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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

February 2017
Zootaxa 4227(1):75-88

February 2017
4227(1):75-88

DOI:10.11646/zootaxa.4227.1.4

Authors:

Edvárd Mizsei

Kiskunság National Park Directorate

Stephanos A. Roussos

Maria Dimaki

The Goulandris Natural History Museum

Show all 7 authorsHide

Meadow vipers (Vipera ursinii–renardi complex) are small-bodied snakes that live in either lowland grasslands or mon-tane 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 phyloge-netic 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.

Sampled localities inside the approximate distribution area of Vipera ursinii–renardi complex in Europe. Circles indicate sampling localities of Vipera ursinii–renardi complex, and triangles show the sampling of outgroup taxa. A diamond indicates the type locality of Vipera graeca stat. nov. This figure is published in colour in the online version, the colour of the patches corresponds to the colour of mitochondrial lineages in Fig. 2A.

…

(A) Current mitochondrial Bayesian phylogenetic hypothesis of Vipera ursinii–renardi complex based on CYT B dataset of Ferchaud et al. (2012) and Zinenko et al. (2015); (B) Phylogenetic reconstruction of the concatenated dataset (mtDNA+nDNA genes) obtained in MrBayes/Maximum likelihood (see Table 1). Sequences of Vipera berus (Vbbe-HU, Vbbo-AL, Vbni-RO) included as outgroup are not shown. Bayesian posterior probabilities/bootstrap pseudoreplicates are shown at nodes; (C) SplitsTree phylogenetic network (Huson & Bryant 2006) of the dataset for five mitochondrial and nuclear loci sequenced in the present study using the neighbornet algorithm. Asterisks in Fig. 2C indicate both phased sequences in one branch. Numbers along the edges are the bootstrap support values from 1000 replicates. The scale bar indicates one substitution per one hundred nucleotide positions. Taxon names of the phylogenetic network correspond with the Table 1. Inset shows a male Greek Meadow Viper from Dhëmbel Mountains, Albania.

…

Nuclear allele networks of the three analysed nuclear loci. Circle sizes are proportional to the number of samples/ sequences, small black circles indicate hypothetical haplotypes (alleles). This figure is published in colour in the online version, the colour of the circles in the network corresponds to the colour of mitochondrial lineages in Fig. 2A.

…

Species tree of the Vipera ursinii–renardi complex (with V. berus as outgroup) as inferred in *BEAST based on two mitochondrial and three nuclear loci (A); species-tree cloudogram of the complex based on 27 000 post-burn-in trees resulting from 3 runs of *BEAST, each producing 10,000 trees from which 10% was discarded as burn-in. Higher colour densities represent higher levels of certainty. Maximum clade credibility tree is superimposed upon the cloudogram in bold violet (B). Values of posterior probabilities are given. This figure is published in colour in the online version, the colours of the branches correspond with the colour of mitochondrial lineages in Fig. 2A.

…

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Accepted by H. Zaher: 24 Nov. 2016; published: 1 Feb. 2017

ZOOTAXA

ISSN 1175-5326 (print edition)

ISSN

1175-5334

(online edition)

Zootaxa 4227 (1): 075

–

088

http://www.mapress.com/j/zt/

Article

https://doi.org/10.11646/zootaxa.4227.1.4

http://zoobank.org/urn:lsid:zoobank.org:pub:02913469-1D04-4A2E-A816-993C2FD1731E

Nuclear markers support the mitochondrial phylogeny of Vipera ursinii–renardi

complex (Squamata: Viperidae) and species status for the Greek meadow viper

EDVÁRD MIZSEI

1,10,*

, DANIEL JABLONSKI

2,*

, STEPHANOS A. ROUSSOS

3,4

, MARIA DIMAKI

YANNIS IOANNIDIS

, GÖRAN NILSON

& ZOLTÁN T. NAGY

8,9

Department of Evolutionary Zoology & Human Biology, University of Debrecen, Egyetem tér 1, 4032 Debrecen, Hungary

Department of Zoology, Comenius University in Bratislava, Mlynská dolina, Ilkovičova 6, 842 15 Bratislava, Slovakia

Department of Biological Sciences, Texas Tech University, Lubbock, Texas, 79409-3131, USA

Department of Biological Sciences, University of North Texas, Denton, Texas, 76203, USA

Goulandris Natural History Museum, 100 Othonos St., 145 62 Kifissia, Greece

Biosphere, Aidiniou 40, 172 36 Ymittos, Greece

Göteborg Natural History Museum, Box 7283, SE-402 35 Göteborg, Sweden

Joint Experimental Molecular Unit, Royal Belgian Institute of Natural Sciences, Rue Vautier 29, B-1000 Brussels, Belgium

Museum für Naturkunde, Invalidenstr. 43, D-10115 Berlin, Germany

Corresponding author. E-mail: edvardmizsei@gmail.com

These authors contributed equally to this work

Abstract

Meadow vipers (Vipera ursinii–renardi complex) are small-bodied snakes that live in either lowland grasslands or mon-

tane 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 unex-

pected relationships within the complex which had taxonomical consequences for the detected lineages. The most surpris-

ing 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 phyloge-

netic position of the graeca clade is a clearly distinguished and distinct lineage separated from all other taxa in the com-

plex. 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.

Key words: Albania, Balkan Peninsula, endemic, Greece, nDNA, Pindos mountains, snake, subspecies

Introduction

Meadow vipers (Vipera ursinii–renardi complex) are small-bodied vipers that live in either lowland grasslands or

montane subalpine-alpine meadows. Most of these taxa have highly fragmented distributions, spanning from

eastern France to western China (Nilson & Andrén 2001). European members of the V. ursinii–renardi complex are

among the most endangered species of the European herpetofauna and their systematics and evolutionary histories

have been in the focus recently (see Nilson & Andrén 2001 for review and Tuniyev et al. 2010; Ferchaud et al.

2012; Gvoždík et al. 2012; Zinenko et al. 2015). Based on an extensive analysis of morphology, three groups have

been detected within the V. ursinii–renardi complex (Nilson & Andrén 2001); the ursinii group (“meadow vipers”:

ursinii, macrops, graeca, moldavica, rakosiensis), the “Transcaucasian–Turkish” group (anatolica, ebneri,

eriwanensis, lotievi), and the renardi group (“steppe vipers”: renardi, altaica, tienshanica, parursinii). Over the

past two decades, some of the formerly recognized subspecies within these groups were elevated to full species

MIZSEI ET AL.

status based on morphological, allozyme or immunological differences (e.g. V. eriwanensis Nilson et al. 1993, V.

renardi Kotenko et al. 1999; Nilson & Andrén 2001, V. anatolica and V. e b n e r i Nilson & Andrén 2001).

The molecular phylogeny of meadow and steppe vipers has not been fully resolved due to limited knowledge

on certain taxa or inadequate sampling in analyses, although great strides have been made and clarified several

evolutionary relationships (Kalyabina-Hauf et al. 2004; Ferchaud et al. 2012; Gvoždík et al. 2012; Zinenko et al.

2015). According to the latest mitochondrial phylogeny, the most recent common ancestor of the complex has

Pliocene origin, located presumably in the region between the eastern Mediterranean and the former Paratethys

(Zinenko et al. 2015). Members of the major mitochondrial clades probably radiated in the Anatolian-

Transcaucasian region (renardi clade) and on the Balkan Peninsula (ursinii clade) with subsequent colonization

patterns to the western and eastern parts of central Europe forming the present geographic distributions (Ferchaud

et al. 2012; Zinenko et al. 2015).

