd 2.6 (Crawford 2010). We also calculated locus-by-locus amovas, hierarchical genetic N, W ¢

¢ variance analysis, as well as linkage disequilibrium as implemented in 04

25 arlequin

3.5.1.2 (Excoﬃer and Lischer 2010). Signiﬁcance was 5

43 obtained after 1023 permutations. NeiÕs standard genetic distances (Nei 1972), neighbor-joining (NJ) phenograms and bootstraps based on 1000 iterations were calculated with PHYLIP (Felsenstein 1989). We assessed population structuring using the simulated annealing N, W ¢ ¢ procedure developed by Dupanloup et al. (2002). This approach 59 45

allows the deﬁnition of groups of geographically homogeneous 5 42 populations that maximize the proportion of total genetic variance because of diﬀerences between population groups. For this, we ran a samova (Spatial Analysis of Molecular Variance) 1.0 using 100 N,

W k

¢ simulated annealing processes for values from 2 to 16. ¢

04 The presence of limited gene ﬂow can be indicated by a pattern of 62

4 isolation by distance across the surveyed populations. Following 43 San Glorio Pajares San Isidro Somiedo Leitariegos Pandetrave La Gle Rousset (1997), we performed a Mantel test as implemented in ibdws 3.15 (Jensen et al. 2005) to assess the correlation between FST ⁄ (1 ) FST) and the geographical distance for both the whole European dataset and the Pyrenean–Cantabrian partition. Signiﬁcance of the Mantel test was evaluated after 10 000 randomizations. We calculated geographical distance (in kilometres) for each pair of Table 1. Geographical and frequency distribution of the 13 mitochondrial haplotypes [cytochrome oxidase I (COI)] of coordinates are indicated below the name of the sampling locality Haplotype name H01H02H03H04H05H06 10 8 1 1 1 7 8 1 1 5 7 10 AY346221 H09 H10 H11 H12 H13 H08 H07

J Zool Syst Evol Res (2011) 49(2), 119–132 2010 Blackwell Verlag GmbH 122 Vila, Marı´-Mena, Guerrero and Schmitt

Table 2. Expected heterozygosity (He), observed heterozygosity (Ho), percentage of polymorphic loci [total (Ptot) and with the most common allele not exceeding 95% (P95)], mean number of alleles per locus (A), sample size (N) for all allozyme loci (including monomorphic) and samples analysed of Erebia euryale. For means of comparison, the average values of ten samples of Erebia euryale from other regions of Europe are given at the bottom of the table. The p values of U-tests between the Cantabrian samples and the other European as well as the south-eastern European samples are presented

Sample site He Ho Ptot [%] P95 [%] AN San Glorio 0.159 0.143 52.9 35.3 2.18 33 Pajares 0.179 0.176 58.8 35.3 2.06 36 San Isidro 0.149 0.145 52.9 47.1 1.94 37 Somiedo 0.145 0.133 47.1 41.2 1.71 35 Leitariegos 0.137 0.103 47.1 29.4 1.71 40 Pandetrave 0.178 0.190 47.1 41.2 1.94 37 Cantabrian mean 0.158 0.148 51.0 38.3 1.92 36.3 ±SD ±0.018 ±0.031 ±4.8 ±6.2 ±0.19 2.3 Mean Schmitt and Haubrich (2008) 0.156 0.152 68.5 41.2 2.33 34.6 ±SD ±0.029 ±0.03 ±14.7 ±12.6 ±0.45 ±9.8 p(U-test) 0.801 0.762 0.018 0.639 0.044 0.571 Mean south-eastern Europe 0.178 0.173 84.3 51.0 2.84 39.7 Schmitt and Haubrich (2008) ±SD ±0.011 ±0.008 ±6.8 ±12.2 ±0.30 ±0.6 p(U-test) 0.197 0.302 0.017 0.085 0.019 0.065 sampling locations with Geographic Distance Matrix Generator 1.2.3 differences (k) were calculated in DNAsp 5.1 (Librado and Rozas (PJ Ersts, http://biodiversityinformatics.amnh.org/open_source/gdmg, 2009). accessed October 19, 2010). The phylogeny of the haplotypes was inferred using the statistical To disentangle the population structure pattern both across Europe parsimony (SP) criterion (Templeton et al. 1992). The 95% SP and within the Pyrenean–Cantabrian Mountains (North Spain), we network was calculated using TCS 1.2 (Clement et al. 2000). To have used the program structure 2.3.3 (Pritchard et al. 2000). This a reference to calibrate the extent of divergence between haplogroups, software traditionally used model-based Bayesian clustering of indi- we rooted the resulting network with an homologous sequence from viduals without using prior population definitions. Version 2.3.3 Erebia ligea (accession number AY346224). incorporates the possibility of using models with informative priors, The demographic history of samples belonging to the Pyrenean– i.e. allowing the software to use information about sampling locations. Cantabrian lineage was inferred using different methods. First, a This may be useful to, for instance, assist the clustering when the signal mismatch distribution of the pairwise genetic differences (Rogers and of structure is relatively weak (e.g. significant FST between sampling Harpending 1992) was conducted, and their goodness-of-fit to a locations, but no detection of structure as from the standard structure sudden expansion model was tested using parametric bootstrap models) (Hubisz et al. 2009). We first used this method on the approaches (1000 replicates). The sum of squared deviations (SSD) European allozyme dataset (592 individuals, 17 polymorphic loci, 16 between the observed and expected mismatch distributions was used to localities) under standard settings and the admixture model, trying the assess the significance of the test. In addition, the significance of assumption of correlated allele frequencies, without considering population expansion was tested using TajimaÕs D and FuÕs Fs information about the population of origin of the samples. We tried neutrality tests. All these calculations were performed in arlequin the admixture model, where the individuals may have mixed ancestry, 3.5.1.2. Mismatch analyses were also used to estimate the approximate as (i) it is recommended as starting point, and (ii) the hypothesis of one timing of expansion of the Pyrenean–Cantabrian lineage of Erebia or several hybrid zones for Erebia euryale seemed plausible at the euryale. We used the relationship s = 2ut (Rogers and Harpending European scale. We used a burn-in of 50 000 iterations followed by 1992) and the approach taken by Horn et al. (2009) and Hammouti 100 000 iterations for parameter estimation. Each simulation was run et al. (2010). Therefore, we performed these calculations using both five times, exploring values for K (the total number of clusters to be l = 1.15*10)8 and l = 5*10)9 per year per site. constructed in a given simulation) ranging from 1 to 17. To select the most appropriate number of groups, we followed Evanno et al. (2005). We then ran structure for the Pyrenean–Cantabrian allozyme Results dataset (258 individuals, 13 polymorphic loci, 7 locations), under the Cantabrian allozyme dataset no admixture model (more powerful at detecting subtle structure standard settings) and using the LOCPRIORS option. The rest of the The study of 218 adult males of Cantabrian Erebia euryale parameters and interpretation of results were set as above, except for revealed 13 of the 17 analysed loci as polymorphic. Loci Fum, the values of K to be explored (from one to eight). Gpdh, Mdh1 and Pk were monomorphic in the six surveyed As there were suspicions of inbreeding in the locality of Leitariegos, localities, as they were in the Pyrenean locality of La Gle` be, pairwise relatedness values between individuals were calculated for this investigated by Schmitt and Haubrich (2008). The highest site using the method by Lynch and Ritland (1999), as implemented in identix 1.1.5 (Belkhir et al. 2002). We estimated relatedness (r) using number of six different alleles per locus was observed for two the multilocus genotypes from the eight loci polymorphic at Leitari- loci (Pgi and Pgm), and the average was 2.94 (±1.75 SD). The egos. After estimation of r and its variance, we performed 1000 allele frequencies of the six Cantabrian populations analysed permutations to tests the null hypothesis of no relatedness. This are shown in Table S2. procedure can be applied to both arithmetic mean and variance of the To describe the extent of genetic diversity and compare it above-mentioned estimator. with the data from Schmitt and Haubrich (2008), we calculated the following estimators for the 218 Cantabrian individuals Statistical analyses: mtDNA dataset based on allele frequencies of the 17 surveyed loci: (i) the mean number of alleles per locus per population (A) ranged from DNA sequences were aligned by eye. Alignments were straightforward. Haplotypes and their frequencies, standard indices of genetic variation 1.71 to 2.18, with a mean of 1.92 (±0.19 SD), (ii) the such as the number of segregating sites (S), nucleotide diversity per percentage of polymorphic loci with the most common allele gene (p), haplotype diversity (h) and the average number of nucleotide not exceeding 95% (P95) ranged from 29% to 47% with a J Zool Syst Evol Res (2011) 49(2), 119–132 2010 Blackwell Verlag GmbH Phylogeography of Erebia euryale 123

mean of 38% (±6 SD), and (iii) the total percentage of average over 13 loci (variance component = 0.111; FST polymorphic loci (Ptot) ranged from 47% to 58%, mean 51% (FST) = 0.062, p = 0.003). This result agreed with the FST (±5 SD); (iv) average expected heterozygosity (He) was 0.158 (h) value = 0.077 (95% CI = 0.043–0.116). The genetic (±0.218 SD) ranging from 0.137 to 0.179, (v) average variance within populations was moderate among individuals observed heterozygosity (Ho) was 0.148 (±0.031 SD) varying (variance component = 0.083, FIS 0.077, p < 0.001), but from 0.103 to 0.176. The means for Ptot and A were relatively high within individuals (variance compo- significantly lower than the ones of the eleven populations nent = 1.246). The unbiased genetic distances between the from other European mountain ranges, whereas the other six Cantabrian populations ranged from 0.007 to 0.038 with a three parameters and the mean number of individuals analysed mean of 0.018 (±0.008 SD). were not. This pattern was more pronounced when compared against the samples from the genetically richest region of south-eastern Europe found by Schmitt and Haubrich (2008). Comparison with published data: European scale Details are shown in Table 2. The NJ tree calculated using the Cantabrian and the samples We repeated the descriptive calculations for the Cantabrian surveyed by Schmitt and Haubrich (Fig. 2) did not show a dataset presented in this work and provided by the 13 straightforward correspondence between its topology and the polymorphic loci. The average of alleles per locus (overall geographical location of the samples. The most closely related populations) was 2.19 (±1.17 SD), ranging from 1.92 ± 0.95 population to the Cantabrian samples was the Pyrenean at Leitariegos to 2.54 ± 1.39 at San Glorio. Data on allelic locality of La Gle` be (distance: 0.031 ± 0.016 SD), followed by richness, a more accurate descriptor as it takes difference in the eastern Alpine sites (distance: 0.048 ± 0.017 SD) and the sample size into account, are provided in Table 3. Overall, all south-eastern European ones (distance: 0.053 ± 0.018 SD); populations and loci conformed to Hardy–Weinberg expecta- the western Alps samples showed the highest genetic distances tions (HWE) (exact test, v2 = 85.76.84, df = 90, p > 0.05). Using the probability tests, we found a significant deviation from HWE in five cases combined over loci and populations. Based on chance alone, we would expect to find three significant results at the 0.05 level out of the 45 tests. None of those five tests were significant after sequential Bonferroni correction. When we tested for HWE using the score U (global) test and heterozygote deficit as null hypothesis, the only locus globally departing from HWE after correction for multiple tests was PGM (Table S3). The only population that significantly deviated from HWE was Leitariegos, which congruently showed a significant inbreeding coefficient (FIS = 0.249, p < 0.001, Table 3, Table S4). Data from Leitariegos did not significantly depart from the null hypothesis of no relatedness (average r = )0.025, variance = 0.143) neither when the test was performed on the means nor the variances of the relatedness coefficient (p > 0.05). The same result was obtained when permutation type was changed from ÔgenotypesÕ to ÔallelesÕ. No significant genetic linkage disequi- Fig. 2. Neighbor-joining phenogram based on NeiÕs standard genetic librium was detected between any pair of loci. There was some distances (Nei 1972) of the six populations of Erebia euryale from the subtle structuring within the Cantabrian dataset, as revealed Cantabrian Mts. Ten populations from the Pyrenees, the Alps, the by the test for population differentiation (log-likelihood G southern Carpathians and the Rila Mountains (Schmitt and Haubrich 2008) were included. Distances were calculated using the results pro- statistic not assuming random mating within samples, vided by 17 allozyme loci. Bootstrap values over 50% are given at the p < 0.0001) (Goudet et al. 1996). nodes. Cantabrian localities are marked in italics. Those localities from The genetic variance among Cantabrian localities was which some individuals were sequenced for the cytochrome oxydase I relatively high (locus-by-locus amova results as a weighted gene appear underlined

