Molecular Phylogenetics and Evolution 38 (2006) 546–552www.elsevier.com/locate/ympev

Short communication

Phylogeography of the asp viper (Vipera aspis) inferred from mitochondrial DNA sequence data: Evidence for multiple

Mediterranean refugial areas

S. Ursenbacher a,b,¤,1, A. Conelli a, P. Golay c, J.-C. Monney d, M.A.L. ZuYe, G. Thiery f,

T. Durand g, L. Fumagalli a

a Laboratoire de Biologie de la Conservation, Département d’Ecologie et Evolution, Bâtiment de Biologie, Université de Lausanne, CH-1015 Lausanne, Switzerland

b School of Biological Sciences, University of Wales, Bangor LL57 2UW, Wales, UKc Cultural Foundation Elapsoïdea, CH-1219 Aire/Geneva, Switzerland

d Centre de Coordination pour la Protection des Amphibiens et des Reptiles de Suisse, KARCH, CH-3005 Berne, Switzerlande Museo di Storia Naturale e del Territorio, Università di Pisa, Via Roma 79, I-56011 Calci, Italy

f 805 Rue du Pré de l’Ane, F-73000 Chambéry, Franceg Saury, F-74210 La Tuile, France

Received 18 April 2005; revised 12 August 2005; accepted 13 August 2005Available online 6 October 2005

1. Introduction

Climatic Xuctuations during the Pleistocene had drasticinXuence on the distribution and the extent of intraspeciWcgenetic diversities of temperate European taxa. Repeatedglacial and interglacial cycles forced species to retreat intorefugia and to expand from these regions during intergla-cial warmings (Hewitt, 1996). Consequently, after recoloni-sation of the whole continent, taxa maintained theirdiVerentiation in several distinct genetic groups, thesedivergences leading in some cases to speciation events(Hewitt, 2001). Three main peninsular refugia have beendeduced from phylogenetic studies for most temperate spe-cies in Europe, namely Iberia, Italy, and the Balkans (seeHewitt, 1996, 2000; Taberlet et al., 1998), but additionalglacial refugia in central and northern Europe have beenrecently proposed (Bilton et al., 1998; Kotlik and Berrebi,2001; Nesbø et al., 1999; Pfenninger and Posada, 2002; Sed-don et al., 2002; Stewart and Lister, 2001).

Most studies on the genetic consequences of past demo-graphic Xuctuations have involved endotherm species withrelatively high dispersal capabilities (e.g., Avise, 2000).

* Corresponding author.E-mail address: s.ursenbacher@bangor.ac.uk (S. Ursenbacher).

1 Present address: School of Biological Sciences, University of Wales,Bangor LL57 2UW, Wales, UK.

1055-7903/$ - see front matter 2005 published by Elsevier Inc.doi:10.1016/j.ympev.2005.08.004

In contrast, few studies have been conducted on terrestrialpoikilotherms with narrow habitat requirements and lim-ited dispersal potentials such as snakes, partly also due totheir secretive habits and to the diYculty to obtain repre-sentative samples. The asp viper (Vipera aspis) is a temper-ate species limited to the southwestern range of theEuropean continent. Five subspecies are currently recogni-sed (Golay et al., 1993; Mallow et al., 2003; but see ZuY,2002): V. a. aspis in France and Switzerland; V. a. atra inthe western part of the Alps; V. a. zinnikeri in Spain, thePyrenees and southwestern France; V. a. francisciredi inCentral and northern Italy as well as extreme south of Swit-zerland; V. a. hugyi in southern Italy. Using sequence datafrom the mitochondrial cytochrome b (cyt b) and ND2genes, a recent molecular phylogeny of the genus Viperasuggested, based on a limited sampling of V. aspis, the pres-ence of two distinct groups for the species, an Italian one(V. a. francisciredi and V. a. hugyi) and a French one (V. a.aspis, V. a. atra, and V. a. zinnikeri) (Garrigues et al., 2005).Moreover, the authors proposed a separation between“neurotoxic” French populations (V. a. aspis) and the otherV. a. aspis, as well as between V. a. atra and V. a. aspis, andsuggested that V. a. zinnikeri and V. a. aspis are clusteredtogether.