Based on these most recent studies, there is a need for taxonomical investigation and reassessments for taxa

with unexpected phylogenetic positions in the whole genus; or more specifically the V. ursinii–renardi complex

(see Ferchaud et al. 2012; Zinenko et al. 2015). In such a case, nuclear DNA loci (nDNA) could help corroborate

the pattern and be used to better resolve the evolutionary relationships and taxonomy of meadow vipers, but this

has yet to be done.

A member of the genus with a surprising phylogenetic placement is the Greek meadow viper, Vipera ursinii

graeca Nilson & Andrén, 1988, which is a rare taxon described three decades ago. The species lives in high

elevation meadows (1600–2300 m) of the central and southwestern Hellenides mountain range (Dimitropoulos

1985; Nilson & Andrén 1988; Korsós et al. 2008; Mizsei et al. 2016). According to the phylogenetic study of

Ferchaud et al. (2012), V. u. graeca is sister to all other clades of ursinii and renardi in the V. ursinii–renardi

complex and forms a distinct, divergent clade sister to the others, isolated in fragmented habitats in the Pindos

mountains. Similar results have been reported by Zinenko et al. (2015) who also included samples from Vi pera

anatolica, and a seemingly relict population of Vipera kaznakovi which is highly divergent. As there is fairly strong

consensus between taxonomic units in reptiles using mitochondrial DNA (mtDNA) analyses (Joger et al. 2007),

Ferchaud et al. (2012) proposed V. u . graeca as a possible candidate for full species status. However, taking a

multilocus approach, in this day and age, is the most widely used and strongly supported data and results when

discerning species and implementing taxonomic assessments (Torstrom et al. 2014). Using molecular methods,

taxonomy is currently experiencing a new boom with many re-evaluated or newly described reptile taxa also in

such well-known region as is Europe (e.g. Dinarolacerta, Malpolon; Carranza et al. 2006; Ljubisavljević et al.

2007 and Speybroeck et al. 2010 for review). Therefore, the aim of our study was to analyse nDNA loci in the V.

ursinii-renardi complex, to see if the results concur with previous mtDNA studies, and resolve the taxonomic

status and phylogenetic placement of V. u. graeca.

Material and methods

Geographic and taxon sampling. Two samples of V. u . graeca from Albania were included in this study (Mizsei

et al. 2016), both representing the same mtDNA haplotype as the published sequences from Greece in previous

studies (CYT B, ND4; Ferchaud et al. 2012). Other specimens investigated were samples representing montane

populations of Vipera ursinii macrops (Albania, Montenegro), V. u. ursinii (Italy), V. ursinii ssp. (an undescribed

lineage from Croatia), lowland vipers V. u. moldavica (Romania) and V. u. rakosiensis (Hungary, Romania), V.

renardi (Crimea) as well as three subspecies of the common adder V. berus; berus (Hungary), bosniensis (Albania)

and nikolskii (Romania) (see Fig. 1 and Table 1 for details) as outgroup taxa.

DNA extraction and sequencing. We selected two mitochondrial markers (CYT B, ND4) and three nuclear

markers (BDNF, NT3, PRLR) shown to be successful in discriminating on various divergence levels among several

reptile species (Joger et al. 2007; Townsend et al. 2008). We used the DNeasy Blood & Tissue Kit (Qiagen) and the

NucleoSpin Tissue kit (Macherey-Nagel) for extracting total genomic DNA. Polymerase chain reaction (PCR)

conditions for amplifying mitochondrial CYT B and ND4 genes were followed protocols outlined in Ferchaud et al.

(2012). The genes were amplified using the primers shown in Table 2. PCR conditions for PRLR and BDNF were

as follows; 180 seconds at 94°C, followed by 40 steps of 94°C (40s), 50°C (30s), 72°C (60s) and a final elongation

step of 7 min at 72°C. PCR conditions for NT3 were 180 seconds at 94°C, followed by 40 steps of 94°C (40s),

48°C (30s), 72°C (60s) and a final elongation step of 7 min at 72°C.

GREEK MEADOW VIPER TAXONOMY

TABLE 1. List of samples used in this study and GenBank accession numbers of sequenced markers.

Taxon ID in this study Country Population mitochondrial DNA nuclear DNA

CYT B ND4 PRLR BDNF NT3

Vipera berus berus Vbbe-HU Hungary Zemplén JN204721 † ** LT220998 ● LT221007 ● LT220962 ● LT220980 ●

Vipera berus bosniensis Vbbo-AL Albania Prokletije LT220959 ● LT220999 ● LT221008 ● LT220963 ● LT220981 ●

Vipera berus nikolskii Vbni-RO Romania Iaşi - LT221005 ● LT221018 ● LT220973 ● LT220991 ●

Vipera ursinii ursinii Vuur-IT Italy Camerino FR727041 ‡ * LT221006 ● LT221024 ● LT220979 ● LT220997 ●

Vipera ursinii ssp. Vurs-HR1 Croatia Velebit FR727052 ‡ FR726984 ‡ LT221009 ● LT220964 ● LT220982 ●

Vipera ursinii ssp. Vurs-HR2 Croatia Velebit FR727053 ‡ FR726985 ‡ LT221010 ● LT220965 ● LT220983 ●

Vipera ursinii macrops Vumc-AL1 Albania Korab LT220961 ● LT221000 ● LT221013 ● LT220968 ● LT220986 ●

Vipera ursinii macrops Vumc-MN1 Montenegro Bjelasica FR727058 ‡ * LT221001 ● LT221014 ● LT220969 ● LT220987 ●

Vipera ursinii macrops Vumc-MN2 Montenegro Bjesalica FR727059 ‡ * LT221002 ● LT221015 ● LT220970 ● LT220988 ●

Vipera ursinii rakosiensis Vura-HU1 Hungary Nagypuszta FR745956 ‡ FR745905 ‡ LT221019 ● LT220974 ● LT220992●

Vipera ursinii rakosiensis Vura-RO Romania Csengerpuszta FR745989 ‡ FR745901 ‡ LT221020 ● LT220975 ● LT220993 ●

Vipera ursinii rakosiensis Vura-HU2 Hungary Fűzfa-szigetek FR745959 ‡ FR745900 ‡ * LT221021 ● LT220976 ● LT220994 ●

Vipera ursinii moldavica Vuml-RO1 Romania Iaşi JN204699 † * LT221003 ● LT221016 ● LT220971 ● LT220989 ●

Vipera ursinii moldavica Vuml-RO2 Romania Iaşi JN204700 † * LT221004 ● LT221017 ● LT220972 ● LT220990 ●

Vipera renardi Vren-CM1 Ukraine Crimea FR745991 ‡ * FR745893 ‡ * LT221022 ● LT220977 ● LT220995 ●

Vipera renardi Vren-CM2 Ukraine Crimea FR745992 ‡ * FR745894 ‡ * LT221023 ● LT220978 ● LT220996 ●

Vipera graeca stat. nov. Vgre-AL1 Albania Dhëmbel LT220960 ● LN835177 ♦ LT221011 ● LT220966 ● LT220984 ●

Vipera graeca stat. nov. Vgre-AL2 Albania Trebeshinë HG940677 ● * HG940672 ♦ LT221012 ● LT220967 ● LT220985 ●

†Gvoždík et al. 2012; ‡Ferchaud et al. 2012; ♦Mizsei et al. 2016; ●present study;

* not the same individual as for nDNA sequencing, but same population;

** not the same individual as for nDNA sequencing, but same taxon; - missing sequence.

MIZSEI ET AL.

TABLE 2. Primers used in this study.

FIGURE 1. Sampled localities inside the approximate distribution area of Vipera ursinii–renardi complex in Europe. Circles

indicate sampling localities of Vipera ursinii–renardi complex, and triangles show the sampling of outgroup taxa. A diamond

indicates the type locality of Vipera graeca stat. nov. This figure is published in colour in the online version, the colour of the

patches corresponds to the colour of mitochondrial lineages in Fig. 2A.