Table 3. Genetic diversity within populations of Erebia euryale as from 13 polymorphic allozyme loci. Mean n, average number of individuals analysed; proportion of missing data per locus for each population; AR, allelic richness (based on 27 diploid individuals); PA, number of private alleles; HWE, deviations from Hardy–Weinberg equilibrium; signiﬁcance after sequential Bonferroni correction is marked in bold. NS, p > 0.05; *p < 0.05; **p < 0.01; ***p < 0.001; HE, unbiased expected and HO, observed heterozygosities; FIS, inbreeding coefﬁcient. Standard errors are given after symbol ±

Mean n Missing data (locus) AR PA HWE HE HO FIS San Glorio 32.54 ± 0.31 0.12 (6Pgdh) 2.45 ± 1.33 1 (G6P) * 0.207 ± 0.247 0.187 ± 0.218 0.096 NS Pajares 36.00 ± 0.00 0 2.28 ± 1.24 0 NS 0.234 ± 0.251 0.231 ± 0.253 0.013 NS San Isidro 36.77 ± 0.17 0 2.15 ± 1.22 0 NS 0.196 ± 0.214 0.19 ± 0.21 0.028 NS Somiedo 34.23 ± 0.23 0.08 (Apk) 1.89 ± 0.93 0 NS 0.19 ± 0.225 0.174 ± 0.2 0.088 NS Leitariegos 39.77 ± 0.12 0 1.82 ± 0.89 0 *** 0.178 ± 0.234 0.134 ± 0.162 0.2491* Pandetrave 35.23 ± 0.96 0.16 (6Pgdh), 2.11 ± 1.11 1 (MD2) NS 0.229 ± 0.241 0.247 ± 0.257 )0.077 NS 0.24 (Mdh), 0.18 (Pgm)

1Signiﬁcant deﬁcit of heterozygotes, based on 1560 randomizations. Indicative adjusted nominal level (5%) was 0.00064. p = 0.0006. J Zool Syst Evol Res (2011) 49(2), 119–132 2010 Blackwell Verlag GmbH 124 Vila, Marı´-Mena, Guerrero and Schmitt

Table 4. Below diagonal: Pairwise FST (Weir and Cockerham 1984) values between the 16 localities of Erebia euryale as revealed by the allozyme dataset (17 loci). Signiﬁcant FST values are displayed in bold. Indicative adjusted nominal level (5%) for multiple comparisons = 0.00042. Above diagonal: Harmonic mean of the estimator of actual differentiation Dest (Jost 2008). Glb, La Gle` be; StM, St. Martin; Vis, Val dÕIse` re; Pat, Partnun; Kal, Kalkstein; Obe, Obertauern; Tra, Trauneralm; Gro, Groapa Seaca; Sin, Sinaia; Ril, Rila Mountains; Glo, San Glorio; Paj, Pajares; Isi, San Isidro; Som, Somiedo; Lei, Leitariegos; Pan, Pandetrave. Diagonal: Population speciﬁc FST indices, in italics Western Alps Eastern Alps East Europe Cantabrian Pyrenean

StM Vis Pat Kal Obe Tra Gro Sin Ril Glo Paj Isi Som Lei Pan Glb

StM 0.209 0.004 0.006 0.003 0.006 0.005 0.012 0.010 0.009 0.014 0.013 0.015 0.009 0.016 0.020 0.026 Vis 0.121 0.214 0.004 0.012 0.014 0.013 0.014 0.014 0.014 0.018 0.019 0.023 0.012 0.019 0.021 0.026 Pat 0.099 0.171 0.209 0.010 0.010 0.010 0.007 0.007 0.007 0.010 0.010 0.023 0.016 0.027 0.025 0.018 Kal 0.205 0.407 0.294 0.209 0.003 0.001 0.011 0.012 0.011 0.007 0.009 0.009 0.008 0.012 0.014 0.013 Obe 0.221 0.381 0.257 0.049 0.207 0.004 0.014 0.014 0.012 0.006 0.010 0.009 0.011 0.017 0.015 0.014 Tra 0.205 0.386 0.278 0.022 0.042 0.208 0.010 0.010 0.008 0.005 0.009 0.012 0.009 0.018 0.016 0.013 Gro 0.196 0.325 0.179 0.175 0.146 0.151 0.207 0.000 0.000 0.012 0.011 0.015 0.011 0.018 0.017 0.007 Sin 0.182 0.315 0.177 0.163 0.131 0.147 )0.002 0.207 0.000 0.011 0.011 0.013 0.012 0.020 0.018 0.010 Ril 0.179 0.315 0.177 0.156 0.133 0.137 0.003 0.003 0.206 0.010 0.009 0.016 0.012 0.021 0.018 0.009 Glo 0.273 0.415 0.272 0.133 0.135 0.144 0.147 0.165 0.151 0.209 0.002 0.006 0.004 0.006 0.003 0.006 Paj 0.270 0.404 0.258 0.142 0.118 0.147 0.128 0.128 0.128 0.045 0.206 0.006 0.002 0.007 0.006 0.003 Isi 0.365 0.509 0.403 0.266 0.285 0.291 0.286 0.295 0.277 0.108 0.151 0.209 0.003 0.004 0.005 0.016 Som 0.261 0.411 0.314 0.137 0.145 0.159 0.151 0.151 0.155 0.063 0.050 0.131 0.209 0.001 0.001 0.004 Lei 0.325 0.463 0.377 0.215 0.234 0.244 0.245 0.246 0.239 0.104 0.077 0.106 0.027 0.209 0.001 0.005 Pan 0.302 0.429 0.336 0.183 0.188 0.202 0.199 0.205 0.194 0.054 0.061 0.088 0.030 0.026 0.209 0.005 Glb 0.331 0.456 0.320 0.222 0.185 0.193 0.148 0.167 0.165 0.099 0.070 0.238 0.097 0.140 0.104 0.208 against the Cantabrian Mountains (distance: 0.096 ± 0.022 SD). Hierarchical variance analyses supported this structure (FCT values against: Pyrenees 0.060, p = 0.134; eastern Alps 0.143, p < 0.0001; south-eastern Europe 0.166, p < 0.001; and western Alps 0.287, p < 0.001). There was significant structuring for the whole European dataset, as revealed by the test for population differentiation (log-likelihood G statistic not assuming random mating within samples, p < 0.0001) and the relative differentiation provided by the 95% confidence interval for FST (0.096–0.35). The pairwise comparisons involving maximum differentiation mostly agreed between FST and Dest estimators (Val dÕIse` re Fig. 3. Results of the spatial analysis of molecular variance showing versus San Isidro: FST = 0.509, Dest = 0.023) (Table 4). The the genetic affinity between the Cantabrian and Pyrenean samples. The minimum differentiation was congruently determined by both most likely subdivision of the whole distribution area into four groups, estimators among the eastern European localities of Sinaia, when the increment of FCT was the largest (DFCT = 0.0124). These Groapa Seaca and Rila Mountains. All sampling sites four groupings corresponded to: [Pyrenean–Cantabrian samples] [W contributed similarly to the average relative differentiation, Alps] [SE Europe], and [E Alps] i.e. FST indices were fairly similar for the 16 localities ranging from 0.206 (Rila Mountains) to 0.214 (Val dÕIse` re) (Table 4). Therefore, no particular evolutionary constraint (e.g. selec- decrease, and (iii) DFCT was the largest (DFCT = 0.012). These tion) left a deep track in the allozyme dataset (Weir and Hill four groupings corresponded to: [La Gle` be plus the six 2002). Cantabrian sites] [St. Martin-Val dÕIse` re-Partnun] [Groapa The European nuclear dataset did not conform to the Seaca-Sinaia-Rila Mountains], and [Kalkstein-Obertauern- isolation-by-distance model, as there was no significant corre- Trauneralm]. The genetic variance among groups was 18.7%, lation between genetic differentiation in relation to geograph- among populations within groups 6.4% and within popula- ical distance (Z = 39647.54, r = 0.15, one-sided p = 0.104) tions 75% with FCT = 0.187 and FSC = 0.079 (all (Fig. S5). p < 0.001). The genetic variance among the 16 European localities was The same four groups were obtained as second most likely relatively high (locus-by-locus amova results as a weighted clustering scheme when applying the Bayesian algorithm average over 17 loci): variance component = 0.342; FST: implemented in structure 2.3.3 to the whole allozyme 0.206, p < 0.001). The genetic variance within populations dataset. Interestingly, the most likely number of clusters (K) was moderate among individuals (variance compo- was two, separating the Pyrenean–Cantabrian localities (W nent = 0.055, FIS 0.042, p = 0.002), but high within individ- Europe) from the remaining surveyed localities. Two localities uals (variance component = 1.262). showed very homogeneous gene pools: Val dÕIse` re (W Alps) The spatial analysis of molecular variance also supported and San Isidro (W Europe) (Fig. 4). the genetic affinity of the Cantabrian and Pyrenean samples. Significant differences in average gene diversity and allelic This analysis indicated a most likely subdivision of the whole richness were found among these four groupings (two-sided distribution area into four groups (K = 4, Fig. 3), when (i) test, 5000 permutations, p = 0.001 for gene diversity, and FCT values approached a plateau, (ii) FST showed a steep p = 0.006 for allelic richness). The average gene diversity was J Zool Syst Evol Res (2011) 49(2), 119–132 2010 Blackwell Verlag GmbH Phylogeography of Erebia euryale 125

(a)

(b)

(c)

(d)

Fig. 4. Two (a) and four (b) most likely groupings of 592 individuals of Erebia euryale as revealed by running the Bayesian structure 2.3.3 with a 17 allozyme loci dataset and under the admixture model and correlated allele frequencies. The Pyrenean population (La Gle` be) always clustered with the Cantabrian localities. Bayesian clustering of 258 individuals of the Pyrenean and Cantabrian localities as revealed by 13 polymorphic loci using the LOCPRIOR option and (c) non-admixture model, correlated allele frequencies, or (d) non-admixture model, independent allele frequencies. Each individual is represented as a bar, divided into K colours, where K is the number of most likely clusters inferred following Evanno et al. (2005)

Table 5. Below diagonal: Pairwise FST (Weir and Cockerham 1984) values between the four major genetic groups of Erebia euryale as revealed by the allozyme dataset. All FST values resulted signiﬁcant after correction for multiple comparisons [indicative adjusted nominal level (5%) = 0.0083 calculated after 120 permutations]. Above diagonal: Harmonic mean of the estimator of actual differentiation Dest across loci (Jost 2008). The numbers after regions correspond to the number of complete multilocus genotypes. Diagonal: average gene diversity and allelic richness across loci

W Alps E Alps E Europe Pyrenean–Cantabrian (89) (103) (117) (229)

