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Quaternary range dynamics and taxonomy of the Mediterranean collared dwarf racer, Platyceps collaris (Squamata: Colubridae)

October 2021
Zoological Journal of the Linnean Society 193(2):655-672

DOI:10.1093/zoolinnean/zlaa151

Authors:

Jiri Smid

National Museum, Prague, Czech Republic

Tatiana Aghova

General University Hospital

Jirí Moravec

National Museum, Prague, Czech Republic

Show all 10 authorsHide

The geological and geographical settings of the Eastern Mediterranean have resulted in complex patterns of intraspecific diversifications and phylogeographical histories that can be observed in squamates. In this study, we examined genetic differentiation of the Collared dwarf racer (Platyceps collaris) using a multilocus genetic dataset with a sampling that covered the entire range of the species. We developed distribution models in current and past climatic conditions to assess the dynamics of the species distribution through time. We sequenced a fragment of the cytochrome b mitochondrial gene of the holotype and eight paratypes of Coluber rubriceps thracius, which is considered a synonym of Platyceps collaris. Our results show that there are two distinct clades within P. collaris, one occupying the Balkans and western and southern Anatolia (termed the Balkan–Anatolian clade), the other in the Levant (termed the Levantine clade). All type specimens of C. r. thracius are genetically identical and cluster within the Balkan–Anatolian clade. Distribution models indicate the presence of two refugia during climatically challenging periods. One was in western Anatolia and served as a source for the colonization of the Balkans and southern Anatolia, and the other was in the northern Levant, from where P. collaris dispersed further south. According to our results, we revise the subspecific taxonomy of P. collaris.

Phylogenetic tree resulting from the Bayesian inference (BI) analysis of three mitochondrial and three nuclear genes concatenated. Nodes were considered supported when Bayesian posterior probability was ≥ 0.95 and maximum likelihood (ML) bootstrap values ≥ 70. Lengths of branches connecting the split between Platyceps collaris and Platyceps najadum and the crown nodes of those species are not proportional to the rest of the tree and the scale, which is indicated by their partial transparency. The two clades, the Balkan-Anatolian and the Levantine, are highlighted in the tree with the red and green shading, respectively. Four species of Telescopus used to root the tree are not shown. Each tree tip is connected by a dashed line with the locality of its sample, which is marked by a number (for details, see Table 1). Type localities are marked with stars: P. collaris collaris in green, P. collaris rubriceps in white and Coluber rubriceps thracius in pink. The potential current distribution of P. collaris based on the species distribution model with the maximum training sensitivity plus specificity threshold applied is shown in blue. Haplotype networks reconstructed for the six markers are on the right. Circles are colour coded according to the clade assignment, and their size is proportional to the number of individuals. Lines represent mutational steps. The network for the cytb gene was constructed using the 148-bp-long fragment that was available for the types of Coluber rubriceps thracius. The position of sample DJ8199 from locality 19, whose phylogenetic placement differed in the maximum likelihood and Bayesian inference analyses, is marked with the locality number in each network.

…

Phylogenetic network from SplitStree, showing the reticulate relationships within the two clades of Platyceps collaris, the Balkan-Anatolian clade (red) and the Levantine clade (green). Numbers in circles are locality numbers shown in Figure 1 and detailed in Table 1. Bootstrap support values for major nodes ≥ 70 are indicated. The specimen depicted is from Ropotamo, Bulgaria.

…

Species distribution models for Platyceps collaris in current conditions (A) and in conditions during the Last Glacial Maximum (B), the Last Interglacial (C), mid-Pleistocene (D) and mid-Pliocene (E). Black points in A indicate the records that were used for the species distribution modelling. The bottom right panel shows reconstructions of global temperature in the last 4 Myr relative to the peak Holocene temperature (Hansen & Sato, 2012), with grey arrows and dashed lines highlighting the temperature in the time periods used in the present study.

…

Figure S1. Phylogenetic tree of Platyceps collaris resulting from the ML analysis of all genes concatenated. Bootstrap support values ≥ 60 are shown above branches. Type specimens of Coluber rubriceps thracius are in red. 0.02

…

Figure S6. Geographic distribution of the Eastern Mediterranean conifer-broadleaf forest ecoregion (Dinerstein et al., 2017).