The present study investigates the phylogeography ofthe asp viper V. aspis across its whole distribution rangeusing mitochondrial DNA control region sequence data.

S. Ursenbacher et al. / Molecular Phylogenetics and Evolution 38 (2006) 546–552 547

In particular, our aims are: (i) to construct a molecular phy-logeny and compare it with the current taxonomic interpre-tations based on morphology; (ii) to locate the circum-Mediterranean Quaternary refugial areas; (iii) to assess thedistribution and extent of genetic variation within andamong these refugia; (iv) to evaluate the potential of poiki-lotherm species restricted to the southern portion of theEuropean continent in revealing the internal structure ofthe major Mediterranean refugial regions.

2. Materials and methods

We sampled a total of 53 Vipera aspis individuals cover-ing the whole geographical range of the species (Fig. 1 andAppendix A). Depending on the source material, DNA wasextracted from blood, slough skin, or tissue. Nose-hornedviper (V. ammodytes) has been used as outgroup. Totalgenomic DNA was extracted using QIAamp DNA MiniKit (Qiagen). A 671 base pairs (bp) portion of the mito-chondrial DNA (mtDNA) control region (CR) wassequenced using primers L16571VA (5�-CTCTTTCCAAGGCCTCTGGCT-3�) and H690 (Kumazawa et al., 1996).Polymerase chain reaction (PCR) was conducted in 25 �lvolumes with 2 �l of DNA template, 1 £ PCR buVer(Qiagen), 2 mg/ml of Q solution (Qiagen), 2 mM of MgCl2,0.2 mM dNTPs, 0.5 �M of each primer and 0.5 units of Taq

polymerase (Qiagen). AmpliWcation conditions consisted of35–45 cycles of denaturation for 30 s at 94 °C, annealing for30 s at 56 °C and extension for 45 s at 72 °C. PCR productswere puriWed using QIAquick PCR PuriWcation Kit(Qiagen). Cycle sequencing was performed in 7.5 �l contain-ing 3–4�l of ampliWed DNA, 0.5–1.5�l of 1–3 �M primerand 3 �l of ABI PRISM Dye Terminator cycle sequenceReady Reaction Kit (Applied Biosystems). Water wasadded up to 7.5�l. Reaction sequences were visualised onan ABI 377 automated sequencer (Applied Biosystems).Sequences were deposited in GenBank (Accession No.DQ168595–DQ168615).

Mitochondrial DNA sequences were aligned using theClustal method (Higgins et al., 1992) implemented inSequenceNavigator software (Applied Biosystems). Weused multiple approaches to examine phylogenetic relation-ships among V. aspis haplotypes: distance (neighbor-join-ing; NJ), maximum parsimony (MP) and maximumlikelihood (ML) analyses, as well as statistical parsimony(Crandall, 1994; Templeton et al., 1992). MP analyses, usingPAUP* 4.0b10 (SwoVord, 2002), were conducted with TBRbranch swapping options and with indels considered as anew state (Wfth base). ML analyses were conducted withPHYML 2.4.4 (Guindon and Gascuel, 2003) and NJ withPAUP*. For the ML and NJ analyses, a total of 56 substi-tution models were evaluated using ModelTest v3.6

Fig. 1. Geographic location of Vipera aspis samples analysed in the present study. The geographic distribution areas of all recognized subspecies are sepa-rated by dotted lines based on ZuY (2002). Question marks indicate regions where the distribution of the subspecies is unknown. Subclade 1A: white cir-cles; subclade 1B: black circles; subclade 2A: black squares; subclade 2B: white squares. Asterisk indicates sample from the region where “neurotoxic” asp

vipers are found (see Garrigues et al., 2005). Sample labels correspond to localities listed in Appendix A.