W Alps 0.118, 1.94 0.005 0.008 0.012 E Alps 0.258 0.160, 2.19 0.011 0.008 E Europe 0.203 0.137 0.178, 2.56 0.013 Pyrenean–Cantabrian 0.294 0.146 0.160 0.157, 1.84 higher in the eastern than in the western Alps groups (one- correlation between genetic differentiation in relation to sided test, 5000 permutations, p = 0.025). It was also higher in geographical distance (Z = 649.47, r = 0.35, one-sided the eastern European gene pool than in the western Alps p = 0.21; Fig. S6), not even when using the logarithm of (p = 0.001) (Table 5). Allelic richness was significantly higher geographical distance (Z = 5.07, r = 0.31, p = 0.23). in eastern Europe than in both the western Alps (p = 0.005) Regarding population-wise comparisons, San Isidro and the Pyrenean–Cantabrian gene pool (p = 0.0006) appeared as the most differentiated population. The extent of (Table 5). significant differentiation was moderate (FST > 0.1) for the four pairs where San Isidro was involved (i.e. San Isidro-San Glorio, San Isidro-Pajares, San Isidro-Somiedo and San Comparison with published data: Pyrenean–Cantabrian lineage Isidro-Leitariegos), reaching 0.238 between San Isidro and There was also a remarkable population structuring among the the Pyrenean locality of La Gle` be. The lowest degree of seven northern Iberian localities surveyed, as revealed by the differentiation resulted between Leitariegos and Somiedo (FST) FST value 0.095 (95% CI = 0.059–0.123), and the test for or Somiedo-Leitariegos-Pandetrave (Dest) (Table 4). population differentiation (log-likelihood G statistic not Exploratory calculations to identify the genetic groups assuming random mating within samples, p < 0.001). within the Pyrenean–Cantabrian Erebia euryale through the The Pyrenean–Cantabrian nuclear dataset did not conform Bayesian clustering implemented in structure 2.3.3 always to the isolation-by-distance model, as there was no significant resulted in a most likely number of clusters (K) of two (data J Zool Syst Evol Res (2011) 49(2), 119–132 2010 Blackwell Verlag GmbH 126 Vila, Marı´-Mena, Guerrero and Schmitt not shown). This was inferred using only genetic information, dismissing the geographical origin of samples and by examin- ing the graphical output from the software and by considering the logarithmic probability of data [lnP(D)] for different numbers of K as well as the statistic DK (Evanno et al. 2005), which considers the rate of change in lnP(D) among successive K values. As some structure was expected (see significant FST values in Table 4), and our dataset might show a relatively weak signal of structure, we ran again the clustering algorithm but using the sampling locations as prior information to assist the grouping (Hubisz et al. 2009). Four most likely clusters were inferred, regardless of using the correlated or independent frequency models. In both cases, the localities of San Isidro and La Gle` be were clearly differentiated (Fig. 4). Interestingly, both La Gle` be and San Isidro appeared as the most homogeneous gene pools within the surveyed localities in northern Iberia. mtDNA dataset Fig. 5. Statistical parsimony network showing abundance and occur- Thirteen haplotypes were obtained for the COI fragment rence of each cytochrome oxidase I haplotype obtained for the 89 analysed in 90 individuals from the six surveyed Cantabrian individuals of Erebia euryale. Circle size reflects the frequency of localities plus one from the Pyrenees (La Gle` be), another one haplotype (maximum: 53; minimum: 1). Solid lines connecting haplo- from western Alps (Val dÕIse` re), two from eastern Alps types represent a single mutational event, regardless of their length. Black rectangles represent missing or theoretical haplotypes. Leitari- (Obertauern and Kalkstein) and one from south-eastern egos (Lei), Somiedo (Som), Pajares (Paj), San Isidro (Isi), Pandetrave Europe (Sinaia) (Table 1). These haplotypes were defined by (Pan), San Glorio (Glo), La Gle` be (Glb), Val dÕIse` re (Vis), Kalkstein 13 variable sites along the analysed COI fragment (816 bp (Kal), Obertauern (Obe), Sinaia (Sin). The Erebia euryale intraspecific length). Five of them corresponded to singleton variable sites, statistical parsimony network (95%) had a connection limit of six. The whereas the remaining eight were parsimony informative (two network was rooted using Erebia ligea as an outgroup variants). Eleven substitutions were transitions. Twelve substitutions involved synonymous changes. The only replace- ment change was a first codon position transition that differentiated samples from Sinaia, Obertauern and Kalkstein haplotype (H01) in the Cantabrian Mountains, was the only from the western populations. Overall, haplotype diversity was one present in the Pyrenean locality (La Gle` be). The Canta- (h) = 0.632 ± 0.055, nucleotide diversity brian haplotypes grouped in a star-like shape. Pajares was the (p) = 0.0021 ± 0.0003, and the average number of nucleotide most diverse locality (four haplotypes, two of them shared differences (k) = 1.7563 (Table 6). with Somiedo), followed by San Isidro, Pandetrave and Figure 5 shows the statistical parsimony network and Somiedo (two haplotypes). Leitariegos (westernmost locality), geographical occurrence of the obtained mitochondrial vari- San Glorio (easternmost Cantabrian site) and the Pyrenean ants. Haplotypes differing by up to 12 mutations (including the population only yielded the most common haplotype. H01 was outgroup) could be connected with 95% confidence as multiple only one substitution apart from the one present in Val dÕIse` re substitutions did not occur at any particular nucleotide (western Alps). Sinaia (SE Europe, three haplotypes) was the position. When focussed, Erebia euryale, the most common only locality containing haplotypes differentiated by more than one mutational step. One of its haplotypes (H11) was phylogenetically closer to haplotype H13 from Obertauern (E Alps) than to the others from Sinaia. In fact, the two populations from the eastern Alps (Kalkstein and Obertauern) Table 6. Measurements of genetic diversity for Erebia euryale as from appeared closer to Sinaia than to each other. the mitochondrial dataset and according to sampling location. n, number of sequenced individuals; S, number of segregating sites; NH, Mismatch analyses for the Pyrenean–Cantabrian lineage number of haplotypes; h, haplotypes diversity; p, nucleotide diversity; were consistent with the sudden expansion model and k, average number of nucleotide differences (SDD = 0.002, p = 0.44) and showed a unimodal distribution that closely fitted the expected distributions (Fig. S6). FuÕs Population n SNH h p k Fs and TajimaÕs D statistics were both negative and significant Leitariegos 5 0 1 0 0 0 (Fs=)6.057, p < 0.001; D = )1.906, p = 0.002). The Somiedo 9 1 2 0.2222 0.0003 0.2222 approximate timing of demographic expansion t was estimated Pajares 9 3 4 0.5833 0.0008 0.6667 for the Pyrenean–Cantabrian lineage assuming an annual cycle San Isidro 8 1 2 0.25 0.0003 0.25 for the species (s = 3.0, 95% CI = 0.35–3.5), and trying two Pandetrave 8 1 2 0.25 0.0003 0.25 San Glorio 10 0 1 0 0 0 mutation rates used by prior literature. Therefore, the expan- La Gle` be 10 0 1 0 0 0 sion of the Pyrenean–Cantabrian lineage was estimated to date Val dÕIse` re 10 0 1 0 0 0 back to either 79 923 years before present (yBP) (95% Kalkstein 5 0 1 0 0 0 CI = 9324–93 243 yBP; calculations using l = 1.15*10)8) Obertauern 6 1 2 0.3333 0.0004 0.3333 or 183 823 yBP (95% CI = 21 446–214 460 yBP; calculations Sinaia 10 3 3 0.3778 0.0007 0.6 using l = 5*10)9).

J Zool Syst Evol Res (2011) 49(2), 119–132 2010 Blackwell Verlag GmbH Phylogeography of Erebia euryale 127