PCR products were purified with High Pure PCR Product Purification Kit (Roche) or on NucleoFast 96 PCR

plates (Macherey-Nagel) using vacuum filtering. DNA sequencing was performed using BigDye v1.1 (Life

Technologies) for cycle sequencing reaction and DNA sequencing was performed on an ABI 3130xl capillary

sequencer (Life Technologies).

Phylogenetic analyses. Sequences were assembled and aligned using CodonCode Aligner v5 (CodonCode

Corp.) and chromatograms were checked visually in order to clean the sequences. Coding gene fragments were

Primer Gene Reference Sequence

L14724Vb CYT B Ursenbacher et al. 2006 5'-GATCTGAAAAACCACCGTTG-3'

H15914Vb CYT B Ursenbacher et al. 2006 5'-AAATAGAAAGTATCATTCTGGTTTAAT-3'

ND4 ND4 Arevalo et al. 1994 5'-CACCTATGACTACCAAAAGCTCATGTAGAAGC-3'

H12763V ND4 Wüster et al. 2008 5'-TTCTATCACTTGGATTTGCACCA-3'

BDNFf BDNF Townsend et al. 2008 5'-GACCATCCTTTTCCTKACTATGGTTATTTCATACTT-3'

BDNFr BDNF Townsend et al. 2008 5'-CTATCTTCCCCTTTTAATGGTCAGTGTACAAAC-3'

NT3-F3 NT3 Noonan & Chippindale 2006 5'-ATATTTCTGGCTTTTCTCTGTGGC-3'

NT3-R4 NT3 Noonan & Chippindale 2006 5'-GCGTTTCATAAAAATATTGTTTGACC-3'

PRLR_f1 PRLR Townsend et al. 2008 5'-GACARYGARGACCAGCAACTRATGCC-3'

PRLR_r3 PRLR Townsend et al. 2008 5'-GACYTTGTGRACTTCYACRTAATCCAT-3'

GREEK MEADOW VIPER TAXONOMY

trimmed and translated into amino acids; no stop codons were observed. For this study, a complete dataset of two

mitochondrial and three nuclear markers was analysed for all samples/localities, except the Vipera berus nikolskii

specimen where CYT B sequence was missing and the gap was replaced by “N” in the analysis.

Heterozygous positions in nuclear genes were manually identified based on the presence of double peaks in

chromatograms. Identified heterozygous loci were coded according to the IUPAC ambiguity codes.

For the purpose of allele network construction, sequences with more than one heterozygous position (detected

in the presence of two peaks of approximately equal height at a single nucleotide site of BDNF, NT3, PRLR) were

resolved in PHASE 2.1.1 (Stephens et al. 2001) for which the input data for PHASE were prepared in SeqPHASE

(Flot 2010). PHASE was run under default settings except the probability threshold, which was set to 0.7.

Haplotype networks of the three nuclear markers (BDNF, NT3, PRLR) were drawn using TCS 1.21 (Clement et al.

2000) with 95% connection limit. New sequences were deposited in the GenBank (Table 1).

The best-fit codon-partitioning schemes and the best-fit substitution models for phylogenetic analyses were

selected using PartitionFinder v1.1.1. (Lanfear et al. 2012) separately for each dataset and methodological

approach (i.e. models available in the used software) with the following parameters: Bayesian approach (BA) -

linked branch length; all models; BIC model selection; greedy schemes search; data blocks by codons for each used

marker. The best partitioning scheme and models of nucleotide substitutions were: 1

position of CYT B (TrN), 2

position of CYT B (HKY + I), 3

position of CYT B (HKY), 1

+ 3

positions of ND4 (HKY + I), 2

position of

ND4 (HKY + G), BDNF (K80), NT3 (K80), PRLR (TrN). A Maximum likelihood (ML) analysis following the

same procedure as above (the best model in this case was the GTR+G+I with a single partition). The number of

variable (V) and parsimony informative (Pi) sites were calculated in DnaSP 5.10 (Librado & Rozas 2009).

To resolve phylogenetic relationships we analysed the own obtained dataset consisting of mitochondrial (1788

bp) and nuclear (1715 bp) sequence data. To show and confirm relationships based on the mtDNA (full sequences

for all taxa of ursinii–renardi complex were available only for CYT B; 1116 bp) we recalculated dataset of

Ferchaud et al. (2012) together with a Vipera anatolica sequence of Zinenko et al. (2015) and our new graeca

sequences. Mitochondrial phylogenetic trees were inferred using the BA performed with MrBayes 3.2.1 (Ronquist

et al. 2012) and ML analysis performed with RAxML 8.0 (Stamatakis 2014). Each codon position treated

separately was selected as the best-fit partitioning scheme for BA (see above). The BA analysis was set as follows;

two separate runs, with four chains for each run, 10 million generations with trees sampled every 100

generation.

First 20% of trees were discarded as the burn–in after inspection for stationarity of log–likelihood scores of

sampled trees in Tracer 1.6 (Rambaut et al. 2013; all parameters had effective sample sizes of > 200). A majority–

rule consensus tree was drawn from the post–burn–in samples and posterior probabilities were calculated as the

frequency of samples recovering any particular clade. The same procedure was performed with the concatenated

dataset of all genes with a final length of 3503 bp. Sequences of nuclear genes were not phased and partitioned by

genes due to low level of divergence. Both protein-coding genes (CYT B, ND4) were partitioned by codon position.

Nodes with posterior probability (pp) values ≥ 0.95 were considered as strongly supported. The ML clade support

was assessed by 1000 bootstrap pseudoreplicates.

The NeighborNet algorithm (Bryant & Moulton 2004) implemented in the software SplitsTree 4.10 (Huson &

Bryant 2006) was used to generate a phylogenetic network of the phased dataset. To assess the support for the

observed structure, bootstrap analysis was performed with 1000 pseudoreplicates. Nodes were considered strongly

supported if they received bootstrap values > 70%. This phylogenetic analysis is a powerful tool for visualizing

conflicting and consistent information present in the dataset (Huson & Bryant 2006).

Species tree estimation. Coalescent-based species tree estimation (STE) was performed with *BEAST v.1.8.0

(Drummond et al. 2012a) with the same dataset used in the phylogenetic network analysis. Because *BEAST

assumes no recombination within loci (Heled & Drummond 2010), we tested for the presence of recombination

within all nuclear loci analysed using RDP4 (Martin et al. 2010). Alignments of both mtDNA and all four nDNA

genes were imported independently into BEAUti 1.7.5 (Drummond et al. 2012a). Nuclear genes were phased prior

to analysis as described above. Three individual runs were performed for 5 × 10

generations with a sampling

frequency of 5000. Appropriate substitution models are specified as above and priors applied are as follows

(otherwise by default): Coalescence–Yule process of speciation; random starting tree, substitution rate fixed to 1;

strict clock; base substitution Uniform (0, 100); alpha Uniform (0, 100); initial = 0.5; clock rate Uniform (0, 1).

Parameter values both for clock and substitution models were unlinked across partitions and trees for the mtDNA

partitions were linked. Each run of STE was analysed in Tracer v.1.6 (Rambaut et al. 2013) to confirm that

MIZSEI ET AL.

stationarity, convergence and effective sample sizes (ESS) were sufficient for all parameters (posterior ESS values

> 300). LogCombiner and TreeAnnotator (both available in *BEAST package) were used to infer the ultrametric

tree after discarding 10% of the samples from each run and the production of the chronogram. A maximum clade

credibility tree from the sampled trees was produced using TreeAnnotator. Apart from producing a maximum clade

credibility tree of the full dataset (mtDNA + nDNA), we visualized all post burn-in sampled trees from all three

runs (27 000 trees) using DensiTree 2.1.11 (Bouckaert 2010), which allows superimposing all the sampled trees.

