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Nuclear markers support the mitochondrial phylogeny of Vipera ursinii–renardi complex (Squamata: Viperidae) and species status for the Greek meadow viper



Meadow vipers (Vipera ursinii–renardi complex) are small-bodied snakes that live in either lowland grasslands or montane subalpine-alpine meadows spanning a distribution from France to western China. This complex has previously been the focus of several taxonomic studies which were based mainly on morphological, allozyme or immunological characters and did not clearly resolve the relationships between the various taxa. Recent mitochondrial DNA analyses found unexpected relationships within the complex which had taxonomical consequences for the detected lineages. The most surprising was the basal phylogenetic position of Vipera ursinii graeca, a taxon described almost 30 years ago from the mountains of Greece. We present here new analyses of three nuclear markers (BDNF, NT3, PRLR; a first for studies of meadow and steppe vipers) as well as analyses of newly obtained mitochondrial DNA sequences (CYT B, ND4).Our Bayesian analyses of nuclear sequences are concordant with previous studies of mitochondrial DNA, in that the phylogenetic position of the graeca clade is a clearly distinguished and distinct lineage separated from all other taxa in the complex. These phylogenetic results are also supported by a distinct morphology, ecology and isolated distribution of this unique taxon. Based on several data sets and an integrative species concept we recommend to elevate this taxon to species level: Vipera graeca Nilson & Andrén, 1988 stat. nov.



Reptilia, Albania, Balkan Peninsula, endemic, Greece, nDNA, Pindos mountains, snake, subspecies

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Arevalo, E., Davis, S.K. & Sites, J.W. (1994) Mitochondrial DNA sequence divergence and phylogenetic relationships among eight chromosome races of the Sceloporus grammicus complex (Phrynosomatidae) in central Mexico. Systematic Biology, 43 (3), 387–418.

Assis, L.C.S. (2009) Coherence, correspondence, and the renaissance of morphology in phylogenetic systematics. Cladistics, 25 (5), 528–544.

Baker, R.J. & Bradley, R.D. (2006) Speciation in Mammals and the Genetic Species Concept. Journal of Mammalogy, 87 (4), 643–662.

Bouckaert, R.R. (2010) DensiTree: making sense of sets of phylogenetic trees. Bioinformatics, 26 (10), 1372–1373.

Bryant, D. & Moulton, V. (2004) NeighborNet: an agglomerative algorithm for the construction of planar phylogenetic networks. Molecular Biology and Evolution, 21 (2), 255–265.

Carranza, S., Arnold, E.N. & Pleguezuelos, J.M. (2006) Phylogeny, biogeography, and evolution of two Mediterranean snakes, Malpolon monspessulanus and Hemorrhois hippocrepis (Squamata, Colubridae), using mtDNA sequences. Molecular Phylogenetics and Evolution, 40 (2), 532–546.

Clement, M., Posada, D. & Crandall, K.A. (2000) TCS: a computer program to estimate gene genealogies. Molecular Ecology, 9 (10), 1657–1659. 10.1046/j.1365-294x.2000.01020.x

Cracraft, J. (1983) Species concepts and speciation analysis. In: Johnston, R.F. (Ed.), Current Ornithology. Vol. 1. Springer US, Boston, pp. 159–187. 10.1007/978-1-4615-6781-3_6

de Queiroz, K. (1998) The general lineage concept of species, species criteria, and the process of speciation: a conceptual unification and terminological recommendations. In: Howard, D.J. & Berlocher, S.H. (Eds.), Endless Forms: Species and Speciation. Oxford University Press, New York, pp. 57–75.

Dimitropoulos, A. (1985) First records of Orsini’s viper, Vipera ursinii (Viperidae) in Greece. Annales Musei Goulandris, 7, 319–323.

Drummond, A.J., Suchard, M.A., Xie, D. & Rambaut, A. (2012a) Bayesian Phylogenetics with BEAUti and the BEAST 1.7. Molecular Biology and Evolution, 29 (8), 1969–1973.

Drummond, A.J., Xie, W. & Heled, J. (2012b) Bayesian Inference of Species Trees from Multilocus Data using *BEAST. Available from: (accessed 20 December 2016)

Ferchaud, A.-L., Ursenbacher, S., Cheylan, M., Luiselli, L., Jelić, D., Halpern, B., Major, Á., Kotenko, T., Keyan, N., Crnobrnja-Isailović, J., Tomović, L., Ghira, I., Ioannidis, Y., Arnal, V. & Montgerald, C. (2012) Phylogeography of the Vipera ursinii complex (Viperidae): mitochondrial markers reveal an east–west disjunction in the Palaearctic region. Journal of Biogeography, 39 (10), 1836–1847.