Discussion results after long-lasting but relatively mild bottlenecks over generally small populations. Such diffuse reductions in popu- Population structure of Cantabrian Erebia euryale lation size mostly affect allele frequencies, but much less The 218 individuals from the Cantabrian Mountains showed genetic diversity. San Isidro is a suitable candidate to match significant structuring as for the nuclear dataset, but they were that case. On the other hand, genetic impoverishment is monomorphic for four out of the 17 surveyed allozyme loci usually produced by strong, but relatively short bottlenecks, (see Appendix S7 for further discussion on the levels of genetic mostly affecting the number of alleles and, to a lesser extent, diversity and resolution of the allozyme dataset). Leitariegos polymorphic loci (England et al. 2003). This might be the case was the only locality departing from HWE due to a significant for Somiedo and Leitariegos. Our nuclear dataset did not track deficit of heterozygotes. However, it is likely that such a any bottleneck (data not shown), so this stochastic scenario deviation was not due to relatedness within the surveyed remains speculative. Future research surveying more northern sample. We rather advocate null alleles as a more likely Iberian populations and using more variable genetic markers is explanation for such an excess of homozygotes (Appendix S7). needed to unravel the processes (stochastic demographic Three Cantabrian clusters were inferred: Leitariegos-Somi- effects and ⁄ or landscape features) shaping the observed genetic edo-Pandetrave, San Isidro, and Pajares-San Glorio. Indeed, pattern for the Cantabrian large ringlets. there was no correlation between genetic and geographical distance among these localities. Such a pattern of genetic differentiation may be the result of genetic drift acting upon Definition of an Evolutionarily Significant Unit in northern several isolated ancestral populations. It is worth noting that Iberia this species occurs in scattered localities along the Cantabrian Both mitochondrial and nuclear markers revealed the genetic Mountains (Fig. 1). Considering the current geographical distinctiveness of a single genetic lineage of Erebia euryale orientation of the sample localities, it could be argued that spanning the Cantabrian Mountains and eastern Pyrenees. the grouping Leitariegos-Somiedo-Pandetrave (northern side, The mitochondrial dataset shows geographical co-occurrence Asturias Province) resulted from a common ancestor that of related haplotypes, i.e. all Pyrenean–Cantabrian localities colonized the northern slope of the Cantabrian Mountains, share the same most common haplotype (H01), whereas the whereas the grouping Pajares-San Glorio (southern side, Leoń other five rare haplotypes H02–H06 are separated from H01 Province) might have resulted from a northwards colonization by only one substitution. From the nuclear perspective, we from the southern slopes. Thus, the large ringlets from San found the maximum differentiation between the Pyrenean– Isidro may be the descendents of a third ancestral gene pool. Cantabrian lineage and the south-western Alps when using Gutie´rrez (1997) found a higher butterfly species richness in NeiÕs distance and FST as estimators (Fig. 2, Table 4). By sheltered gorges of the National Park of Picos de Europa contrast, the Pyrenean–Cantabrian grouping and eastern Alps (sampled also in the present study, i.e. Pandetrave) than on (H11) ⁄ eastern Europe (H13) were the most differentiated northern slopes. Differentiation depending on slope orienta- (congruently with geography) as from the mitochondrial tion was found for populations of Pinus sylvestris in the perspective and the actual differentiation estimator (Dest) northern Meseta and peripheral mountain chains (central and applied on the nuclear dataset (Fig. 5, Table 4). These north-western Iberian Peninsula). Robledo-Arnuncio et al. findings, together with the monophyly of these populations (2005) showed that Scots pine isolates growing on disjunct inferred from the allozyme dataset, prove that Cantabrian and mountain blocks, but on slopes flowing to the same basin were Pyrenean E. euryale share a most common recent ancestor. genetically closer than populations growing on different slopes We also confirmed the strong decline of the genetic diversity of the same mountain chain, flowing to different basins. from East to West detected by Schmitt and Haubrich (2008) Genetic differentiation between populations from northern (Tables 2 and 3). For instance, allelic richness decreased from and southern Pyrenean sides was reported for the lizard E Europe (2.56) to the Pyrenean–Cantabrian lineage (1.84) Zootoca vivipara (Jacquin, 1787) (Guillaume et al. 2000). (Table 5). Such a gradient has also been detected in vertebrate Gutie´rrez (1997) also suggested that current species richness species, e.g. the Pyrenean capercaillie (Tetrao urogallus, and composition of butterfly assemblages in the Picos de Rodrı´guez-Mun˜ oz et al. 2007) and a woodland plant (Polyg- Europa area might be the consequence of differential coloni- onatum verticillatum; Kramp et al. 2009). Richer East-poorer zation of refuges during the Quaternary climatic changes. West gradients, in terms of genetic diversity, seem to indicate Again, the evolutionary history of Z. vivipara (Guillaume larger effective population sizes for those taxa in populations et al. 2000) illustrates how heterogeneous colonization of outside the Iberian Peninsula and within the framework of glacial Pyrenean ⁄ Iberian refugia may have caused genetic Quaternary history and postglacial expansions (revised by differentiation within them. Other reports for the Cantabrian Schmitt 2009). area suggested the existence of at least two glacial refugia for Our data support the hypothesis of a distinct Pyrenean some species (e.g. Garcıá-Parı´s et al. 2003; Vialette et al. 2008), lineage within Erebia euryale put forward by Schmitt and in agreement with the fine-scale pattern of Ôrefugia within the Haubrich (2008). The present work extends this finding to Iberian refugeÕ reviewed by Go´mez and Lunt (2007). show that the Pyrenean locality of La Gle` be and the six West Nevertheless, we cannot rule out the possibility that the Cantabrian localities newly genotyped formed a single genetic current Cantabrian allozyme pattern is simply caused by lineage, opposite to what we expected in the light of classical stochastic effects, i.e. strong geographical isolation of all these taxonomy and genetic findings in other northern Iberian populations over the postglacial period and quick random montane species (e.g. Pe´rez et al. 2002; Pauls et al. 2006). The allele shifts in these isolates. In this context, populations discovery of a distinct Cantabrian–Pyrenean lineage is an suffering stronger bottlenecks might be either genetically more important knowledge for conservation managers, as all distant from the other demes and ⁄ or genetically impoverished. the Cantabrian sampling sites are within protected areas The evolution of genetically more distant populations typically (Ministerio de Medioambiente, Medio Rural y Marino, http:// J Zool Syst Evol Res (2011) 49(2), 119–132 2010 Blackwell Verlag GmbH 128 Vila, Marı´-Mena, Guerrero and Schmitt www.mma.es/portal/secciones/biodiversidad/rednatura2000/ This is because the wide temporal intervals may imply opposite rednatura_espana/index.htm, accessed October 19, 2010) con- displacement scenarios: during glacial stages large ringlets sidering the general interest in knowing the extent of genetic likely sought to open wide lowlands for refuge, whereas during structuring of species occurring in different protected areas. interstadials populations are expected to become fragmented As a distinct Pyrenean–Cantabrian lineage was clearly as they gain elevation. We acknowledge that montane species revealed by both nuclear and mitochondrial results, we may seek refuge in three types of areas during cold stages: support the definition of the Pyrenean–Cantabrian lineage of nunatak, peripheral and lowland refugia (Holderegger and Erebia euryale as an ESU which is reinforced by the following Thiel-Egenter 2009). However, the current Cantabrian eleva- two arguments. First, the DK statistic of Evanno et al. (2005) tional range (700 to 1650 m a.s.l., H. Mortera, pers. comm.) showed the largest variation (Fig. S8b) when the data clustered makes plausible the peripheral refugia during glacial stages. in two groups (K = 2). This indicates that the most likely We therefore postulate that the obtained expansion of division for the European allozyme dataset is the separation of E. euryale corresponds to an environmental cooling event the Pyrenean–Cantabrian lineage from all the other surveyed and the down-slope shift of montane forests. samples. There was a second interesting change in DK when the A deeper understanding of the time when Erebia euryale clustering determined four groupings (K = 4), which also split from its sister taxon or when the splitting of the major made geographical sense, as those four entities essentially genetic groups occurred is beyond the scope of this paper. Our grouped samples according to their major regions of origin markers have insufficient resolution for these questions, while (Fig. 4b). This split between lineages endemic (or predomi- a caveat is also imposed for applying a strict molecular clock at nant) at the Pyrenean–Cantabrian region and the rest of the intraspecific level when (i) population sizes can be Europe is in accordance with the evolutionary history inferred demonstrated not to be constant (as in our case from the for, e.g. Pinus sylvestris (Cheddadi et al. 2006) or Zootoca heterozygosity deficit text, data not shown, e.g. expansions in vivipara (Guillaume et al. 2000). Second, there might be some West and East Alps, as well as East Europe), and (ii) it is differentiation between the habitat occupied by the Cantabrian invalid to extrapolate molecular rate of change across different Erebia euryale and the populations of this species surveyed by evolutionary timescales because of the time dependency of Schmitt and Haubrich (2008). Their populations are located molecular rate estimates (Ho et al. 2005). However, it is near coniferous forests, which are scarce in the Cantabrian encouraging that the above-mentioned tentative time frame for Mountains. Cantabrian large ringlets currently use non-conif- the Pyrenean–Cantabrian lineage agrees with the Upper erous tree species to find shelter, as well as some southernmost Pleistocene formation of divergent lineages within species of Alpine, Apennine and Balkan populations of this species (F. Erebia (Martin et al. 2002; Vila et al. 2005; Albre et al. 2008). Cupedo, pers. comm.). As the Cantabrian pine tree forests severely declined since the Late Holocene (Rubiales et al. 2008) and particularly since the 19th century (Quevedo et al. Mitochondrial versus nuclear patterns 2006), one might argue that the patchy distribution of Despite the continuum along the Pyrenean–Cantabrian local- E. euryale in the Cantabrian Mountains relates to the intensive ities revealed by the mtDNA, there was significant differenti- use of land and deforestation. ation between some Cantabrian localities and the Pyrenean site of La Gle` be (i.e. maximum differentiation with San Isidro: FST = 0.238, Dest = 0.016). Such a nuclear differentiation Population fluctuations in northern Iberia most probably resulted from genetic drift (causing quick The expansion of the Pyrenean–Cantabrian lineage of Erebia random allele shifts) acting upon different demes that became euryale likely occurred during the Upper Pleistocene. Using the isolated after the population expansion of the Pyrenean– same substitution rate as Horn et al. (2009), the pine-depen- Cantabrian ancestral population. A similar pattern due to dent beetle Tomicus piniperda (Linnaeus, 1758) and E. euryale postglacial events has also been observed in the violet copper show a somehow coincident time (90, 80 kyBP, respectively) Lycaena helle (Denis and Schiffermu¨ ller, 1775) (Habel et al. for expansion of their Iberian mitochondrial lineages 2010). Another factor that may contribute to this discrepancy (Appendix S7). Both dates correspond to the marine oxygen- between mitochondrial and nuclear results would be a sex- isotope stage (MIS) 5 (Gibbard and van Kolfschoten 2004). biased dispersal. One might argue that these dates represent the transition To the best of our knowledge, there are no published data between the interglacial MIS5e (Eemian chronostratigraphic on the dispersal pattern of Erebia euryale. However, we cannot subage) and the Late Glacial Maximum (MIS 2, Late rule out dispersal in this species to be male-biased, as it occurs Weschelian ⁄ Wu¨ rm). However, at fine scale, MIS5 also showed in some congeneric species [e.g. Erebia epiphron (Knoch, 1783) climatic oscillations (Shackleton et al. 2003) and 80 kyBP and Erebia sudetica Staudinger, 1861, Kuras et al. 2003], but likely corresponded to a warmer stage than 90 kyBP (Potter not in others (e.g. Erebia epipsodea Butler, 1868, Brussard and et al. 2004). In both species, their Iberian lineages have not Ehrlich 1970). contributed to the current genetic diversity found in other Our allozyme and mitochondrial results agree in indicating European regions. However, computing the expansion time by four major European lineages (Fig. S8). The degree of means of the average rate given by Hammouti et al. (2010), the differentiation we infer among the four major groupings must growth of the Pyrenean–Cantabrian lineage (183 kyBP) would be regarded as provisional, because genetic information from fall into the transition between MIS 7 (interglacial) and 6 Italian populations is lacking. Samples from the Italian (glacial), both within the Saalian ⁄ Riss. The temporal discrep- peninsula (Ligurian Alps as well as Central Apennines) will ancy because of the use of different mutation rates as well as likely reveal at least a fifth distinct genetic lineage, as suggested the wide and overlapping confidence intervals for those by the diverging morphology of Italian large ringlets and also estimates prevent further discussion on the palaeoenvironmen- studies on other organisms (e.g. Nazari and Sperling 2007; tal scenario when such a population expansion took place. Ansell et al. 2008; Rousselet et al. 2010). J Zool Syst Evol Res (2011) 49(2), 119–132 2010 Blackwell Verlag GmbH Phylogeography of Erebia euryale 129