Nodes were considered strongly supported if they received posterior probability (pp) values > 0.95.

Results

Our dataset included 18 specimens of the genus Viper a; three samples of Vipera berus for outgroup (subspecies

berus, bosniensis and nikolskii) as a species phylogenetically very close to the Vipera ursinii–renardi complex, two

samples of Vipera renardi complex and 13 samples of Vipera ursinii complex (subspecies graeca, macrops,

moldavica, rakosiensis, ursinii and ursinii ssp. from Croatia; see Ferchaud et al. 2012, Zinenko et al. 2015 for

details about mitochondrial phylogeny). The dataset included mitochondrial gene fragments of CYT B (1043 bp, V

= 142, Pi = 107) and ND4 (747 bp, V = 101, Pi = 80) and nuclear gene fragments of BDNF (663 bp, V = 5, Pi = 4),

NT3 (497 bp, V = 14, Pi = 10) and PRLR (553 bp, V = 17, Pi = 15) totalling to 3503 bp (Table 1). No evidence of

recombination was detected within the nuclear loci.

Phylogenetic reconstructions and allele networks. Phylogenetic analyses (BA, ML) and phylogenetic

network (Fig. 2) constructed from the mitochondrial as well as from the concatenated dataset resulted in a topology

concordant with main clades as observed in the mtDNA phylogeny of Ferchaud et al. (2012) and Zinenko et al.

(2015) (Fig. 2). High Bayesian posterior probabilities (≥ 0.95; Fig. 2A, 2B) and bootstrap support values (> 70; Fig.

2A,B,C) were noted for the graeca clade as well as for most of other included clades (Fig. 2). Concatenated

mtDNA + nDNA data set revealed four deeply divergent clades within the ursinii–renardi complex, with a high

degree of structure corresponding to divergent lineages (see Fig. 2). There is strong support for the graeca clade

and for clade covering the V. u. moldavica, V. u. rakosiensis and V. u. macrops subclades. Similarly to mtDNA tree,

a clade covering V. u. ursinii and V. ursinii ssp. was not supported by BA analysis. The clade covering Vi per a

renardi–eriwanensis subclades is not supported in concatenated dataset probably due to missing data of

eriwanensis subclade.

The networks constructed for the phased haplotypes of the full length nuclear markers BDNF (6 unique

haplotypes), NT3 (9 haplotypes) and PRLR (12 haplotypes) are presented in Fig. 3. A very low level of haplotype

variability was detected for the BDNF marker. The NT3 marker was more variable with five haplotypes within the

ursinii group. The most variable marker was the PRLR with six haplotypes in the ursinii group. This marker shows

Vumc-MN1b allele among two alleles of Croatian population probably due to incomplete lineage sorting (=

ancestral polymorphism). Alleles of the graeca clade belong to distinct haplotypes in all three nuclear loci,

however, they share a haplotype of BDNF with Italian V. u. ursinii. These results indicate that alleles of NT3 and

PRLR are clearly unique for graeca, and in case of NT3, the haplotype of graeca is highly diverged from all other

taxa by six mutation steps (Fig. 3). Furthermore, alleles of the graeca lineage always represent one particular

haplotype which support evolutionary distinction among the taxa analysed including V. b e r u s.

Species tree. All three independent *BEAST runs converged, ESS values of all parameters in all runs

exceeded 200, a critical value suggested by the *BEAST manual (Drummond et al. 2012b) indicating adequate

mixing of the MCMC analyses. The ESS of the likelihoods was > 4000. The topology inferred from the maximum

clade credibility species tree based on the mitochondrial and nuclear loci was the same as the mtDNA gene tree of

Ferchaud et al. (2012), and similar to the topology Zinenko et al. (2015) (Fig. 2A). The graeca lineage is sister to

all other members of the ursinii–renardi group, and V. renardi is the sister lineage to the ursinii group. Within the V.

ursinii group, the montane vipers of V. u. ursinii and V. ursinii ssp. from Croatia form a clade sister to a lowland-

montane clade of V. u. rakosiensis, V. u. moldavica and V. u. macrops. Most relationships were highly supported (>

0.95) except the relationship between V. u. ursinii and V. ursinii ssp.. Considering the focus of this study, the graeca

lineage was confirmed as a highly supported and basal lineage within the V. ursinii–renardi complex according to

the *BEAST analysis (Fig. 4, pp = 1.00).

GREEK MEADOW VIPER TAXONOMY

FIGURE 2. (A) Current mitochondrial Bayesian phylogenetic hypothesis of Vipera ursinii–renardi complex based on CYT B

dataset of Ferchaud et al. (2012) and Zinenko et al. (2015); (B) Phylogenetic reconstruction of the concatenated dataset

(mtDNA+nDNA genes) obtained in MrBayes/Maximum likelihood (see Table 1). Sequences of Vipera berus (Vbbe-HU,

Vbbo-AL, Vbni-RO) included as outgroup are not shown. Bayesian posterior probabilities/bootstrap pseudoreplicates are

shown at nodes; (C) SplitsTree phylogenetic network (Huson & Bryant 2006) of the dataset for five mitochondrial and nuclear

loci sequenced in the present study using the neighbornet algorithm. Asterisks in Fig. 2C indicate both phased sequences in one

branch. Numbers along the edges are the bootstrap support values from 1000 replicates. The scale bar indicates one substitution

per one hundred nucleotide positions. Taxon names of the phylogenetic network correspond with the Table 1. Inset shows a

male Greek Meadow Viper from Dhëmbel Mountains, Albania.

MIZSEI ET AL.

FIGURE 3. Nuclear allele networks of the three analysed nuclear loci. Circle sizes are proportional to the number of samples/

sequences, small black circles indicate hypothetical haplotypes (alleles). This figure is published in colour in the online version,

the colour of the circles in the network corresponds to the colour of mitochondrial lineages in Fig. 2A.

GREEK MEADOW VIPER TAXONOMY

FIGURE 4. Species tree of the Vipera ursinii–renardi complex (with V. berus as outgroup) as inferred in *BEAST based on

two mitochondrial and three nuclear loci (A); species-tree cloudogram of the complex based on 27 000 post-burn-in trees

resulting from 3 runs of *BEAST, each producing 10,000 trees from which 10% was discarded as burn-in. Higher colour

densities represent higher levels of certainty. Maximum clade credibility tree is superimposed upon the cloudogram in bold

violet (B). Values of posterior probabilities are given. This figure is published in colour in the online version, the colours of the

branches correspond with the colour of mitochondrial lineages in Fig. 2A.

Systematics

In accordance with the evolutionary (Wiley 1978), general lineage (de Queiroz 1998), integrative taxonomic

(Miralles et al. 2010), phylogenetic (Cracraft 1983) and genetic species concepts (i.e. genetic species is a group of

genetically compatible interbreeding natural populations that is genetically isolated from other such groups; Baker

& Bradley 2006), we propose a full species rank for the Greek Meadow Viper to resolve the polyphyly in the

Vipera ursinii–renardi complex. This is supported by morphology, distribution, ecology and genetics (i.e.

multilocus approaches) as is described below.

MIZSEI ET AL.

Vipera graeca Nilson & Andrén, 1988 stat. nov.

Greek meadow viper

Vipera ursinii graeca Nilson & Andrén, 1988

Vipera macrops graeca Welch, 1994: 123

Holotype. Göteborg Natural History Museum, GNM Re. ex. 4942. Leg. Nilson & Andrén 1988.