548 S. Ursenbacher et al. / Molecular Phylogenetics and Evolution 38 (2006) 546–552

(Posada and Crandall, 1998) and PAUP*. The best model,selected by the Akaike Information Criterion (AIC), wasthe HKY + I model (freq. A D 0.2797; freq. C D 0.2488; freq.G D 0.1254; freq. T D 0.3460; ti/tv ratio D 1.7042; proportionof invariable sitesD 0.7859). The robustness of the branch-ing pattern of the trees was tested by 10,000 bootstrap repli-cates for ML, NJ, and MP analyses. While NJ, MP, andML have diYculty resolving relationships among closelyrelated haplotypes, statistical parsimony more convenientlyallows the display of relationships among sequences with alow number of mutational diVerences, and yields a quanti-tative assessment of the reliability of connections. Resultsof statistical parsimony showing genealogical relationshipsamong haplotypes were represented graphically using anetwork with TCS 1.13 software (Clement et al., 2000). Dis-tances (uncorrected distance) between subspecies or cladeswere calculated using MEGA 2.1 (Kumar et al., 2001). Alikelihood ratio test was performed to determine whetherevolutionary rates among clades were statistically diVerent(see Muse and Weir, 1992). For all subclades regroupingmore than 25 samples (to avoid weak results) we studiedpairwise mismatch distributions to detect evidence of popu-lation growth from low-diversity founder populations. Dis-tributions were also plotted and tested for a goodness-of-Wtusing parametric bootstrapping (1000 replicates) withARLEQUIN (Schneider et al., 2000). Moreover, an excessof rare alleles, which is indicative of a recent expansion, wastested with Fu’s FS test (Fu, 1997). The timing of thisexpansion was inferred from the mode of the mismatch (�)and the mean number of pairwise nucleotide diVerences (m)(Rogers, 1995; Rogers and Jorde, 1995), considering a 5years generation time for V. aspis.

Calibration of the molecular clock based on the CR isnot possible in V. aspis due to a lack of fossils. However,calibration for the cyt b and ND4 regions is available inViperidae based on geological events (evolution rate:1.4% Myr; 95% conWdence limits: 1.09–1.77; see Wüsteret al., 2002). To get a rough approximation of the rate ofdivergence in the CR, we compared the substitution rates ofcyt b and CR in V. aspis by sequencing 600 bp of the cyt bfor six samples representative of the species distributionrange. Comparison of p-distances between the two regionsshowed that CR substitution rate is 1.33 lower than cyt b.Using this approximation, the rate of divergence of CR inV. aspis can be estimated at about 1.05%/Myr (95% conW-dence limit: 0.82–1.33).

Genetic diVerentiation was determined by conducting ananalysis of molecular variance and calculating �-ST indices(AMOVA in Arlequin 2.0) at diVerent (mtDNA) levels:among groups (corresponding to the inferred clades) andwithin each group. Finally, we have tested, using the Shi-modaira–Hasegawa (HS) test (Shimodaira and Hasegawa,1999) implemented in PAUP*, whether the most likelihoodtree obtained in our analysis was signiWcantly diVerentfrom the three following hypotheses: (i) Garrigues et al.(2005): V. a. aspis neurotoxic is the sistergroup of the cladecomposed by V. a. atra and V. a. aspis + V. a. zinnikeri,

whereas V. a. francisciredi is the sisterclade to V. a. hugyi;(ii) ZuY (2002): V. a. atra, V. a. zinnikeri, V. a. hugyi areconsidered as sistergroup of the clade V. a. aspis + V. a.francisciredi; (iii) the taxonomic cladogram: the Wve sub-species separated in the same time.