The nuclear differentiation of the Pyrenean–Cantabrian H. Fernańdez Vidal, Toma´s Latasa, Hugo Mortera, Gloria Robles, lineage and other major European geno-phenotypic groups of Hugo Robles, and Georges Verhulst contributed to fieldwork with this species is high (Table 5). This also holds true for genetic their advice, assistance, and equipment. This work benefited from the discussions held with Frans Cupedo, Graciela Este´vez, and Roger Vila lineages of other Erebia species (e.g. E. epiphron, Schmitt et al. as well as the revisions by Zoltań Varga and two anonymous referees. 2006) and even other satyrine butterflies such as Melanargia An earlier version of this manuscript was greatly improved after sp. (Habel et al. 2005). However, the major genetic groups reviewed by David C. Lees. David Romero assisted us in preparing revealed by the nuclear dataset of Erebia euryale are not artwork. This investigation was performed under permits from all largely differentiated at the mitochondrial level (Fig. 5). In environmental authorities concerned. It was financially supported by fact, only one substitution differentiates the most frequent grants from Xunta de Galicia (PGIDIT06PXIB103258PR) and the Pyrenean–Cantabrian haplotype from the one present in the University of A Corun˜ a. MV was funded by Xunta de Galicia (Isidro Parga Pondal programme). NMM was funded by the Spanish Ministry south-western Alps; a lesser extent of differentiation than of Science (FPI programme, ref. BES-2008-002571). shown by the same mitochondrial marker for other subspecies or divergent lineages of Erebia, e.g. Erebia triaria pargaponda- lense Fernańdez Vidal, 1984 (Vila et al. 2005), western Resumen Pannonian populations of Erebia medusa (Denis and A algunas mariposas no les importa demasiado la topografıá: un uńico Schiffermu¨ ller, 1775) (Hammouti et al. 2010) or other lepid- linaje gene´tico de Erebia euryale (Nymphalidae) a lo largo de las opterans (Vandewoestijne et al. 2004; Nazari and Sperling montanãs septentrionales de la Penıńsula Ibe´rica 2007; Kerdelhueét al. 2009). Schmitt and Haubrich (2008) suggested that according to La filogeografıá de especies de montan˜ a ha revelado frecuentemente their allozyme and morphological data, the most ancient split una fuerte diferenciacioń gene´tica entre especies que habitan distintos sistemas montan˜ osos. Tanto la morfologıá cla´sica como estudios in European E. euryale occurred between the western Alpine gene´ticos previos sugerıán la distincioń de las poblaciones pirenaicas E. euryale adyte and all the other populations. Our mitochon- de la mariposa Erebia euryale, aunque esta hipo´tesis precisaba ser drial results rather point to a first split of an eastern group with confirmada mediante el estudio de las poblaciones ma´s occidentales del haplotypes currently present in East Europe (Sinaia) and the area de distribucioń de esta especie: la Cordillera Canta´brica. En este eastern Alps (Obertauern) (Fig. 5). trabajo se pretende describir la estructura gene´tica de las poblaciones The population sample from Val dÕIse` re used for the mtDNA de Erebia euryale de dicha a´rea, donde la especie esta´distribuida en ´ analyses seems genetically impoverished at nuclear level (see localidades dispersas. Para ello, se estimaron la diversidad genetica y la diferenciacioń presentadas por 218 individuos muestreados en Table 3 at Schmitt and Haubrich 2008). In fact, its commonest seis localidades de la Cordillera Canta´brica (Norte de Espan˜ a) y allele is fixed at several loci, but allele frequencies are also genotipados para 17 loci alozı´micos. Asimismo, se secuenciaron 816 pb strongly distorted at some other loci. The signal is the same in del gen mitocondrial COI en 49 individuos de dichas localidades the other western Alps samples (St. Martin, Partnun) but to a Canta´bricas y 41 especı´menes procedentes de otras cinco poblaciones europeas. Los datos mitocondriales apoyaron la division en cuatro lesser extent. This pattern results in higher FST values (Table 4) and a long branch in the NJ tree (Fig. 2). Future research shall grupos gene´ticos principales para las poblaciones no ibe´ricas previa- mente sugeridos por otros autores en base a datos alozı´micos. Los determine whether the COI haplotype found in this valley is resultados procedentes de los marcadores mitocondriales y nucleares representative of the surrounding populations or not, as Val revelaron la distinciońdeunuńico linaje gene´tico Canta´brico- dÕIse` re might correspond to a relatively isolated population. Pirenaico de Erebia euryale. La falta de estructura geogra´fica, ası´ Populations currently occurring in the south-western Alps como la topologıá en forma de estrella mostrada por los haplotipos tracked a glacial refuge in peripheral areas also used by other mitocondriales indicaron una expansioń demogra´fica en la poblacioń species such as the European Scots pine Pinus sylvestris ancestral a las actualmente distribuidas en el Norte peninsular. Dicha expansioń probablemente estuvo relacionada con las oscilaciones (Cheddadi et al. 2006) and the caddisfly Rhyacophila pubescens clima´ticas acaecidas durante el Pleistoceno tardıó. Dentro del linaje Pictet, 1834 (Engelhardt et al. 2008). This refugial area in some Canta´brico-Pirenaico, los datos nucleares indicaron una clara estruc- cases provided forebears of present Pyrenean populations. tura gene´tica para las muestras procedentes de la Cordillera Canta´- Schmitt (2009) revised the links between the Pyrenees and south- brica. Los individuos procedentes de San Isidro fueron los que western Alpine populations of several taxa, but the present mostraron una mayor diferenciacioń con respecto a las otras cinco study reveals that this is unlikely to be the case for Erebia localidades canta´bricas, estructuradas a su vez en dos grupos euryale. Rather, the phylogeographical pattern of the Pyre- diferentes. En base a los resultados obtenidos, se propone y argumenta la definicioń de una unidad evolutiva significativa para el linaje nean–Cantabrian lineage supports the isolation of northern Canta´brico-Pirenaico de Erebia euryale. Nuestros resultados ilustran Iberian Erebia euryale from remaining European populations. que la historia evolutiva de una especie puede ser moldeada por procesos Comparative phylogeographical surveys are still scarce and indetectables utilizando uńicamente ADNmt. mostly compared distantly related species (Pauls et al. 2009). The pattern obtained for Erebia euryale can be compared to the one exhibited by E. epiphron and the closely related species pair References Erebia palarica–E. meolans distributed along the Cantabrian Albre J, Gers C, Legal L (2008) Molecular phylogeny of the Erebia Mountains and the Pyrenees. The present work and that of Vila tyndarus (Lepidoptera, Rhopalocera, Nymphalidae, Satyrinae) et al. (2006) pave the way for a future comparative phylogeo- species group combining CoxII and ND5 mitochondrial genes: a case study of a recent radiation. Mol Phylogenet Evol 47:196– graphical study aiming at disentangling the evolutionary 210. factors shaping the genetic structure of populations of Erebia *Ali M, Tunçman G, Cross NCP, Vidailhet M, Bo¨ kesoy I, species occurring in the northern Iberian mountain ranges. Gitzelmann R, Cox TM (1994) Null alleles of the aldolase B gene in patients with hereditary fructose intolerance. J Med Genet 31:499–503. Acknowledgements *A´lvarez-Lao D, Garcıá N (2010) Chronological distribution of Pleistocene cold-adapted large mammal faunas in the Iberian We thank Nuria Remoń and Nadja Stabenow for their valuable help in Peninsula. Quat Int 212:120–128. the laboratory. Vittorio Baglione, Almudena Fernańdez Briera, Eliseo

J Zool Syst Evol Res (2011) 49(2), 119–132 2010 Blackwell Verlag GmbH 130 Vila, Marı´-Mena, Guerrero and Schmitt

Ansell SW, Grundmann M, Russell SJ, Schneider H, Vogel JC (2008) Go´mez A, Lunt DH (2007) Refugia within refugia: patterns of Genetic discontinuity, breeding-system change and population phylogeographic concordance in the Iberian Peninsula. In: Weiss S, history of Arabis alpina in the Italian Peninsula and adjacent Alps. Ferrand N (eds), Phylogeography of Southern European Refugia. Mol Ecol 17:2245–2257. Springer, Dordrecht, The Netherlands, pp 155–188. Belkhir K, Castric V, Bonhomme F (2002) IDENTIX, a software to Goudet J (1995) FSTAT version 1.2: a computer program to calculate test for relatedness in a population using permutation methods. Mol F-statistics. J Hered 86:485–486. Ecol Notes 2:611–614. Goudet J, Raymond M, de-Meeus T, Rousset F (1996) Testing *Bjo¨ rnerfeldt S, Hailer F, Nord M, Vila` C (2008) Assortative mating Differentiation in Diploid Populations. Genetics 144:1933–1940. and fragmentation within dog breeds. BMC Evol Biol 8:28. *Grandal dÕAnglade A, Lo´pez-Gonza´lez F, Vidal Romanı´JR (1997) Brussard PF, Ehrlich PR (1970) The population structure of Erebia Condition factors in the distribution of large mammals from Galicia epipsodea (Lepidoptera: Satyrinae). Ecology 51:119–129. (NW Iberian Peninsula) during the Upper Quaternary. Cad Lab Centeno-Cuadros A, Delibes M, Godoy JA (2009) Phylogeography of Xeol Laxe 22:43–66. Southern Water Vole (Arvicola sapidus): evidence for refugia within Guillaume C-P, Heulin B, Arrayago MJ, Bea A, Bran˜ a F (2000) the Iberian glacial refugium? Mol Ecol 18:3652–3667. Refuge areas and suture zones in the Pyrenean and Cantabrian Cheddadi R, Vendramin GG, Litt T, François L, Kageyama M, regions: geographic variation of the female MPI sex-linked alleles Lorentz S, Laurent J-M, de Beaulieu J-L, Sadori L, Jost A, Lunt D among oviparous populations of the lizard Lacerta (Zootoca) (2006) Imprints of glacial refugia in the modern genetic diversity of vivipara. Ecography 23:3–10. Pinus sylvestris. Glob Ecol Biogeogr 15:271–282. Gutie´rrez D (1997) Importance of historical factors on species richness Clement M, Posada D, Crandall KA (2000) TCS: a computer program and composition of butterfly assemblages (Lepidoptera: Rhopalo- to estimate gene genealogies. Mol Ecol 9:1657–1660. cera) in a northern Iberian mountain range. J Biogeogr 24:77–88. Cooper SJB, Ibrahim KM, Hewitt GM (1995) Postglacial expansion Habel JC, Schmitt T, Mu¨ ller P (2005) The fourth paradigm pattern of and genomy subdivision in the European grasshopper Chorthippus post-glacial range expansion of European terrestrial species: the parallelus. Mol Ecol 4:49–60. phylogeography of the Marbled White butterfly (Satyrinae, Lepi- Crawford NG (2010) SMOGD: software for the measurement of doptera). J Biogeogr 32:1489–1497. genetic diversity. Mol Ecol Resour 10:556–557. *Habel JC, Meyer M, El Mousadik A, Schmitt T (2008) Africa goes *Cupedo F (2007) Geographical variation and Pleistocene history of Europe: the complete phylogeography of the Marbled White the Erebia pandrose – sthennyo complex (Nymphalidae; Satyrinae). butterfly species complex Melanargia galathea ⁄ lachesis. Org Divers Nota Lepid 30:329–353. Evol 8:121–129. Cupedo F (2010) A revision of the infraspecific structure of Erebia Habel JC, Schmitt T, Meyer M, Finger A, Ro¨ dder D, Assmann T, euryale (Esper, 1805) (Nymphalidae: Satyrinae). Nota Lepid 33:85– Zachos FE (2010) Biogeography meets conservation: the genetic 106. structure of the endangered lycaenid butterfly Lycaena helle (Denis Deffontaine V, Ledevin R, Fontaine MC, Que´re´J-P, Renaud S, Libois & Schiffermu¨ ller, 1775). Biol J Linn Soc Lond 101:155–168. R, Michaux JR (2009) A relict bank vole lineage highlights the Hammouti N, Schmitt T, Seitz A, Kosuch J, Veith M (2010) biogeographic history of the Pyrenean region in Europe. Mol Ecol Combining mitochondrial and nuclear evidences: a refined evolu- 18:2489–2502. tionary history of Erebia medusa (Lepidoptera: Nymphalidae: *Dick CW, Heuertz M (2008) The complex biogeographic history of a Satyrinae) in Central Europe based on the COI gene. J Zoolog Syst widespread tropical tree species. Evolution 62:2760–2774. Evol Res 48:115–125. Dupanloup I, Schneider S, Excoffier L (2002) A simulated annealing Hewitt GM (1996) Some genetic consequences of ice ages, and their approach to define the genetic structure of populations. Mol Ecol role in divergence and speciation. Biol J Linn Soc Lond 58:247–276. 11:2571–2581. Ho SYW, Phillips MJ, Cooper A, Drummond AJ (2005) Time Engelhardt CHM, Pauls SU, Haase P (2008) Population genetic dependency of molecular rate estimates and systematic overestima- structure of the caddisfly Rhyacophila pubescens, Pictet 1834, north tion of recent divergence times. Mol Biol Evol 22:1561–1568. of the Alps. Fundam Appl Limnol 173:165–176. Holderegger R, Thiel-Egenter C (2009) A discussion of different types England PR, Osler GHR, Woodworth LM, Montgomery ME, Briscoe of glacial refugia used in mountain biogeography and phylogeog- DA, Frankham R (2003) Effects of intense versus diffuse population raphy. J Biogeogr 36:476–480. bottlenecks on microsatellite genetic diversity and evolutionary Horn A, Stauffer C, Lieutier F, Kerdelhue´ C (2009) Complex potential. Conserv Genet 4:595–604. postglacial history of the temperate bark beetle Tomicus piniperda Evanno G, Regnaut S, Goudet J (2005) Detecting the number of L. (Coleoptera, Scolytinae). Heredity 103:238–247. clusters of individuals using the software STRUCTURE: a simula- Hubisz MJ, Falush D, Stephens M, Pritchard JK (2009) Inferring tion study. Mol Ecol 14:2611–2620. weak population structure with the assistance of sample group Excoffier L, Lischer HEL (2010) Arlequin suite ver 3.5: a new series of information. Mol Ecol Resour 9:1322–1332. programs to perform population genetics analyses under Linux and Jensen JL, Bohonak AJ, Kelley ST (2005) Isolation by distance, web Windows. Mol Ecol Resour 10:564–567. service. BMC Genet 6:1–6. Felsenstein J (1989) PHYLIP – Phylogeny Inference Package (Version Jost L (2008) G(ST) and its relatives do not measure differentiation. 3.2). Cladistics 5:164–166. Mol Ecol 17:4015–4026. Fraser DJ, Bernatchez L (2001) Adaptive evolutionary conservation: KerdelhueĆ, Zane L, Simonato M, Salvato P, Rousselet J, Roques A, towards a unified concept for defining conservation units. Mol Ecol Battisti A (2009) Quaternary history and contemporary patterns in a 10:2741–2752. currently expanding species. BMC Evol Biol 9:220. Garcıá-Barros E, Munguira ML, Martıń Cano J, Romo Benito H, Kramp K, Huck S, Niketic M, Tomovic G, Schmitt T (2009) Multiple Garcia-Pereira P, Maravalhas E (2004) Atlas of the Butterflies of the glacial refugia and complex postglacial range shifts of the obligatory Iberian Peninsula and Balearic Islands (Lepidoptera: Papilionoidea woodland plant Polygonatum verticillatum (Convallariaceae). Plant & Hesperioidea). Sociedad Entomolo´gica Aragonesa, Zaragoza, Biol 11:392–404. Spain, pp 126–135. Kuras T, Benes J, Fric Z, Konvicka M (2003) Dispersal patterns of Garcıá-Parı´s M, Alcobendas M, Buckley D, Wake DB (2003) endemic alpine butterflies with contrasting population structures: Dispersal of viviparity across contact zones in Iberian populations Erebia epiphron and E. sudetica. Popul Ecol 45:115–123. of fire salamanders (Salamandra) inferred from discordance of Lafranchis T (2000) Les papillons de jour de France, Belgique et genetic and morphological traits. Evolution 57:129–143. Luxembourg et leurs chenilles. E´ditions Biotope, Me` ze, France, 448 Gibbard P, van Kolfschoten T (2004) The Pleistocene and pp. Holocene Epochs. In: Gradstein FM, Ogg JG, Smith AG (eds), *Langley CH, Voelker RA, Brown AJL, Ohnishi S, Dickson B, A Geologic Time Scale. Cambridge University Press, Cambridge, Montgomery E (1981) Null allele frequencies at allozyme loci in pp 441–452. natural populations of Drosophila melanogaster. Genetics 99:151–156.