Paratypes. GNM Re. ex. 6823 (six newborn), GNM Re. ex. 6849 (ZIG 146), GNM Re. ex. 6850 (ZIG 147),

GNM Re. ex. 6851 (ZIG 142) + GNM ZIG 145. Leg. Nilson & Andrén 1988.

Terra typica. Peristeri, Lakmos Mountains in the central Pindos mountain range, 1900 m altitude, Greece

(Nilson & Andrén 1988).

Morphological Diagnosis. This taxon differs from all other members of V. ursinii–renardi complex by having

the following combination of morphological characters (Nilson & Andrén 1988; Nilson & Andrén 2001; Mizsei et

al. 2016): small body size (for males a snout to vent length (SVL) max. 40.6 cm, and tail length is 5.4 cm, and for

females a SVL max. 44.3 cm, and tail length is 4.1 cm); non-bilineate body ground colour pattern; white or bright

brownish-grey ventral colour; no dark spots on labial, lateral and dorsal sides of head except occipital and

postorbital stripes; dorsal zigzag pattern tagged with pointed corners at windings, or consisting of a narrow

vertebral line only; 45–58 dorsal windings; nasal divided into two plates or united with nasorostralia; rostral as

high as broad; 2–8 loreals; 13–20 circumoculars; upper preocular not separated from nasal; 7–20 crown scales;

more fragmented parietals; 12–15 supralabials (sum of right and left sides); first three supralabials two times larger

than the following ones; third supralabial below orbit; 14–19 sublabials (sum of right and left sides); 3–5 mental

scales; early dorsal scale row reduction; 120–129 ventrals for males, 119–133 ventrals for females; lowest number

of subcaudals in the complex: 21–29 subcaudals for males, 13–26 subcaudals for females.

Molecular Diagnosis. The works of Ferchaud et al. (2012) and Zinenko et al. (2015) showed divergence of

Vipe ra graeca stat. nov. based on mtDNA datasets. Its distinction is supported by phylogenetic position (basal

taxon for all other species of the ursinii-renardi complex), time of divergence (the Middle Pliocene) and value of

uncorrected pairwise p–distances (4.5% in view of ursinii clade) as is defined by Ferchaud et al. (2012). All our

analyses based both on mitochondrial and nuclear loci support these results. Specimens in the study of Ferchaud et

al. (2012) originated from Stavros area in the Vardoussia Mts., Greece), but in the present study we used samples

from Albania. The specimens used here share the same CYT B and ND4 haplotypes as the previously analysed

Greek samples of Ferchaud et al. (2012). A single ND4 haplotype is presented by the southernmost (Stavros area,

Vardoussia Mts., Greece) and the northernmost populations (Tomorr Mts., Albania; Mizsei et al. 2016). Therefore,

a single haplotype is very likely to be present throughout most of the distribution area of this species. Because the

sequence variability of the nDNA regions is, in general, much less variable than mtDNA (Townsend et al. 2008),

we could use specimens originated from another locality than the type locality as they represent the same

phylogenetic pattern and position.

Distribution. The species occurs in the subalpine meadows of the Hellenides mountain system of southern

Albania and central Greece (Dimitropoulos 1985; Nilson & Andrén 1988; Nilson & Andrén 2001; Korsós et al.

2008; Mizsei et al. 2016). These localities are Koziakas, Lakmos (Peristeri; type locality), Metsovon, Oiti, Tsouka

Karali, Tzoumerka (Athamanika), Tymfristos, Vardoussia (including Stavros area) mountains in Greece, and

Dhëmbel, Llofiz, Lunxhërisë, Griba, Nemerçkë (crossborder mountain, called Nemertzika in Greek), Shëndelli,

Tomorr and Trebeshinë mountains in Albania. The entire distribution is extremely fragmented and each mountain

population is completely isolated by a large matrix of unsuitable habitat for the taxon consisting of deep valleys

and plains.

Ecology and habitats. A mosaic of open or closed grass and shrub communities formed on limestone

characterizes the main habitats of the taxon. Annual mean temperatures are about ~6°C, and the meadows are

partially covered by snow until early summer (May-June. South-facing slopes are usually more open and rocky

than north-facing slopes. Different species of Festuca, Poa and Sesleria dominate the open grasslands, and

characteristic shrubs are Juniperus sabina, Daphne oleoides and Astragalus creticus. Most of the observed vipers

were found close to shrubs or stone piles in south-facing habitat patches. The diet of the species consists mainly of

Orthoptera (97%) species, of which Stenobothrus rubicundulus, Platycleis sp., Decticus verrucivorus is the most

frequent prey (Mizsei et al. in prep.). The abundance of Orthopterans is high from June to September (Lemonnier-

Darcemont et al. 2015.). Known predators of the species are Vulpes vulpes, Falco tinnunculus and Circaetus

gallicus.

GREEK MEADOW VIPER TAXONOMY

Discussion

Our results of analysing two mitochondrial and three nuclear gene fragments support the distinct position of Vi per a

graeca stat. nov. first observed by the mtDNA results of Ferchaud et al. (2012). This was found in all four

analytical approaches used, i.e. gene phylogenetic reconstruction, phylogenetic network, allele (haplotype)

networks and coalescent species tree. Furthermore, the results support the uniqueness of the taxonomically

unrecognized meadow viper lineage from Croatia, which needs a formal description and further investigation. The

amendation of the subspecies status for V. graeca stat. nov. may influence the taxonomy of other taxa within V.

ursinii–renardi complex, and call to attention the revision of the taxonomic entities in this geographically and

evolutionary polytypic complex. It is important to use multiple loci in molecular approaches, and/or integrate other

morphological approaches to taxonomical assessments (e.g. using hemipenes or skull morphology) to resolve

complex relationships among taxa of meadow vipers.

The meristic morphology of V. graeca stat. nov. is very similar to V. u. macrops, as well as to other montane

populations of V. u r s i n i i s. l. Therefore it is not surprising that it was first described as a subspecies of V. ursinii

when it was discovered (Nilson & Andrén 1988). Albeit morphological characteristics function for the

determination of the taxon V. graeca stat. nov., there is a conflict between morphological and molecular evidence

(compare Nilson & Andrén 2001 and Ferchaud et al. 2012). Among European reptiles, incongruence between

morphological and molecular data is common in their phylogeny (see Pisani et al. 2007; Assis 2009; Gvoždík et al.

2010; Kindler et al. 2013 and references therein). Based on the hypothesis if suggested colonization and

diversification of the V. ursinii–renardi complex (Ferchaud et al. 2012; Zinenko et al. 2015), the shifts of steppe

like grassland habitats following climatic oscillations were not only latitudinal dispersion, but in the Hellenides

mountain complex also vertical (altitudinal) shifts. Thus, the meadow viper ancestors in the Balkans dispersed less

compared to other lineages, and the morphological similarity could be explained by the presence and higher

prevalence of plesiomorphic characteristics in taxa close to the radiation centre.

The extant distribution of V. graeca stat. nov. is severely fragmented (Mizsei et al. 2016), because the

populations are currently in interglacial refugial “sky-islands” of the Pindos mountains, surrounded by a sea of

coniferous/deciduous forests that make up the unsuitable habitat in the deep valleys below. This viper is threatened

because populations are completely isolated from each other in small patches, intentionally killed by local people

and are at risk over extinction vortices. Populations are probably very small due to microhabitat preferences, highly

susceptible to inbreeding depression and genetic drift. Climate change is a contemporary concern as warming

temperatures in the Mediterranean threaten the species’ habitat by tree encroachment and/or secondary

submediterranean grasslands (Kunstler et al. 2007). Following the IUCN Red List criteria the conservation status

of this taxon should be Endangered (B2abiii). Most suitable habitats are used as sheep, goat or cattle pastures, and

overgrazing has a direct negative effect on habitat structure in these meadows (Papanastasis et al. 2002). Our study

not only helped resolve some uncertainties within the V. ursinii–renardi complex but significantly contributes to

assessing the conservation status of V. graeca stat. nov., laying a platform for which future conservation efforts

may be initiated for this region.