3. Results and discussion

A total of 20 haplotypes among the 53 samples analysedwere obtained. There were 41 (6.1%) variable sites (15.8%including the outgroup taxon), of which 35 (5.2%) werephylogenetically informative under MP criteria. One indeloccurred within V. aspis, with another 11 indels amongingroup and the outgroup taxon. The sequence divergence(uncorrected p-distance) between subspecies ranged from0.66 to 4.4%, with the exception of V. a. aspis and V. a. atra,which share identical haplotypes.

NJ, MP, and ML analyses consistently found two well-supported clades of V. aspis and produced similar topolo-gies (Fig. 2a): (i) a Western clade comprised all samplesfrom Spain, France, Switzerland with the exception of itsextreme southern portion, and northwestern Italy (V. a.aspis, V. a. atra, and V. a. zinnikeri); (ii) an Eastern cladecomprised all individuals from the Italian peninsula exceptits northwestern part (V. a. hugyi and V. a. francisciredi).Within the Western clade, two groups can be diVerentiatedin the analyses: a southern subclade (1A, comprising vipersfrom Spain and southwestern France, all samples beingrecognised as V. a. zinnikeri) and a northern subclade (1B,with most of Switzerland, France, and extreme northwest-ern Italy, all samples corresponding to V. a. aspis and V. a.atra). The Eastern clade can also be split in a southerngroup (2A, with V. a. hugyi), and a central and northerngroup (2B, with V. a. francisciredi). Our results conWrm thesystematics of the species, except for V. a. atra which is notdistinct from V. a. aspis, and refute the suggestions of Gar-rigues et al. (2005) about the position of V. a. zinnikeri(however, the HS test is not signiWcant; p D 0.596) as well asthe proposal of ZuY (2002) to consider V. a. atra as a sepa-rate species (HS test strongly rejected this hypothesis,p < 0.001).

The statistical parsimony analysis conWrmed the resultsof the phylogenetic analyses, but provided more resolutionamong haplotypes within clades. Exact parsimonious prob-abilities for the linkages spanning from one to as many aseleven single mutational steps were >95% (i.e., 21 of 22 link-ages showed a statistically signiWcant level of conWdencethat no multiple hits have occurred). This resulted in theconstruction of two minimum spanning networks ofmtDNA haplotypes (Fig. 2b) which discriminate three lin-eages with no geographical overlap, corresponding to subc-lade 1A, subclade 1B, and subclades 2A + 2B.

The likelihood ratio test did not reject the null hypothesisof homogeneous rate across lineages (¡2log�D20.8;pD0.34), suggesting that the genetic distance between haplo-types is linked to the geological events. Using the rate ofdivergence approximation of the CR, rough dates of split

S. Ursenbacher et al. / Molecular Phylogenetics and Evolution 38 (2006) 546–552 549

between the diVerent clades can be estimated. These date esti-mates should be taken with caution and only as an indicationof the geological periods when genetic splits occurred. There-fore, only conWdence intervals are given. The split betweenthe two clades is estimated to have occurred in the MiddlePliocene (95% conWdence limit: 2.8–4.5 Myr). The splitbetween the subclades occurred more recently (1.4–2.2 Myrbetween 1A and 1B, and 0.8–1.2 Myr between 2A and 2B).The clade 1B (the only one that was tested since it regroupsenough samples) show a genetic signature of an expansion(Fs D¡5.14, p < 0.0001; mismatch distribution test of good-ness-of-Wt, pD0.39). The expansion time consequently hasbeen estimated to 0.11–0.29 Myr for subclade 1B (estimations

Fig. 2. (a) Maximum likelihood tree for the 20 haplotypes of V. aspisbased on 671 bp of the mtDNA control region. Values of bootstrap sup-port are shown for nodes found in more than 50% of 10,000 trees forneighbour joining, maximum parsimony and likelihood analyses (fromtop to bottom, respectively); (b) Parsimony networks obtained using TCS(Clement et al., 2000), based on 671 bp of the mtDNA control region of 20distinct haplotypes of Vipera aspis. Black circles indicate the number ofmutational changes between haplotypes. Asterisk indicates sample fromthe region where “neurotoxic” asp vipers are found (see Garrigues et al.,2005). The size of the haplotype is proportional to its frequency. The hap-lotype in a square has the biggest outgroup weight.

based on m and �). The AMOVA showed that most of thetotal variance (94%, p < 0.0001) was explained by diVerencesamong the four subclades, whereas within-subclade variationaccounted for only 6% of the total variance.