J Zool Syst Evol Res (2011) 49(2), 119–132 2010 Blackwell Verlag GmbH Phylogeography of Erebia euryale 131

Librado P, Rozas J (2009) DnaSP v5: a software for comprehensive demographic history of the pine processionary moth, Thaumetopoea analysis of DNA polymorphism data. Bioinformatics 25:1451–1452. pityocampa (Lepidoptera: Notodontidae). J Biogeogr 37:1478–1490. Lunt DH, Zhang DX, Szymura JM, Hewitt GM (1996) The insect Rousset F (1997) Genetic differentiation and estimation of gene flow cytochrome oxidase I gene: evolutionary patterns and conserved from F-statistics under isolation by distance. Genetics 145:1219– primers for phylogenetic studies. Insect Mol Biol 5:153–165. 1228. Lynch M, Ritland K (1999) Estimation of pairwise relatedness with Rousset F (2008) GenepopÕ007: a complete reimplementation of the molecular markers. Genetics 152:1753–1766. Genepop software for Windows and Linux. Mol Ecol Resour 8:103– Martin JF, Gilles A, Descimon H (2000) Molecular phylogeny and 106. evolutionary patterns of the European satyrids (Lepidoptera: Rubiales JM, Garcıá-Amorena I, GarcıáA´lvarez S, Go´mez Manz- Satyridae) as revealed by mitochondrial gene sequences. Mol aneque F (2008) The Late Holocene extinction of Pinus sylvestris in Phylogenet Evol 15:70–82. the western Cantabrian Range (Spain). J Biogeogr 35:1840–1850. Martin JF, Gilles A, Lortscher M, Descimon H (2002) Phylogenetics Schmitt T (2009) Biogeographical and evolutionary importance of the and differentiation among the western taxa of the Erebia tyndarus European high mountain systems. Front Zool 6:9. group (Lepidoptera : Nymphalidae). Biol J Linn Soc Lond 75:319– Schmitt T, Haubrich K (2008) The genetic structure of the mountain 332. forest butterfly Erebia euryale unravels the late Pleistocene and Naydenov K, Senneville S, Beaulieu J, Tremblay F, Bousquet J (2007) postglacial history of the mountain coniferous forest biome in Glacial vicariance in Eurasia: mitochondrial DNA evidence from Europe. Mol Ecol 17:2194–2207. Scots pine for a complex heritage involving genetically distinct refugia Schmitt T, Seitz A (2004) Low diversity but high differentiation: the at mid-northern latitudes and Asia Minor. BMC Evol Biol 7:233. population genetics of Aglaope infausta (Zygaenidae: Lepidoptera). Nazari V, Sperling FAH (2007) Mitochondrial DNA divergence and J Biogeogr 31:137–144. phylogeoraphy in western Palaearctic Parnassiinae (Lepidoptera: Schmitt T, Hewitt GM, Mu¨ ller P (2006) Disjunct distributions during Papilionidae): how many species are there? Insect Syst Evol 38:121– glacial and interglacial periods in mountain butterflies: Erebia 138. epiphron as an example. J Evol Biol 19:108–113. Nei M (1972) Genetic distances between populations. Am Nat *Semerikov VL, Semerikov LF, Lascoux M (1999) Intra- and 106:283–291. interspecific allozyme variability in European Larix Mill. Species. Pauls SU, Lumbsch HT, Haase P (2006) Phylogeography of the Heredity 82:193–204. montane caddisfly Drusus discolor: evidence for multiple refugia and Shackleton NJ, Sańchez-Gon˜ i MF, Pailler D, Lancelot Y (2003) periglacial survival. Mol Ecol 15:2153–2169. Marine Isotope Substage 5e and the Eemian Interglacial. Global Pauls SU, Theissinger K, Ujvarosi L, Balint M, Haase P (2009) Planet Change 36:151–155. Patterns of population structure in two closely related, partially Siegismund HR (1993) G-Stat, Version 3, Genetical Statistical sympatric caddisflies in Eastern Europe: historic introgression, Programs for the Analysis of Population Data. The Arboretum, limited idspersal, and cryptic diversity. J North Am Benthol Soc Royal Veterinary and Agricultural University, Horsholm, Denmark. 28:517–536. Sonderegger P (2005) Die Erebien der Schweiz (Lepidoptera: Satyri- Pen˜ a C, Nylin S, Walhberg N (in press) The radiation of Satyrini nae, genus Erebia). Selbstverlag (=private edition), Biel ⁄ Bienne, butterflies (Nymphalidae: Satyrinae): a challenge for phylogenetic Switzerland, 712 pp. methods. Zool J Linn Soc. van Swaay C, Cuttelod A, Collins S, Maes D, Lo´pez Munguira M, Pe´rez T, Albornoz J, Domıńguez A (2002) Phylogeography of chamois Sasic M, Settele J, Verovnik R, Verstrael T, Warren M, Wiemers M, (Rupicapra spp.) inferred from microsatellites. Mol Phylogenet Evol Winhoff I (2010) European Red List of Butterflies. Publications 25:524–534. Office of the European Union, Luxembourg, pp 35. Petit RJ, Hampe A, Cheddadi R (2005) Climate changes and tree Templeton AR, Crandall KA, Sing CF (1992) A cladistic analysis of phylogeography in the Mediterranean. Taxon 54:877–885. phenotypic association with haplotypes inferred from restriction Potter E-K, Esat TM, Schellmann G, Radtke U, Lambeck K, endonuclease mapping and DNA sequence data. III. Cladogram McCulloch MT (2004) Suborbital-period sea-level oscillations dur- estimation. Genetics 132:619–633. ing marine isotope substages 5a and 5c. Earth Planet Sci Lett Tennent WJ (2008) A checklist of the satyrine genus Erebia (Lepi- 225:191–204. doptera) (1758–2006). Zootaxa 1900:1–109. Pritchard JK, Stephens M, Donnelly P (2000) Inference of population Tolman T, Lewington R (1997) Butterflies of Britain and Europe. structure using multilocus genotype data. Genetics 155:945–959. Harper Collins, London, pp 210. Pyha¨ ja¨ rvi T, Salmela MJ, Savolainen O (2008) Colonization routes of Vandewoestijne S, Baguette M, Brakefield PM, Saccheri IJ (2004) Pinus sylvestris inferred from distribution of mitochondrial DNA Phylogeography of Aglais urticae (Lepidoptera) based on DNA variation. Tree Genet Genomes 4:247–254. sequences of the mitochondrial COI gene and control region. Mol *Queney G, Ferrand N, Weiss S, Mougel F, Monnerot M (2001) Phylogenet Evol 31:630–646. Stationary distributions of microsatellite loci between divergent *Varga Z (1998) Die Erebien der Balkanhalbinsel und Karpaten IV. population groups of the European rabbit (Oryctolagus cuniculus). U¨ bersicht der subspezifischen Gliederung und der verbreitung der Mol Biol Evol 18:2169–2178. Erebia-Arten in der Balkanhalbinsel und in den Karpaten (Lepi- Quevedo M, Ban˜ uelos MJ, Saéz O, Obeso JR (2006) Habitat selection doptera, Nymphalidae, Satyrinae). Entomol Rom 3:15–29. by Cantabrian capercaillie Tetrao urogallus cantabricus at the edge *Verity R (1927) Notes sur quelques Rhopaloce´res dÕEspagne [Lep.]. of the speciesÕ distribution. Wildl Biol 12:267–276. Bull Soc Entomol France 1927:172–176. *RaspeÓ, Jacquemart A-L (1998) Allozyme diversity and genetic Vialette A, Guiller A, Bellido A, Madec L (2008) Phylogeography and structure of European populations of Sorbus aucuparia L. (Rosa- historical demography of the Lusitanian snail Elona quimperiana ceaee: Maloideae). Heredity 81:537–545. reveal survival in unexpected separate glacial refugia. BMC Evol Robledo-Arnuncio JJ, Collada C, Alıá R, Gil L (2005) Genetic Biol 8:339. structure of montane isolates of Pinus sylvestris L. in a Mediterra- Vila M, Bjo¨ rklund M (2004) The utility of the neglected mitochondrial nean refugial area. J Biogeogr 32:595–605. control region for evolutionary studies in Lepidoptera (Insecta). J Rodrı´guez-Mun˜ oz R, Mirol PM, Segelbacher G, Fernańdez A, Mol Evol 58:280–290. Tregenza T (2007) Genetic differentiation of an endangered caper- Vila M, Vidal-Romanı´JR, Bjo¨ rklund M (2005) The importance of caillie (Tetrao urogallus) population at the Southern edge of the time scale and multiple refugia: Incipient speciation and admixture species range. Conserv Genet 8:659–670. of lineages in the butterfly Erebia triaria (Nymphalidae). Mol Rogers AR, Harpending H (1992) Population growth makes waves in Phylogenet Evol 36:249–260. the distribution of pairwise genetic differences. Mol Biol Evol 9:552– Vila M, Cassel Lundhagen A, Thuman KA, Stone JR, Bjo¨ rklund M 569. (2006) A new conservation unit in the butterfly Erebia triaria Rousselet J, Zhao R, Argal D, Simonato M, Battisti A, Roques A, (Nymphalidae) as revealed by nuclear and mitochondrial markers. KerdelhueĆ (2010) The role of topography in structuring the Ann Zool Fenn 43:72–79.