Acknowledgements

The authors would like to thank B. Halpern (Hungary), O. Zinenko (Ukraine), D. Jelić (Croatia), J. Crnobrnja-

Isailović (Serbia), and A. Strugariu (Romania) for providing tissue samples. Many thanks are given to J. Šmíd

(Czech Republic) for his kind comments to the analyses and to Z. Varga (Hungary) and G. Kardos (Hungary) for

their suggestions to the manuscript. Financial support of EM was provided by the Balassi Institute, Hungary (B2/

1SZ/12851) and DJ was supported by the Comenius University grants UK/20/2014, UK/37/2015 and by the Slovak

Research and Development Agency under the contract no. APVV-0147-15. We are grateful to the three anonymous

referees for their valuable comments and suggestions.

References

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,

MIZSEI ET AL.

43 (3), 387–418.

https://doi.org/10.1093/sysbio/43.3.387

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

25 (5), 528–544.

https://doi.org/10.1111/j.1096-0031.2009.00261.x

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

643–662.

https://doi.org/10.1644/06-MAMM-F-038R2.1

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

https://doi.org/10.1093/bioinformatics/btq110

Bryant, D. & Moulton, V. (2004) NeighborNet: an agglomerative algorithm for the construction of planar phylogenetic

networks. Molecular Biology and Evolution, 21 (2), 255–265.

https://doi.org/10.1093/molbev/msh018

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.

https://doi.org/10.1016/j.ympev.2006.03.028

Clement, M., Posada, D. & Crandall, K.A. (2000) TCS: a computer program to estimate gene genealogies. Molecular Ecology,

9 (10), 1657–1659.

https://doi.org/ 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.

https://doi.org/ 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.

https://doi.org/10.1093/molbev/mss075

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

Available from: http://beast.bio.ed.ac.uk/tutorials (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.

https://doi.org/10.1111/j.1365-2699.2012.02753.x

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.

https://doi.org/10.1111/j.1755-0998.2009.02732.x

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.

https://doi.org/10.1016/j.ympev.2010.01.007

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.

https://doi.org/10.1016/j.ympev.2011.12.001

Heled, J. & Drummond, A.J. (2010) Bayesian inference of species trees from multilocus data. Molecular Biology and

Evolution, 27 (3), 570–580.

https://doi.org/10.1093/molbev/msp274

Huson, D.H. & Bryant, D. (2006) Application of phylogenetic networks in evolutionary studies. Molecular Biology and

Evolution, 23 (2), 254–267.

https://doi.org/10.1093/molbev/msj030

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.

https://doi.org/10.1016/j.jcz.2007.09.002

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)

GREEK MEADOW VIPER TAXONOMY

Mitochondrial phylogeography, contact zones and taxonomy of grass snakes (Natrix natrix, N. megalocephala). Zoologica

Scripta, 42 (5), 458–472.

https://doi.org/10.1111/zsc.12018

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

Herpetologica, 3 (2), 167–173.

https://doi.org/10.13128/Acta_Herpetol-2683

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: 10

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.

https://doi.org/10.1139/x06-225

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.

https://doi.org/10.1093/molbev/mss020

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.

https://doi.org/10.1093/bioinformatics/btp187

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.

https://doi.org/10.1093/bioinformatics/btq467

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..

https://doi.org/10.1111/j.1463-6409.2010.00453.x

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.

https://doi.org/10.1163/15685381-00003031

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.

https://doi.org/10.1111/j.1463-6409.1988.tb00106.x

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.

https://doi.org/10.1163/156853893x00183

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.

https://doi.org/10.1016/j.ympev.2006.03.010

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.

https://doi.org/10.1007/s10441-007-9015-8

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

beast.bio.ed.ac.uk/Tracer (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.

https://doi.org/10.1093/sysbio/sys029

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.

https://doi.org/10.1093/bioinformatics/btu033

MIZSEI ET AL.

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.

https://doi.org/10.1086/319501

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.

https://doi.org/10.1016/j.ympev.2014.03.011

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.

https://doi.org/10.1016/j.ympev.2008.01.008

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.

https://doi.org/10.1111/j.1365-294X.2006.03031.x

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.

https://doi.org/10.2307/2412809

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.

https://doi.org/10.1016/j.ympev.2008.08.019

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.

https://doi.org/10.1016/j.ympev.2014.12.005

Well-known species, unexpected results: high genetic diversity in declining Vipera ursinii in central, eastern and southeastern Europe

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Nov 2022
AMPHIBIA-REPTILIA

The Meadow and Steppe viper, Vipera ursinii-renardi complex is a well-studied group that is divided into several morphological subspecies. In this study, we combine the analyses of two mitochondrial genes with 9 microsatellite markers to compare both phylogenetic signals. Whereas the signal is similar between both genomes within most subspecies, the relative relationships between subspecies are more differentiated. Moreover, the nuclear phylogenetic reconstruction supports genetic homogeneity within V. u. macrops (in contrast to mtDNA). Both genetic portions show an unexpected differentiation between a population from Bistra Mountain and other V. u. macrops populations. Globally, the microsatellite markers suggest high genetic diversity in most subspecies, even in V. u. rakosisensis which is highly threatened; only V. u. macrops showed a limited genetic diversity. Within lowland subspecies, the differentiation between populations is globally limited compared to the distance between them (except in some populations of V. u. moldavica). The limited differentiation might be the consequence of a recent isolation (few decades) of previously large populations. Nevertheless, the only way to maintain this genetic diversity and to avoid an increase in genetic differentiation between populations in the future is to recreate suitable habitats and reconnect the populations.

Microhabitat selection of meadow and steppe vipers enlightened by digital photography and image processing to describe grassland vegetation structure

Article

Full-text available

Nov 2023
J ZOOL

Understanding animals' selection of microhabitats is important in both ecology and biodiversity conservation. However, there is no generally accepted methodology for the characterization of microhabitats, especially for vegetation structure. We studied microhabitat selection of three Vipera snakes by comparing grassland vegetation structure between viper occurrence points and random points in three grassland ecosystems: V. graeca in mountain meadows of Albania, V. renardi in loess steppes of Ukraine and V. ursinii in sand grasslands in Hungary. We quantified vegetation structure in an objective manner by automated processing of images taken of the vegetation against a vegetation profile board under standardized conditions. We developed an R script for automatic calculation of four vegetation structure variables derived from raster data obtained in the images: leaf area (LA), height of closed vegetation (HCV), maximum height of vegetation (MHV) and foliage height diversity (FHD). Generalized linear mixed models revealed that snake occurrence was positively related to HCV in V. graeca, to LA in V. renardi and to LA and MHV in V. ursinii, and negatively to HCV in V. ursinii. Our results demonstrate that vegetation structure variables derived from automated image processing significantly relate to viper microhabitat selection. Our method minimizes the risk of subjectivity in measuring vegetation structure, enables the aggregation of adjacent pixel data and is suitable for comparison of or extrapolation across different vegetation types or ecosystems.

Voluntary thermal maximum of grassland vipers ( Vipera spp.): environmental drivers and local adaptation

Article

Sep 2023

The thermal tolerance of ectotherms is a critical factor that influences their distribution, physiology, behaviour, and, ultimately, survival. Understanding the factors that shape thermal tolerance in these organisms is, therefore, of great importance for predicting their responses to forecasted climate warming. Here, we investigated the voluntary thermal maximum (VTmax) of nine grassland viper taxa and explored the factors that influence this trait. The small size of these vipers and the open landscape they inhabit render them particularly vulnerable to overheating and dehydration. We found that the VTmax of grassland vipers is influenced by environmental temperature, precipitation, short-wave flux, and individual body size, rather than by phylogenetic relatedness. Vipers living in colder environments exhibited a higher VTmax, contradicting the hypothesis that environmental temperature is positively related to VTmax. Our findings emphasize the importance of considering local to regional adaptations and environmental conditions when studying thermal physiology and the evolution of thermal tolerance in ectotherms.