The asp viper V. aspis displays two major clades sepa-rated by the Alpine barrier as previously suggested by Gar-rigues et al. (2005). This important split has already beenwell documented in a great number of diverse taxa, such as,shrews of the Sorex araneus group (Taberlet et al., 1994),newts of the genus Triturus (Wallis and Arntzen, 1989), thetrout Salmo trutta (Bernatchez et al., 1992), the honeybeeApis mellifera (Garnery et al., 1992) and the grasshopperChorthippus parallelus (Cooper et al., 1995).

Although the presence of two (or more) refugial areas inItaly has previously been proposed for some species (Deme-sure et al., 1996; Podnar et al., 2005), the presence of subc-lades 2A and 2B as shown by the MP and the ML analysesis questionable. Indeed, the number of samples in southernItaly is very limited and the number of mutational diVer-ences between these two subclades is low (4 substitutions).Moreover, restricted sampling might be the reason for thelack of detection of intermediate haplotypes. Within theWestern clade, two distinct refugial areas have been occu-pied by asp vipers. By contrast to the Italian clade, the highnumber of substitutions between subclades 1A and 1B(mean: 1.77%) might not be explained by a sampling bias,since the contact region between both phylogroups hasbeen sampled. Whereas Spain was an important refugialarea for numerous animal and plant species (see Taberletet al., 1998), our data are consistent with the hypothesis ofan additional refugium for V. aspis in southern France dur-ing the last glaciations. The estimation of the expansionbased on the mismatch distribution conWrms a recent dis-persal of this subclade. This refugial area has already beensuggested for lizards (Guillaume et al., 2000), barbels (Kot-lik and Berrebi, 2001), snails (Pfenninger and Posada,2002), and for some plants (Palme and Vendramin, 2002;Vogel et al., 1999), although with a variable degree of reso-lution concerning its precise location. These taxa, charac-terised by a low dispersion rate and a high tolerance tosurvive in cold and humid environments, might have per-sisted in this area during the coldest periods. SouthernFrance has never been covered by ice sheets during theQuaternary glacial periods, and was characterised by thepresence of a steppe vegetation (Elenga et al., 2000). Fur-thermore, predicted sea levels along the French Mediterra-nean coast before and during last glacial maximum (18,000years BP) were considerably lower than today (more than100 m below present level), allowing taxa such as asp vipersto inhabit dry coastal areas which today are submerged(Lambeck and Bard, 2000). Moreover, the higher (albeitnot signiWcant) genetic diversity found in the southeasterngeographical portion of this subclade suggests that the refu-gial area was in the south and that snakes recolonised cen-tral France more recently.

The results of our phylogenetic analyses indicate thatV. aspis is separated in four distinct groups, corresponding