J Zool Syst Evol Res (2011) 49(2), 119–132 2010 Blackwell Verlag GmbH 132 Vila, Marı´-Mena, Guerrero and Schmitt

*Von der Goltz DH (1930) In: Seitz A (ed.), Die Gross-Schmetterlinge Appendix S7. Genetic diversity and resolution of the allozyme der Erde. Supplement 1: 147. Alfred Kerner, Stuttgart, pp 147 and markers. plate 9. Figure S8. (a) Log-likelihood of the allozyme data on 16 Erebia Weir BS, Cockerham CC (1984) Estimating F-statistics for the analysis euryale populations given K clusters that were obtained after of population structure. Evolution 38:1358–1370. Weir BS, Hill WG (2002) Estimating F-statistics. Annu Rev Genet ﬁve runs of the STRUCTURE algorithm; (b) Corresponding 36:721–750. DK statistic (Evanno et al. 2005) showing peaks at K ¼ 2 and K ¼ 4. Table S9. Proportion of missing data (italics) and private Supporting Information alleles (bold) for the eleven populations and 17 loci of Erebia Additional Supporting Information may be found in the online euryale analysed by Schmitt and Haubrich (2008). version of this article: Figure S10. Approximate distribution area (grey surface) and Appendix S1. Phylogeography of Erebia euryale and intraspe- sampling locatilities surveyed by Schmitt and Haubrich (2008) ciﬁc taxonomy. (bold) and the present work. Table S2. Allele frequencies of all 13 polymorphic allozyme Table S11. Sampling information. loci of six populations of Erebia euryale from the Cantabrian Please note: Wiley-Blackwell are not responsible for the Mts. content or functionality of any supporting materials supplied Table S3. Single and multilocus genetic diversity estimates for by the authors. Any queries (other than missing material) 13 polymorphic loci. should be directed to the corresponding author for the article. Table S4. FIS values per locus and sampling locality. Figure S5. Results of the Isolation-By-Distance analyses. Figure S6. Unimodal mismatch distribution for the Pyrenean- Cantabrian lineage consistent with the expected sudden expansion model.

J Zool Syst Evol Res (2011) 49(2), 119–132 2010 Blackwell Verlag GmbH Supporting Information S1 . Phylogeography of Erebia euryale and intraspecific taxonomy . S.1.1. Iberian phylogeography The hypothesis of a distinct genetic lineage for the Cantabrian and Pyrenean populations of Erebia euryale is supported by classical intraspecific taxonomy. Erebia euryale antevortes *Verity (1927) at the Hautes-Pyrénées (southwestern France); ssp. pyreaenaeicola *von der Goltz (1930) at the eastern Pyrenees; and ssp. cantabricola *Verity (1927) in the Cantabrian Mountains (northern Spain). Although the genus Erebia is one of the more nomenclaturally controversial genera among the Holarctic Lepidoptera (Albre et al. 2008) due to the high number of species and intraspecific taxa, recent morphological work supports the significant differentiation between the subspecies antevortes and pyraenaeicola . However, individuals from some Pyrenean localities also present intermediate features. Cantabrian populations may also be very variable concerning wing pattern, somewhat like those intermediate populations found in the Pyrenees (F. Cupedo, pers. comm. ). A recent morphological study surveying not only wing traits, but also male genitalia, classified the subspecies antevortes , pyraenaeicola , and cantabricola (as well as five other subspecies) as belonging to a single group: Erebia euryale (group euryale , see below) (Cupedo 2010). Interestingly, there is fairly high morphological variability within the Cantabrian large ringlets from San Glorio (n = 28, F. Cupedo pers.comm .). It is worth mentioning that the description of the subspecies cantabricola (*Verity 1927) was based on individuals from Pajares, a locality surveyed by the present study. West-East Pyrenean differentiation was found in several other Erebia species, either from the morphological (e.g., Erebia pandrose – sthennyo complex; *Cupedo 2007) or genetic perspective (e.g., Erebia epiphron ; Schmitt et al. 2006). We acknowledge that our study lacked samples from the subspecies antevortes (W Pyrenees), and therefore, some new lineages might appear when surveying additional northern Iberian localities. In fact, the occurrence of a Basque/SW French/W Pyrenean lineage in such divergent organisms as the land snail Elona quimperana (Vialette et al. 2008) or the bank vole Myodes glareolus (Deffontaine et al. 2009) opens the possibility of a second genetic lineage in the western Pyrenees. Such a hypothesis is triggered by the classic circumscription of the morphological subspecies E. euryale pyraenaicola in the western Pyrenees. However, in the light of ongoing research, the intraspecific classification of the northern Iberian Erebia euryale into three separate subspecies remains controversial (F. Cupedo, pers. comm. ).

1 We postulate that the distinctiveness of the Pyrenean-Cantabrian lineage found in Erebia euryale may have resulted from an ancestral population spreading from France into North Spain from the West and East fringes of the Pyrenees. The Pyrenees acted as a barrier for many species crossing from France to Iberia and vice-versa . However, the areas between this mountain range and the Bay of Biscay and the Mediterranean were suitable habitats for the migration of many animal taxa. This is inferred, for instance, from the type of dated fossils found in the large number of paleontological sites in the northeastern Spanish province of Girona and all along the so-called “northern-corridor” (strip between the Cantabrian Mountains and the Bay of Biscay) further down to Portugal (*Grandal d’Anglade et al. 1997; *Álvarez-Lao and García 2010). Our hypothesis of a East-West entrance of Erebia euryale into Spain at some point in the Upper Pleistocene does not preclude the existence of one or more ancient populations already present in Iberia, as there is a wide geographic gap between San Glorio and La Glèbe not covered by the present study.

S.1.2. Implications for intraspecific taxonomy Our results at European scale partially supported a four group genetic division, congruent with the results by Schmitt and Haubrich (2008). Our allozymes and mitochondrial results indicated (i) a Pyrenean-Cantabrian lineage (Pyrenean lineage of isarica ), (ii) a western Alps grouping ( adyte ), (iii) an eastern Alps cluster ( isarica from Obertauern and Trauneralm plus ocellaris from Kalkstein), and (iv) a final eastern Europe clade ( syrmia ). The subspecies o cellaris was considered and ecotype of ssp. isarica by Schmitt and Haubrich (2008), which is supported by our nuclear data (Fig. 2), but not by the mitochondrial results (Fig. 5). In the morphology based intraspecific taxonomy suggested by Cupedo (2010) the subspecies pyraenaeicola , cantabricola , isarica , ocellaris , and syrmia were clustered into the euryale group. Further support to synonymise E. euryale isarica with E. euryale euryale (*Varga 1998) comes from the fact that the nominotypic population “Riesengebirge” is only a marginal isolate of the northern Alpine-Carpathian genetic lineage (Z. Varga, pers. comm. ). The taxonomic status of E. euryale syrmia Fruhstorfer, 1910 from eastern/southern Carpathian – Balkan area also needs further work, as this subspecies was described from Bosnia (Trebević) and the eastern Balkan populations show some morphological differentiation (Z. Varga, pers. comm. ). Both Schmitt and Haubrich (2008) and Cupedo (2010) defined an adyte group. This is in line with our nuclear data, as the sample from Val d’Isère (assigned to the subspecies adyte by Schmitt and Haubrich 2008) showed a significant differentiation from the other two

2 localities from the West Alps (St. Martin and Partnun). However, the resolution of the markers used in the present study prevented us from drawing any further conclusion about the differentiation within this West Alpine group. To conclude, the intraspecific taxonomy of Erebia euryale deserves further molecular research, as there are several issues that need to be clarified. For instance, the degree of differentiation of the kunzi -group, confined to a restricted Italian part of the southern Alps (Cupedo 2010).

3 S2. Allele frequencies of all 13 polymorphic allozyme loci of six populations of Erebia euryale from the Cantabrian Mts.

Loc Allele San Glorio Paj San Isidro Somi Leitarieg Pandetra us ares edo os ve 6Pg 1 0.069 0 0.216 0 0 0 dh 2 0.466 0.6 0 0.647 0.462 0.435 67 3 0.466 0.3 0.784 0.353 0.526 0.548 33 4 0 0 0 0 0.013 0.016 Apk 3 0.985 1.0 1.000 1.000 1.000 1.000 00 4 0.015 0 0 0 0 0 G6P 1 0.136 0.3 0.216 0.309 0.513 0.392 dh 06 2 0 0 0 0.015 0 0 3 0.818 0.6 0.784 0.676 0.487 0.608 81 4 0 0.0 0 0 0 0 14

4 7 0.045 0 0 0 0 0 Gap 1 0 0.0 0 0 0 0 dh 83 2 1.000 0.9 1.000 1.000 1.000 1.000 17 Aat1 4 1.000 0.9 0.986 1.000 1.000 1.000 58 5 0 0.0 0.014 0 0 0 42 Aat2 2 0.016 0 0 0 0 0.162 5 0.547 0.5 0.865 0.853 0.850 0.662 83 6 0.016 0 0 0 0 0 7 0.422 0.4 0.135 0.147 0.150 0.176 17 Idh1 2 1.000 0.9 0.944 0.882 0.962 1.000 72 4 0 0 0 0.029 0 0 8 0 0.0 0.056 0.088 0.038 0 28 Idh2 4 0.030 0 0 0 0 0

5 5 0.970 1.0 1.000 1.000 0.988 1.000 00 9 0 0 0 0 0.013 0 Mdh 1 0.061 0.0 0.014 0 0 0.036 2 14 2 0.848 0.9 0.900 1.000 1.000 0.804 58 3 0.015 0.0 0.014 0 0 0.054 28 4 0.076 0 0.071 0 0 0.089 5 0 0 0 0 0 0.018 Me 2 1.000 1.0 1.000 0.986 1.000 0.959 00 3 0 0 0 0.014 0 0.041 Pep 1 0.015 0.0 0.081 0.074 0.013 0.081 14 2 0.985 0.9 0.919 0.926 0.988 0.919 86 Pgi 4 0 0 0 0 0 0.014 5 0.045 0.0 0.230 0 0 0 28

6 6 0.030 0.0 0.014 0 0 0 69 7 0.894 0.5 0.703 0.871 0.837 0.824 69 8 0.030 0.3 0.054 0.129 0.150 0.162 06 9 0 0.0 0 0 0.013 0 28 Pgm 1 0.016 0.0 0.095 0 0 0 14 2 0.047 0.0 0.041 0.044 0.100 0 28 3 0.172 0.2 0.338 0.412 0.475 0.500 50 5 0.438 0.4 0.432 0.309 0.375 0.250 86 7 0.328 0.2 0.095 0.235 0.050 0.233 22 8 0 0 0 0 0 0.017

7 S3. Single and multilocus genetic diversity estimates for 13 polymorphic loci. Calculations performed across the six Cantabrian populations of Erebia euryale . AR = allelic richness (based on 27 individuals), HWE = deviations from Hardy-Weinberg equilibrium (score U (global) test, H 1 = heterozygote deficit) significance after sequential Bonferroni correction is marked in bold. NS = p >0.5, * = p < 0.05, ** = p < 0.01, *** = p < 0.001. Average F-statistics and their standard errors (after symbol ±) were calculated using a jacknifing-over-populations procedure. The G-test for population differentiation was calculated not assuming random mating within samples. Significance obtained after 10,000 randomisations.