Phylogenomic insights into the diversity and evolution of Palearctic vipers

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Full-text available

Aug 2024
MOL PHYLOGENET EVOL

Despite decades of molecular research, phylogenetic relationships in Palearctic vipers (genus Vipera) still essentially rely on a few loci, such as mitochondrial barcoding genes. Here we examined the diversity and evolution of Vipera with ddRAD-seq data from 33 representative species and subspecies. Phylogenomic analyses of ∼ 1.1 Mb recovered nine major clades corresponding to known species/species complexes which are generally consistent with the mitochondrial phylogeny, albeit with a few deep discrepancies that highlight past hybridization events. The most spectacular case is the Italian-endemic V. walser, which is grouped with the alpine genetic diversity of V. berus in the nuclear tree despite carrying a divergent mitogenome related to the Caucasian V. kaznakovi complex. Clustering analyses of SNPs suggest potential admixture between diverged Iberian taxa (V. aspis zinnikeri and V. seoanei), and confirm that the Anatolian V. pontica corresponds to occasional hybrids between V. (ammodytes) meridionalis and V. kaznakovi. Finally, all analyzed lineages of the V. berus complex (including V. walser and V. barani) form vast areas of admixture and may be delimited as subspecies. Our study sets grounds for future taxonomic and phylogeographic surveys on Palearctic vipers, a group of prime interest for toxinological, ecological, biogeographic and conservation research.

Evolutionary history and systematics of European blind mole rats (Rodentia: Spalacidae: Nannospalax): Multilocus phylogeny and species delimitation in a puzzling group

Article

Oct 2023
MOL PHYLOGENET EVOL

Species delimitation is a powerful approach to assist taxonomic decisions in challenging taxa where species boundaries are hard to establish. European taxa of the blind mole rats (genus Nannospalax) display small morphological differences and complex chromosomal evolution at a shallow evolutionary divergence level. Previous analyses led to the recognition of 25 ‘forms’ in their distribution area. We provide a comprehensive framework to improve knowledge on the evolutionary history and revise the taxonomy of European blind mole rats based on samples from all but three of the 25 forms. We sequenced two nuclear-encoded genetic regions and the whole mitochondrial cytochrome b gene for phylogenetic tree reconstructions using concatenation and coalescence-based species-tree estimations. The phylogenetic analyses confirmed that Aegean N. insularis belongs to N. superspecies xanthodon, and that it represents the second known species of this superspecies in Europe. Mainland taxa reached Europe from Asia Minor in two colonisation events corresponding to two superspecies-level taxa: N. superspecies monticola (taxon established herewith) reached Europe c. 2.1 million years ago (Mya) and was followed by N. superspecies leucodon (re-defined herewith) c. 1.5 Mya. Species delimitation allowed the clarification of the taxonomic contents of the above superspecies. N. superspecies monticola contains three species geographically confined to the western periphery of the distribution of blind mole rats, whereas N. superspecies leucodon is more speciose with six species and several additional subspecies. The observed geographic pattern hints at a robust peripatric speciation process and rapid chromosomal evolution. The present treatment is thus regarded as the minimum taxonomic content of each lineage, which can be further refined based on other sources of information such as karyological traits, crossbreeding experiments, etc. The species delimitation models also allowed the recognition of a hitherto unnamed blind mole rat taxon from Albania, described here as a new subspecies.

Are vipers prototypic fear-evoking snakes? A cross-cultural comparison of Somalis and Czechs

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Full-text available

Oct 2023

Snakes are known as highly fear-evoking animals, eliciting preferential attention and fast detection in humans. We examined the human fear response to snakes in the context of both current and evolutionary experiences, conducting our research in the cradle of humankind, the Horn of Africa. This region is characterized by the frequent occurrence of various snake species, including deadly venomous viperids (adders) and elapids (cobras and mambas). We conducted experiments in Somaliland and compared the results with data from Czech respondents to address the still unresolved questions: To which extent is human fear of snakes affected by evolutionary or current experience and local culture? Can people of both nationalities recognize venomous snakes as a category, or are they only afraid of certain species that are most dangerous in a given area? Are respondents of both nationalities equally afraid of deadly snakes from both families (Viperidae, Elapidae)? We employed a well-established picture-sorting approach, consisting of 48 snake species belonging to four distinct groups. Our results revealed significant agreement among Somali as well as Czech respondents. We found a highly significant effect of the stimulus on perceived fear in both populations. Vipers appeared to be the most salient stimuli in both populations, as they occupied the highest positions according to the reported level of subjectively perceived fear. The position of vipers strongly contrasts with the fear ranking of deadly venomous elapids, which were in lower positions. Fear scores of vipers were significantly higher in both populations, and their best predictor was the body width of the snake. The evolutionary, cultural, and cognitive aspects of this phenomenon are discussed.

A fossil viper (Serpentes: Viperidae) from the Early Pleistocene of the Crimean Peninsula

Article

Aug 2023

Climatic adaptation explains responses to Pleistocene oscillations and diversification in European vipers

Article

Full-text available

Jul 2023
J BIOGEOGR

Aim Allopatric speciation is the primary mode of diversification in the Mediterranean Basin. However, the contribution of climatic adaptation during this process is contradictory. In this work, we investigate the eco-evolutionary processes that drove diversification in this region, using European vipers as a case study. We describe the climatic requirements of different lineages to compare their responses to the Pleistocene climatic oscillations and tackle the evolutionary mechanisms underlying their diversification. Location Eurasia and North Africa. Taxon European vipers (genus Vipera). Methods We used ecological niche modelling (ENM) to identify the climatic requirements of 24 Vipera lineages and infer past range dynamics associated with their diversification during the Pleistocene. To test whether climatic niches varied across lineages, we calculated the phylogenetic signal of different climatic variables and examined the relationship with phylogenetic relatedness. To investigate climatic niche evolution and test for phylogenetic niche conservatism (PNC), we quantified pairwise niche overlap in sister phylogenetic units under a 3D hypervolume approach. Results ENM identified temperature annual range, precipitation of wettest month and precipitation of driest quarter as the most important climatic variables related to the distribution of most lineages, validating Pelias clade as cold-adapted, and Vipera 1 and Vipera 2 as warm-adapted clades. Projections to past conditions varied among clades, with Pelias and Vipera 1 having more similar responses, while Vipera 2 exhibited greater variability. We found significant phylogenetic signal in one temperature-related and two humidity-related climatic variables and detected high complexity in ecological niche evolution across the phylogeny, both rejecting the hypothesis of PNC. Main Conclusions Climatic adaptation played a significant role in driving diversification among European vipers. Cold-adapted and warm-adapted lineages presented similar climatic requirements and remarkable responses to Pleistocene stages, resulting in an intricate pattern of niche divergence along the phylogeny that favours local adaptation rather than PNC.