550 S. Ursenbacher et al. / Molecular Phylogenetics and Evolution 38 (2006) 546–552

to V. a. aspis, V. a. zinnikeri, V. a. hugyi, and V. a. francisci-redi. These results are partially in contradiction with severalsuggestions made by Garrigues et al. (2005). In our analy-ses, the sample from the “neurotoxic” asp viper region(although neurotoxic activity of the venom has not beentested) cannot be genetically diVerentiated from other V. a.aspis or V. a. atra. It is likely that the presence of severaldiVerent haplotypes revealed by our analysis in southernFrance (haplotypes H1, H2, and H4) and the limited num-ber of V. aspis samples analysed by Garrigues et al. (2005)(only eight animals in France), allowed these authors toconclude that several distinct groups were present within V.a. aspis. Moreover, the unexpected phylogenetic relation-ship of V. a. zinnikeri with the V. a. aspis group (belongingto two distinct subclades in our analysis) could beexplained by a hybrid origin of the single specimen ana-lysed by these authors, which is located within the contactzone between these two subspecies. A higher mutation ratein the CR cannot explain the discrepancy between bothstudies, since the CR evolve slower than ND2 or cyt b insnakes (Ashton and de Queiroz, 2001; Burbrink et al., 2000;this study). In addition, sequence data from a portion of thecyt b of one sample from both subclades 1A and 1B (fromSoria, Spain, and Kandersteg, Switzerland, see AppendixA) reveal a divergence of 2.4% (out of 1040 bp; data notshown), demonstrating that substantial divergence betweensubclades 1A and 1B also occurs for cyt b.

The data presented here show the utility of analysingspecies with particular ecological requirements such as rep-tiles, to shed light on the inner structure of regional refugialregions and the extent to which diVerent populations per-sisted in these areas during glacial periods and recolonisedit in postglacial times. Overall, the results illustrate thepotential for the occurrence of multiple, small refugia inaddition to the main Mediterranean peninsular refugialareas usually considered for European temperate species.

Acknowledgments

This work was funded by grants from the Swiss NationalScience Foundation (Grant No. 3100-059132.99/1). Weacknowledge M. Cocquio (Saltrio, Italy), B. and A. Conelli(Arzo, Switzerland), O. Lourdais (Chizé, France), S. Dum-mermuth (Oberdorf, Switzerland), J. Ferrez (BTVS, Portu-gal), E. Garcia Franquesa (MZB, Barcelona, Spain), J.Garzoni (Lausanne, Switzerland), P. Geniez & M. Chelyan(EPHE Montpellier, France), E. Lasne (Angers, France), A.Meyer (KARCH, Bern, Switzerland), M. Nembrini (Bellin-zona, Switzerland), J.-M. Pillet (Ravoire, Switzerland), M.Poggesi (MZUF, Firenze, Italy), B. and L. Rechsteiner (Arzo,Switzerland), R. Rosoux and G. Baron (La Rochelle, France)for providing samples and help in the Weld. We also thank J.Parker, N. Salamin, P. Taberlet and three anonymous review-ers for helpful comments on earlier drafts of this paper.

Appendix A

Vipera aspis specimens used in the present study, with localisation and haplotype designation for the mitochondrial control region and GeneBank Acces-sion Numbers

Taxon Label Locality Voucher No. or collector

CR haplotype GeneBank No.

V. a. aspis Bg Val Bregaglia (Switzerland) DEE-LBC H1 DQ168596Bg Val Bregaglia (Switzerland) DEE-LBC H1JV Jura vaudois (Switzerland) DEE-LBC H1Ge Genève (Switzerland) DEE-LBC H1Bl Balm (Switzerland) S. Dummermuth H1SQ Mont Saint-Quentin, Loraine (France) DEE-LBC H1SS Saint Sauveur en Rue, Massif Central (France) DEE-LBC H1MP Massif du Pilat (France) DEE-LBC H1Pr Prapic, Haute Alpes (France) DEE-LBC H1Ve Vendée (France) O. Lourdais H1MH Mont de l’Hortus, Herault (France) DEE-LBC H1Ca Cabrials, Hérault (France) M. Cheylan, BEV. 7794 H1Ch La Choumette, (France) E. Lasne H1Co Cournonterral (France) M. Cheylan H1MV Mont Ventoux (France) DEE-LBC, Garzoni H1HL Hospitalet du Larzac (France) M. Cheylan; BEV-845 H1BR Breil sur Roya, Alpes Maritimes (France) DEE-LBC H2 DQ168597Ve Vendée (France) O. Lourdais H3 DQ168598Or Orcières, Hautes Alpes (France) DEE-LBC H4 DQ168599