…

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Zoological Journal of the Linnean Society, 2021, 193, 655–672. With 3 figures

Quaternary range dynamics and taxonomy of the

Mediterranean collared dwarf racer, Platyceps collaris

(Squamata: Colubridae)

JIŘÍ ŠMÍD1,2,*,, TATIANA AGHOVÁ3, DOUBRAVKA VELENSKÁ2, JIŘÍ MORAVEC1,

PETR BALEJ4,, BORISLAV NAUMOV5, GEORGI POPGEORGIEV6, NAZAN ÜZÜM7,

AZIZ AVCI7 and DANIEL JABLONSKI8,

1Department of Zoology, National Museum, Cirkusová 1740, Prague, Czech Republic

2Department of Zoology, Faculty of Science, Charles University, Viničná 7, Prague, Czech Republic

3Centre of Oncocytogenomics, Institute of Medical Biochemistry and Laboratory Diagnostics, General

University Hospital and First Faculty of Medicine, Charles University, U Nemocnice 499/2, 128 08,

Prague, Czech Republic

4Department of Applied Geoinformatics and Spatial Planning, Faculty of Environmental Sciences, Czech

University of Life Sciences Prague, Kamýcká 129, Praha—Suchdol, 165 00, Czech Republic

5Institute of Biodiversity and Ecosystem Research, Bulgarian Academy of Sciences, 2 Gagarin Street,

1113 Sofia, Bulgaria

6National Museum of Natural History, Bulgarian Academy of Sciences, 1 Tzar Osvoboditel Boulevard,

1000 Sofia, Bulgaria

7Department of Biology, Faculty of Science and Arts, Aydın Adnan Menderes University, Aydın, Turkey

8Department of Zoology, Comenius University in Bratislava, Ilkovičova 6, Mlynská dolina, 842 15

Bratislava, Slovakia

Received 4 May 2020; revised 13 October 2020; accepted for publication 18 October 2020

The geological and geographical settings of the Eastern Mediterranean have resulted in complex patterns of

intraspecific diversifications and phylogeographical histories that can be observed in squamates. In this study, we

examined genetic differentiation of the Collared dwarf racer (Platyceps collaris) using a multilocus genetic dataset

with a sampling that covered the entire range of the species. We developed distribution models in current and past

climatic conditions to assess the dynamics of the species distribution through time. We sequenced a fragment of

the cytochrome b mitochondrial gene of the holotype and eight paratypes of Coluber rubriceps thracius, which is

considered a synonym of Platyceps collaris. Our results show that there are two distinct clades within P. collaris, one

occupying the Balkans and western and southern Anatolia (termed the Balkan–Anatolian clade), the other in the

Levant (termed the Levantine clade). All type specimens of C. r. thracius are genetically identical and cluster within

the Balkan–Anatolian clade. Distribution models indicate the presence of two refugia during climatically challenging

periods. One was in western Anatolia and served as a source for the colonization of the Balkans and southern

Anatolia, and the other was in the northern Levant, from where P. collaris dispersed further south. According to our

results, we revise the subspecific taxonomy of P. collaris.

ADDITIONAL KEYWORDS: biogeography – Colubroidea – Near East – reptiles – Serpentes – systematics.

INTRODUCTION

The Eastern Mediterranean is a region of complex

geological and biological history. As a result of its

position at the crossroads of Europe, Asia and Africa,

the fauna of the Eastern Mediterranean shows a

wide spectrum of biogeographical patterns and

phylogeographical scenarios. Many recent studies

have indicated that the diversity of squamates of the

Eastern Mediterranean is underestimated and that

*Corresponding author. E-mail: jiri.smd@gmail.com

applyparastyle “fig//caption/p[1]” parastyle “FigCapt”

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656 J. ŠMÍD ET AL.

traditionally recognized species described on the basis

of morphological characters often encompass cryptic

lineages (e.g. Bellati et al., 2015; Jandzik et al., 2018;

Šmíd et al., 2019). Although considerable interest

has been attracted to the islands (Kyriazi et al.,

2013; Poulakakis et al., 2013), differentiation and

phylogeography of the fauna of coastal Mediterranean

Turkey and the Levant are also being studied

increasingly (Sindaco et al., 2014; Kotsakiozi et al.,

2018; Kornilios et al., 2019). However, little is known

in this respect about some of the widespread and

common taxa. The colubrid genus Platyceps Blyth,

1960 is one such example.

Racers of the genus Platyceps are medium-sized,

slender and wary colubrid snakes. Their distribution

spans from south-eastern Europe across the Middle

East to central Asia and southwards across the

Arabian Peninsula to north-eastern Africa (Sindaco

et al., 2013). South-eastern Europe, the Eastern

Mediterranean and the Levant are occupied by two

species, Platyceps collaris (Müller, 1878) and Platyceps

najadum (Eichwald, 1831). Morphological data

indicate that these two species are distinct from other

congeners in having unpaired apical pits (Schätti

et al., 2014), and genetic evidence supports their sister

relationship (Schätti & Utiger, 2001; Schätti, 2004).

The taxonomic history of P. collaris has been

turbulent. It was originally described as a subspecies

(originally a variety) of Zamenis dahlii (Fitzinger,

1826) (currently Platyceps najadum) in the late 19th

century on the basis of two specimens, an adult male

from Beirut, Lebanon [Naturhistorisches Museum,

Basel, Switzerland (NHMB) 1166], and a juvenile from

the vicinity of Tel Aviv, Israel (NHMB 1167; Müller,

1878; Schätti et al., 2001). In the beginning of the

20th century, another subspecies (originally a variety),

Zamenis dahlii rubriceps Venzmer, 1919, was described

from the Taurus Mountains in southern Turkey

[Venzmer, 1919; between Pozanti and Tarsus (37°12′N,

34°48′E) according to Schätti et al., 2001]. The colubrid

genera were later revised by Inger & Clark (1943), and

the genus Platyceps was proposed for several Eurasian

racers, including Coluber najadum (Eichwald, 1831).