Comparative mitochondrial phylogeography of water frogs (Ranidae: Pelophylax spp.) from the southwestern Balkans

Article

Full-text available

May 2023

The genus Pelophylax (water frogs) includes relatively common, widely distributed, and even invasive species, but also endemic taxa with small ranges and limited knowledge concerning their ecology and evolution. Among poorly studied species belong endemics of the southwestern Balkans, namely Pelophylax shqipericus, P. epeiroticus and P. kurtmuelleri. In this study, we focused on the genetic variability of these species aiming to reveal their phylogeographic patterns and Quaternary history. We used 1,088 published and newly obtained sequences of the mitochondrial ND2 gene and a variety of analyses, including molecular phylogenetics and dating, historical demography, and species distribution modeling (SDM). We revelated the existence of two mitochondrial lineages within P. epeiroticus and P. shqipericus that diverged at ~ 0.9 Mya and ~ 0.8 Mya, respectively. Contrarily, no deeply diverged lineages were found in P. kurtmuelleri. Pelophylax kurtmuelleri also shows a close phylogenetic relationship with widely distributed P. ridibundus, suggesting that both represent one evolutionary clade called here P. ridibundus/kurtmuelleri. The estimated split between both lineag-es in the clade P. ridibundus/kurtmuelleri date back to ~ 0.6 Mya. The divergence between the ridibundus and kurtmuelleri lineages on the ND2 gene is thus lower than the divergence between the two lineages found in P. epeiroticus and P. shqipericus. According to haplotype networks, demographic analyses, and SDM, endemic water frogs survived the last glacial maximum (LGM) in Balkan in the Balkans microrefugia, and their distribution has not changed significantly or even retracted since the LGM. Haplotypes of the kurtmuelleri lineage were also found in northern parts of Europe, where haplotype diversity is however much lower than in the Balkans, suggesting the possible hypothesis of their postglacial expansion to the north.

Molecular phylogeny of the common wall lizard Podarcis muralis (Squamata: Lacertideae) in the southern Balkans

Article

Apr 2024

The lacertid wall lizards of the genus Podarcis (Wagler, 1830) originate from Western Europe and are divided into 24-25 species (Speybroeck et al., 2020). Despite the abundance of phylogenetic studies referring to the genus, the relationships among certain species, as well as the taxonomic status of some genealogical lineages, remain unclear due to great genetic diversity. The common wall lizard Podarcis muralis has a relatively wide distribution and despite the existence of various molecular studies focused on this species, its Greek populations had never been thoroughly sampled until now. To fill in this geographical gap, we sampled the species’ Greek distribution and constructed phylogenies that uncovered the presence of at least five monophyletic lineages that correspond to different geographic regions. Furthermore, species delimitation analyses assigned all lineages to a single species diversifying during the early Pleistocene (c. 1.93 mya). The strong association of the genetic lineages with specific geographical regions coupled with the Pleistocene diversification of the group imply the presence of multiple refugia within Greece and, by extension, the Balkan peninsula, supporting a refugia-within-refugia scenario. Finally, in an effort to clarify the position of these new samples within the larger phylogeny of P. muralis , a larger phylogeny was constructed which indicated that the Greek populations cluster with the central European, Italian and Turkish populations of the species.

Bayesian phylogenetics with BEAUti and the BEAST 1.7

Article

Full-text available

Jan 2012
MOL BIOL EVOL

First overview of the south Albanian Orthoptera fauna

Article

Full-text available

Jul 2015

The Orthoptera fauna in southern Albania is still largely unexplored. In the summer and autumn of 2014, several entomological field trips were conducted in various habitats from the coast to the mountains. A total of 87 species were noted. Among them were some first records for the region.

A new species of viper (Reptilia, Viperidae) from the Altay and Saur Mountains, Kazakhstan

Article

Full-text available

Jan 2010

A new species of genus Vipera (Reptilia: Viperidae) belonging to the subgenus Pelias based on several speci-mens from the Altay and Saur mountain region in eastern Kazakhstan is described. It is a small species belonging to the renardi lineage.

RAxML Version 8: A Tool for Phylogenetic Analysis and Post-Analysis of Large Phylogenies

Article

Full-text available

Jan 2014
BIOINFORMATICS

Alexandros Stamatakis

Phylogenies are increasingly used in all fields of medical and biological research. Moreover, because of the next generation sequencing revolution, datasets used for conducting phylogenetic analyses grow at an unprecedented pace. RAxML (Randomized Axelerated Maximum Likelihood) is a popular program for phylogenetic analyses of large datasets under maximum likelihood. Since the last RAxML paper in 2006, it has been continuously maintained and extended to accommodate the increasingly growing input datasets and to serve the needs of the user community. I present some of the most notable new features and extensions of RAxML, such as, a substantial extension of substitution models and supported data types, the introduction of SSE3, AVX, and AVX2 vector intrinsics, techniques for reducing the memory requirements of the code and a plethora of operations for conducting post-analyses on sets of trees. In addition, an up-to-date, 50 page user manual covering all new RAxML options is available. The code is available under GNU GPL at https://github.com/stamatak/standard-RAxML. Alexandros.Stamatakis@h-its.org.

The General Lineage Concept of Species, Species Criteria, and the Process of Speciation: A Conceptual Unification and Terminological Recommendations

Article

Jan 1998

Kevin de Queiroz

Species distribution modelling leads to the discovery of new populations of one of the least known European snakes, Vipera ursinii graeca, in Albania

Article

Feb 2016

Vipera ursinii graeca is a restricted-range, endemic snake of the Pindos mountain range in the southwestern Balkans. The subspecies was previously reported from eight localities in Greece and one locality in southern Albania. We used species distribution modelling based on climate data from known localities in Greece to estimate the potential distribution of the subspecies. The model predicted suitable areas for eleven mountains in southern Albania, which we visited in ten field expeditions in four years. Based on 78 live individuals and 33 shed skins, we validated the presence of the snake on eight of the eleven mountains. Six populations (Dhëmbel, Llofiz, Griba, Shendelli, Tomorr and Trebeshinë Mountains) are reported here for the first time. Morphological characters undoubtedly supported that all individuals found at these new localities belong to V. u. graeca. Genetic analysis of mitochondrial DNA sequences also confirmed the identity of the snakes as V. u. graeca and a low number of identified haplotypes suggested low genetic variability among populations despite significant spatial isolation. All localities were subalpine-alpine calcareous meadows above 1600 m. These high montane habitats are separated by deep valleys and are threatened by overgrazing, soil erosion, and a potential increase in the elevation of the tree line due to climate change. Our surveys increased the number of known populations by 60% and the known geographical range of the subspecies by approximately 30%. Our study serves as a baseline for further ecological research and for conservation measures for one of the least known European viperid snakes.

Genetic And Morphological Differentiation Of Mosor Rock Lizards, Dinarolacerta Mosorensis (Kolombatović, 1886), With The Description Of A New Species From The Prokletije Mountain Massif (Montenegro) (Squamata: Lacertidae)

Article

Oct 2007
ZOOTAXA

A new species of lacertid lizard of the genus Dinarolacerta is described from the Prokletije Mountain Massif, Montenegro. This new species, Dinarolacerta montenegrina sp. nov., is characterized by its relatively small size, by usually having only one postnasal scale on one or on both sides of the head, a relatively lower number of temporal and postocular scales and a relatively high number of ventral scales. Osteologically, it is mainly characterized by the complete absence of the anteromedial process in the postocular bone, and more reduced supraocular osteoderms. The phylogenetic analysis using partial sequences of the mitochondrial 12S rRNA gene supports the specific status of D. montenegrina sp. nov. and shows that it represents an old independent lineage that separated from its sister species, D. mosorensis, in the late Miocene. The Morača river canyon may have acted as a geomorphological and climatic barrier causing the speciation between the two species of Dinarolacerta. The discovery of this new species endemic to the Balkan Peninsula highlights the importance of the Dinarides as one of the main European hotspots of biodiversity. This high level of endemicity in the Dinaric region is probably the result of both its geographic situation and its complex geological history and morphology.

The Meadow and Steppe vipers of Europe and Asia – The Vipera (Acridophaga) ursinii complex

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