V. a. atra Ka Kandersteg (Switzerland) DEE-LBC H1Gz Grimentz (Switzerland) DEE-LBC H1Ri Ritorto, Val Bavona (Switzerland) DEE-LBC H1Be Val Bedretto (Switzerland) J. Garzoni H1Cp Chapelle d’Abondance, Haute Savoie (France) DEE-LBC H1CR Cormet de Rosland, Savoie (France) DEE-LBC H1

S. Ursenbacher et al. / Molecular Phylogenetics and Evolution 38 (2006) 546–552 551

Note. Museum and Institution acronyms: DEE-LBC, Départment d’Ecologie et Evolution –Laboratoire de Biologie de la Conservation, Univer-sity of Lausanne (Suisse); MCSNF, Museo Civico di Storia Naturale di Ferrara (Italy); BTVS, Banco de Tecidos de Vertebrados Selvagens—Insti-tuto da Conservação da Natureza, Gerês (Portugal); MZUF, Museo Zoologico Università di Firenze (Italy); RE, Muséum d’Histoire Naturelle deLa Rochelle (France); MZB, Museu de Zoologia de Barcelona (Spain); BEV, EPHE Biologie & Ecologie des Vertébrés, Monpellier (France).

Appendix A (continued )

Taxon Label Locality Voucher No. or collector

CR haplotype GeneBank No.

Gr Gressoney, Val d’Aosta (Italy) MZUF 30520 H1Pi Piedicavallo, Biella (Italy) DEE-LBC H5 DQ168600

V.a. zinnikeri Pa Pancorbo, Burgos (Spain) DEE-LBC H6 DQ168601Cm Camales, La Rioja (Spain) DEE-LBC H6So Soria (Spain) A. Meyer H6So Soria (Spain) A. Meyer H7 DQ168602EL Emiron du Larzac, Gard (France) DEE-LBC H8 DQ168603CB Caune de Blandas, Gard (France) DEE-LBC H9 DQ168604AA Alt Aneu, Catalunya (Spain) MZB98-1468 H10 DQ168605Le Lescu, Pyrenees (France) DEE-LBC H11 DQ168606VT Val de Ter, Catalunya (Spain) DEE-LBC H12 DQ168607Py Pyrénées Atlantiques (France) R. Rossoux, RE H13 DQ168608

V.a. franscisiredi Ma Macerata, Marche (Italy) MZUF 38298 H14 DQ168609Tr Trento, Trentino Alto-Adige (Italy) MZUF 31765 H15 DQ168610GS Gran Sasso (Italy) DEE-LBC H15Po Populonia, Toscana (Italy) DEE-LBC H16 DQ168611Mo Monte Vemtasso (Italy) S. Mazzotti, MCSNF H16LB Lago Brasion (Italy) S. Mazzotti, MCSNF H16Ar Arzo (Switzerland) DEE-LBC H16Ar Arzo (Switzerland) DEE-LBC H16Ct Cantello, Varese (Switzerland) M. Cocquio H16VP Val Poschiavo (Switzerland) DEE-LBC H16Li Livergnano, Pianoro (Italy) RE0282 H17 DQ168612Ap Alpi Apuane (Italy) DEE-LBC H18 DQ168613

V. a. hugyi SG Sila Grande (Italy) M. A. L. ZuY H19 DQ168614Mt Monte Pollino, Calabria (Italy) J.-M. Pillet H20 DQ168615Mt Monte Pollino, Calabria (Italy) DEE-LBC, Garzoni H20Tb Trebisacce, Calabria (Italy) J.-M. Pillet H20

V. ammodytes Selo Brod —Crna Trava (Serbia) L. Tomovic, S. Tome & J. Crnobrnja-Isailovic

DQ168595

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