Flot, J.F. (2010) Seqphase: A web tool for interconverting phase input/output files and fasta sequence alignments. Molecular Ecology Resources, 10 (1), 162–166.

Gvoždík, V., Jandzik, D., Lymberakis, P., Jablonski, D. & Moravec, J. (2010) Slow worm, Anguis fragilis (Reptilia: Anguidae) as a species complex: Genetic structure reveals deep divergences. Molecular Phylognetics and Evolution, 55 (2), 460–472.

Gvoždík, V., Jandzik, D., Cordos, B., Rehák, I. & Kotlík, P. (2012) A mitochondrial DNA phylogeny of the endangered vipers of the Vipera ursinii complex. Molecular Phylogenetics and Evolution, 62 (3), 1019–1024.

Heled, J. & Drummond, A.J. (2010) Bayesian inference of species trees from multilocus data. Molecular Biology and Evolution, 27 (3), 570–580.

Huson, D.H. & Bryant, D. (2006) Application of phylogenetic networks in evolutionary studies. Molecular Biology and Evolution, 23 (2), 254–267.

Joger, U., Fritz, U., Guicking, D., Kalyabina-Hauf, S.A., Nagy, Z.T. & Wink. M. (2007) Phylogeography of western Palaearctic reptiles – Spatial and temporal speciation patterns. Zoologischer Anzeiger – A Journal of Comparative Zoology, 246 (4), 293–313.

Kalyabina-Hauf, S.A., Schweiger, S., Joger, U., Mayer, W., Orlov, N. & Wink, M. (2004) Phylogeny and systematics of adders (Vipera berus complex). In: Joger, U. & Wollesen, R. (Eds.), Mertensiella. Vol. 15. Verbreitung, Ökologie und Schutz der Kreuzotter (Vipera berus [Linnaeus, 1758]). Deutsche Gesellschaft für Herpetologie und Terrarienkunde, pp. 7–16.

Kindler, C., Böhme, W., Corti, C., Gvoždík, V., Jablonski, D., Jandzik, D., Metallinou, M. Široký, P. & Fritz, U. (2013) Mitochondrial phylogeography, contact zones and taxonomy of grass snakes (Natrix natrix, N. megalocephala). Zoologica Scripta, 42 (5), 458–472.

Korsós, Z., Barina, Z. & Pifkó, D. (2008) First record of Vipera ursinii graeca in Albania (Reptilia: Serpentes, Viperidae). Acta Herpetologica, 3 (2), 167–173.

Kotenko, T., Morozov-Leonov, S.Y. & Mezhzherin, S.V. (1999) Biochemical genetic differentiation of the steppe viper (Vipera ursinii group) in Ukraine and Romania. In: 10th Ordinary General Meeting of the Societas Europaea Herpetologica. Natural History Museum of Crete, Irakleio. pp. 88–90.

Kunstler, G., Chaduf, J., Klein, E.K., Curt, T., Bounchaud, M. & Lepart, J. (2007) Tree colonization of sub-Mediterranean grasslands: effects of dispersal limitation and shrub facilitation. Canadian Journal of Forest Research, 37 (1), 103–115.

Lanfear, R., Calcott, B., Ho, S.Y.W. & Guindon, S. (2012) PartitionFinder: combined selection of partitioning schemes and substitution models for phylogenetic analyses. Molecular Biology and Evolution, 29 (6), 1695–1701.

Lemonnier-Darcemont, M., Puskás, G. & Darcemont, C. (2015) First overview of the south Albanian Orthoptera fauna. Articulata, 30, 63–80.

Librado, P. & Rozas, J. (2009) DnaSP v5: A software for comprehensive analysis of DNA polymorphism data. Bioinformatics, 25 (11), 1451–1452.

Ljubisavljević, K., Arribas, O., Džukić, G. & Carranza, S. (2007) Genetic and morphological differentiation of Mosor rock lizard, Dinarolacerta mosorensis (Kolombatović, 1886), with the description of a new species from the Prokletije Mountain Massif (Montenegro) (Squamata: Lacertidae). Zootaxa, 1613, 1–22.

Martin, D.P., Lemey, P., Lott, M., Moulton, V., Posada, D., & Lefeuvre, P. (2010) RDP3: a flexible and fast computer program for analysing recombination. Bioinformatics, 26 (19), 2462–2463.

Miralles, A., Vasconcelos, R., Perera, A., Harris, D.J. & Carranza, S. (2010) An integrative taxonomic revision of the cape Verdean skinks (Squamata, Scincidae). Zoologica Scripta, 40 (1), 16–44..

Mizsei, E., Üveges, B., Vági, B., Szabolcs, M., Lengyel, S., Pfliegler, W.P., Nagy, Z.T. & Tóth, J.P. (2016) Species distribution modelling leads to the discovery of new populations of one of the least known European snakes, Vipera ursinii graeca, in Albania. Amphibia-Reptilia, 37 (1), 55–68.