Locus AR HWE FIS FST FIT G- test 6Pgdh 3.189 * +0.130 ± 0.174 ± +0.284 ± *** 0.097 0.132 0.153*** Apk 1.126 - -0.002 ± 0.003 ± 0.000 ± 0.000 0.000 0.001 G6Pdh 2.581 NS +0.072 0.060 ± +0.131 ± 0.137 *** ±0.118 0.041 Gapdh 1.550 NS +0.524 ± 0.123 ± +0.611 ± 0.278 0.238 0.056 Aat1 1.413 * -0.034 ± 0.026 ± -0.008 ± 0.005 0.021 0.019 Aat2 2.953 NS -0.018 ± 0.094 ± +0.078 ± 0.075 *** 0.072 0.018 Idh1 2.107 NS -0.067 ± 0.030 ± -0.036 ± 0.015 0.026 0.024 Idh2 1.356 NS -0.016 ± 0.013 ± -0.003 ± 0.002 0.012 0.012 Mdh2 3.133 NS +0.067 ± 0.037 ± +0.102 ± 0.099 0.107 0.023 Me 1.412 NS -0.033 ± 0.025 ± -0.008 ± 0.006 0.019 0.017 Pep 1.934 NS -0.063 ± 0.014 ± -0.048 ± 0.018 0.009 0.009

8 Pgi 4.057 NS -0.101 ± 0.03 0.084 ± -0.009 ± 0.037 *** 0.033 Pgm 4.777 *** +0.167 ± 0.037 ± +0.198 ± *** 0.054 0.013 0.061*** ALL 2.43 ± ** +0.068 ± 0.078 ± +0.140 ± *** 1.39 0.045 0.023 0.044***

9 S4. FIS values per locus and sampling locality. Estimation of p-values was based on 1560 randomisations. Indicative adjusted nominal level (0.05) for one table was =

0.00064. NA: non applicable. The proportion of randomisations that gave a larger FIS (i.e., significant deficit of heterozygotes) resulted in a single significant value (in bold).

There was no significant FIS value when testing for excess of heterozygotes.

SanGlorio Pajares SanIsidro Somiedo Leitariegos Pandetrave 6Pgdh -0.087 0.014 0.216 0.496 0.259 -0.124 Apk 0.000 NA NA NA NA NA G6Pdh -0.158 0.074 0.216 0.093 0.395 -0.292 Gapdh NA 0.286 NA NA NA NA Aat1 NA -0.029 0.000 NA NA NA Aat2 0.237 -0.129 -0.143 -0.158 0.032 -0.058 Idh1 NA -0.014 -0.045 -0.091 -0.027 NA Idh2 -0.016 NA NA NA 0.000 NA Mdh2 0.230 -0.019 0.085 NA NA -0.114 Me NA NA NA 0.000 NA -0.029 Pep 0.000 0.000 -0.075 -0.065 0.000 -0.075 Pgi -0.062 -0.095 -0.187 -0.133 0.016 -0.090 Pgm 0.267 0.118 0.059 0.060 0.367 0.121 All 0.096 0.013 0.028 0.088 0.249 -0.077

10 S5. Results of the Isolation-By-Distance analyses. (a) The European nuclear dataset did not conform to the isolation-by-distance model, as there was no significant correlation between genetic differentiation in relation to geographic distance (Z = 39647.54, r = 0.15, one-sided p = 0.09); (b) the Pyrenean-Cantabrian dataset did not conform either to the isolation-by-distance model (Z = 649.47, r = 0.35, one-sided p = 0.21). (a)

(b)

11 S6. Unimodal mismatch distribution for the Pyrenean-Cantabrian lineage consistent with the expected sudden expansion model (SDD=0.00246, p = 0.44).

12 S7. Genetic diversity and resolution of the allozyme markers. The six Cantabrian populations analysed in the present study were monomorphic for four out of the 17 surveyed allozyme loci. Those markers were polymorphic for other samples of Erebia euryale , as revealed by Schmitt and Haubrich (2008), who reported that the total percentage of polymorphic loci ranged from 47.1 (La Glèbe, souteastern France) to 88.2 (Groapa Seaca and Sinaia, two Romanian localities). The Pyrenean population of La Glèbe was also monomorphic for the same four loci as the Cantabrian samples, as well as for five other markers (Apk, Gapdh, Idh1, Idh2, and Mdh2). Taking into account the 13 polymorphic loci, our study on 218 adult males of Cantabrian large ringlets revealed an average of 1.92 alleles per locus. The proportion of missing data ranged from 0.08 (locus 6Pgdh at Somiedo) to 0.24 (locus Mdh2 at Pandetrave). The only locus globally departing from HWE was Pgm. This pattern indicated that the deviation from HWE was local, a locus-specific phenomenon, and might be caused by processes such as selection, assortative mating or genotyping problems. We cannot exclude any of those hypotheses, but we acknowledge the presence of null alleles as the most likely explanation (see below). Leitariegos was the only locality departing from HWE due to a significant deficit of heterozygotes. Such an excess of homozygotes might be due to processes such as under-dominant selection, unintentional mixing of separate breeding units (i.e., Wahlund effect), inbreeding or genotyping errors (e.g., null alleles). Homozygote excess was found in two out of the eight loci that resulted polymorphic in that locality, and none of them remained significant after Bonferroni correction, so selection does not appear as a plausible explanation. The Wahlund effect is unlikely, as each locality was sampled in plots less than one hectare in size and over one or two consecutive days. However, it would be still possible to find intra-locality genetic structuring, as different morphs (i.e., putative subspecies) of Erebia euryale occur sympatrically but do not hybridise in some European localities (F. Cupedo, pers. comm. ). The results from the Bayesian clustering of individuals implemented in STRUCTURE 2.3.3 did not support the sub-structuring within Leitariegos (Fig. 4). This might, in turn, favour inbreeding as an explanation. Inbreeding should have affected similarly to all markers, causing a large proportion of loci affected by significant positive FIS values (e.g., 74% of polymorphic loci in *Björnerfeldt et al. 2008). This did not seem to be the case of Leitariegos (Table S4). Nevertheless, we tested whether the surveyed individuals from Leitariegos

13 belonged to kin groups, instead of representing a random mating deme. We calculated the average ( r = -0.025) and dispersion (variance = 0.143, SD = 0.379) of the relatedness coefficient r (Lynch and Ritland 1999) from the output provided by IDENTIX for every possible pair of individuals sampled at Leitariegos (data available on request). Data from Leitariegos did not significantly depart from the null hypothesis of no relatedness neither when the test was performed on the means nor the variances of the relatedness coefficient. Finally, we cannot exclude the excess of homozygotes in Leitariegos to be due to the presence of null alleles and/or genotyping errors. We consider the first possibility as more likely. Null alleles are usually a major matter of concern when working with microsatellites in Lepidoptera, but they also occur in allozyme loci as no protein product or a protein product that is enzymatically non-functional (*Langley et al. 1981 and references therein). It is noteworthy that semi-null alleles have been also reported in allozymes, as protein activity may differ when extracted from different tissues (*Semerikov et al. 1999). Concerning lack of product, it was surprising the high proportion of missing data for three loci at Pandetrave: 6Pgdh (0.16), Mdh2 (0.24), and Pgm (0.18). This is a much higher level of missing data than found by Schmitt and Haubrich (2008), which ranged from zero (most cases) to 0.12 (locus Pgm at Val d’Isère, France) (Table S9). To the best of our knowledge, the preservation of the Cantabrian samples should not be the reason for this allelic pattern, as individuals were kept alive until arrival to the -80ºC freezing facilities and after five months transported to Germany (where stored in liquid nitrogen until analysis) in dry ice in less than 16 hours. This same transport protocol and personnel produced more accurate results in other allozymic studies in butterflies from far distant localities (e.g., *Habel et al. 2008). We would like to highlight several facts concerning null alleles that may be of importance when interpreting a homozygote excess such as the one found at Leitariegos. A single null allele may lead a population to depart from HWE (e.g., *Queney et al. 2001). Also, the assumed codominant mode of inheritance for a set of allozyme loci may be disturbed by recessive null alleles (e.g., *Raspé and Jacquemart 1998). An interesting idea that might be applicable in this scenario would be that the putative null alleles causing Leitariegos not to adjust to HWE were private, exclusive to that locality. For instance, a single highly polymorphic microsatellelite locus (out of a panel of 18 markers) failed to amplify in two Iberian localities which belonged to a distinct genetic lineage of the fish Gasterosteus aculeatus (Vila et al. unpublished).

14 Leitariegos and San Glorio were the only Cantabrian localities showing one private allele each, so it might as well be that a private null allele is preventing Leitariegos to adjust to HWE. Unfortunately, this idea remains as speculative in the present study, as mitochondrial results do not support a distinct lineage for Leitariegos. Lastly, the presence of allozyme null alleles has also been linked to deleterious traits expressed as a result of inbreeding (*Ali et al. 1994). To conclude, we postulate that null alleles may play a role in some Cantabrian localities, particularly at Leitariegos, and therefore the results of the presented allozyme data should be interpreted with caution. Demonstration of the presence of null alleles in the Cantabrian populations of Erebia euryale , as well as if they are related to a putative genetic distinctiveness of the westernmost part of the species distribution area (i.e., private null alleles) or to inbreeding opens the possibility for a future study in combination with other genetic markers.

15 S8. (a) Log-likelihood of the allozyme data on 16 Erebia euryale populations given K clusters that were obtained after five runs of the STRUCTURE algorithm; (b) Corresponding ∆K statistic (Evanno et al. 2005) showing peaks at K = 2 and K = 4, which indicate that those are the best solutions for K given the data. Multimodal distributions for ∆K are not unusual (e.g., *Dick and Heuertz 2008) and typically indicate a hierarchical structure. (a)

-6000.00

-6500.00

-7000.00

-7500.00 average L(K) +- SD -8000.00

-8500.00 1 3 5 7 9 11 13 15 17 K

(b)

250.00

200.00

150.00

100.00 DELTA(K)

50.00

0.00 1 3 5 7 9 11 13 15 17

16 S9. Proportion of missing data (italics) and private alleles (bold) for the eleven populations and 17 loci of Erebia euryale analysed by Schmitt and Haubrich (2008). F: France, CH: Switzerland, A: Austria, Ro: Romania.

Population Proportion of missing data (locus) Locus Allele Freq La Glèbe (F) - - - - St Martin (F) 0.08 (6Pgdh) Aat2 4 0.015 Val d'Isère (F) 0.10 (6Pgdh), 0.12 (Pgm) Idh2 4 0.013 Partnun (CH) 0.06 (Fum) - - - Kalkstein (A) 0.08 (Pgm) Idh1 4 0.014 Obertauern (A) - Aat1 5 0.028 Obertauern (A) Idh2 6 0.014 Schöneck (A) - - - - Trauneralm (A) - Pgi 9 0.013 Trauneralm (A) Pgm 10 0.013 Groapa Seaca (Ro) - Idh1 5 0.013 Groapa Seaca (Ro) Idh2 9 0.013 Groapa Seaca (Ro) Mdh1 4 0.013 Groapa Seaca (Ro) Mdh1 5 0.013 Groapa Seaca (Ro) Pk 5 0.013 Sinaia (Ro) - G6Pdh 6 0.013 Sinaia (Ro) Gapdh 4 0,013 Sinaia (Ro) Idh1 7 0,013

17 S10. Approximate distribution area (grey surface) and sampling locatilities surveyed by Schmitt and Haubrich (2008) (bold) and the present work (cf. Fig. 1). The names of subspecies of Erebia euryale cited in the text appear in italics and are placed near the areas where they occur (adapted from Tolman and Lewington (1997) and Cupedo (2010)). GLB: La Glèbe, STM: St. Martin, VIS: Val d’Isère, PAT: Partnun, KAL: Kalkstein, OBE: Obertauern, TRA: Trauneralm, GRO: Groapa Seaca, SIN: Sinaia, RIL: Rila Mountains, GLO: San Glorio, PAJ; Pajares, ISI: San Isidro, SOM: Somiedo, LEI: Leitariegos, PAN: Pantedrave

18 S11. Sampling information. Sample site Geographic N Elevation (meters above sea Date of

coordinates level) collection

San Glorio 43º04’N, 4º62’W 33 1609 July 18, 2006

Pajares 42º59’N, 5º45’ W 36 1378 July 16, 2006

San Isidro 43º04’N, 5º25’W 37 1520 July 11, 2006

Somiedo 43º01’N, 6º13’W 35 1486 July 14, 2006

Leitariegos 42º59’N, 6º24’W 40 1525 July 6, 2006

Pandetrave 43º06’N, 4º32’W 37 1562 July 23, 2006