Nilson, G. & Andrén, C. (1988) A new subspecies of the subalpine meadow viper, Vipera ursinii (Bonaparte) (Reptilia, Viperidae), from Greece. Zoologica Scripta, 17 (3), 311–314.

Nilson, G. & Andrén, C. (2001) The Meadow and Steppe Vipers of Europe and Asia - The Vipera (Acridophaga) ursinii complex. Acta Zoologica, 47 (2–3), 87–267.

Nilson, G., Andrén, C. & Joger, U. (1993) A re-evaluation of the taxonomic status of the Moldavian steppe viper based on immunological investigations, with a discussion of the hypothesis of secondary intergradation between Vipera ursinii rakosiensis and Vipera (ursinii) renardi. Amphibia-Reptilia, 14 (1), 45–57.

Noonan, B.P. & Chippindale, P.T. (2006) Dispersal and vicariance: The complex evolutionary history of boid snakes. Molecular Phylogenetics and Evolution, 40 (2), 347–358.

Papanastasis, V.P., Kyriakakis, S. & Kazakis, G. (2002) Plant diversity in relation to overgrazing and burning in mountain mediterranean ecosystems. Journal of Mediterranean Ecology, 3 (2–3), 53–63.

Pisani, D., Benton, M.J. & Wilkinson, M. (2007) Congruence of morphological and molecular phylogenies. Acta Biotheoretica, 55 (3), 269–281.

Rambaut A., Suchard, M.A., Xie, W. & Drummond, A.J. (2013) Tracer v1.6. 796 the BEAST site. Available from: (accessed 20 December 2016)

Ronquist, F., Teslenko, M., van der Mark, P., Ayres, D.L., Darling, A., Höhna, S., Larget, B., Liu, L., Suchard, M.A. & Huelsenbeck, J.P. (2012) MrBayes 3.2: efficient Bayesian phylogenetic inference and model choice across a large model space. Systematic Biology, 61 (3), 539–542.

Speybroeck, J., Beukema, W. & Crochet, P.A. (2010) A tentative species list of the European herpetofauna (Amphibia and Reptilia) – an update. Zootaxa, 2492, 1–27.

Stamatakis, A. (2014) RAxML version 8: a tool for phylogenetic analysis and post-analysis of large phylogenies. Bioinformatics, 30, 1312–3.

Stephens, M., Smith, N.J. & Donnelly, P. (2001) A new statistical method for haplotype reconstruction from population data. American Journal of Human Genetics, 68 (4), 978–989.

Torstrom, S.M., Pangle K.L. & Swanson, B.J. (2014) Shedding subspecies: The influence of genetics on reptile subspecies taxonomy. Molecular Phylogenetics and Evolution, 76, 134–143.

Townsend, T.M., Alegre, R.E., Kelley, S.T., Weins, J.J. & Reeder, T.W. (2008) Rapid development of multiple nuclear loci for phylogenetic analysis using genomic resources: an example from squamate reptiles. Molecular Phylogenetics and Evolution, 47 (1), 129–142.

Tuniyev, B., Nilson, G. & Andrén, C., (2010) A new species of viper (Reptilia, Viperidae) from the Altay and Saur Mountains, Kazakhstan. Russian Journal of Herpetology, 17 (2), 110–120.

Ursenbacher, S., Carlsson, M., Helfer, V., Tegelström, H. & Fumagalli, L. (2006) Phylogeography and Pleistocene refugia of the adder (Vipera berus) as inferred from mitochondrial DNA sequence data. Molecular Ecology, 15 (11), 3425–3437.

Welch, K.R.G. (1994) Snakes of the world. A Checklist I. Venomous snakes. KCM Books, Somerset, 135 pp.

Wiley, E.O. (1978) The evolutionary species concept reconsidered. Systematic Zoology, 27 (1), 17–26.

Wüster, W., Peppin, L., Pook, C.E. & Walker, D.E. (2008) A nesting of vipers: Phylogeny and historical biogeography of the Viperidae (Squamata: Serpentes). Molecular Phylogenetics and Evolution, 49 (2), 445–459.

Zinenko, O., Stümpel, N., Mazanaeva, L., Bakiev, A., Shiryaev, K., Pavlov, A., Kotenko, T., Kukushkin, O., Chikin, Y., Duisebayeva, T., Nilson, G., Orlov, N.L., Tuniyev, S., Ananjeva, N.B., Murphy, R.W. & Joger, U. (2015) Mitochondrial phylogeny shows multiple independent ecological transitions and northern dispersion despite of Pleistocene glaciations in meadow and steppe vipers (Vipera ursinii and Vipera renardi). Molecular Phylogenetics and Evolution, 84, 85–100.



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