However, most subsequent authors did not follow

this adjustment and applied the genus name Coluber

Linnaeus, 1758 to most Eurasian racers (e.g. Mertens &

Wermuth, 1960; Schätti, 1993). The trinomen Coluber

najadum rubriceps was used until Baran (1976)

elevated Coluber rubriceps to a full species. Rehák

(1985) described specimens from the Black Sea coast

of Bulgaria as a distinct subspecies, Coluber rubriceps

thracius Rehák, 1985, which was characterized by its

low number of ventral and subcaudal scales and a

relatively short tail. The type series of 11 specimens was

originally placed in the collection of the Department of

Systematic Zoology (currently Department of Zoology),

Charles University, Prague, Czech Republic (DZCHU).

Owing to organizational changes at the department,

the specimens were transferred to the herpetological

collections of the National Museum in Prague (NMP)

in 2014, where they are deposited under museum

catalogue numbers NMP-P6V 75257 (holotype), NMP-

P6V 75258, 75259/1–5 and 75260–62 (paratypes;

Table 1). One paratype was sent to Bonn [Zoologisches

Forschungsmuseum Alexander Koenig (ZFMK)],

where it is catalogued under the number ZFMK 46091

(Böhme, 2014).

In a detailed taxonomic treatment, Schätti et al.

(2001) revised the taxonomy of Coluber collaris (Müller,

1878) and C. rubriceps. They recognized these two taxa

to be conspecific, which resulted in the relegation of the

name C. rubriceps to a junior synonym of C. collaris.

The authors also designated the adult male specimen

from Beirut as a lectotype of the species. With respect

to the subspecies C. r. thracius, Schätti et al. (2001)

pointed out that Rehák (1985) based his comparisons

on published data instead of examining the specimens

himself. Also, the variance of the diagnostic characters

of C. r. thracius falls within the range of Asian

populations according to Schätti et al. (2001). Based

on these grounds, they synonymized the subspecies

C. r. thracius with the nominotypical one, which

resulted in the species becoming monotypic. This has

been followed by most recent authors (Sindaco et al.,

2013; Wallach et al., 2014; Geniez, 2018), although

some still use the subspecific designation (Stojanov

et al., 2011).

The last nomenclatural adjustment of the species

had stemmed from the phylogenetic results of Schätti

& Utiger (2001), who confirmed earlier findings of

Inger & Clark (1943) and placed the Eurasian racers,

including C. collaris and C. najadum, in the genus

Platyceps.

The distribution of P. collaris, as currently

recognized, spans from south-eastern Bulgaria along

the entire Mediterranean coast of Turkey through the

Mediterranean part of Syria and Lebanon to Israel

and western Jordan (Sindaco et al., 2013; Geniez,

2018). Isolated populations have been recorded from

central and southern Anatolia (Baran, 1982; İğci et al.,

2015), but the identification of these specimens needs

to be verified because the localities are geographically

disparate from the rest of the range and lie well within

the range of the closely related and morphologically

similar P. najadum.

In this paper, we explore the genetic diversity and

phylogeography of P. collaris across its entire range

using a multilocus dataset of three mitochondrial

and three nuclear markers. Using a new approach of

multistep polymerase chain reaction (PCR) and Illumina

amplicon sequencing, we sequence a fragment of the

cytochrome b (cytb) mitochondrial gene of the holotype

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PHYLOGEOGRAPHY OF COLLARED DWARF RACER 657

and eight paratypes of the nominal taxon Coluber

rubriceps thracius. We conduct a species distribution

modelling analysis for the present climatic conditions and

conditions during the last 3 Myr to assess the limits of the

current potential distribution for the species, its historical

range dynamics and location of potential refugia.

MATERIAL AND METHODS

Sampling, Dna extraction anD amplification of

recent material

For the phylogenetic analysis, we assembled a dataset

of 35 samples of P. collaris from 26 localities in

Bulgaria, Israel, Syria and Turkey, covering the entire

range of the species (Table 1). We supplemented this

dataset with GenBank sequences of three individuals,

two from Israel and one from Jordan (Schätti & Utiger,

2001; Nagy et al., 2004).

Genomic DNA was extracted from 96% ethanol-

preserved tissue samples using an Invisorb Spin

Tissue Kit (STRATEC), following the manufacturer’s

instructions. We PCR-amplified six genetic markers:

three from the mitochondrial DNA [12S ribosomal

RNA (12S), cytochrome b (cytb) and cytochrome c

oxidase I (coi)] and three from the nuclear DNA

[oocyte maturation factor MOS (cmos), neurotrophin-3

(nt3) and recombination activating gene 1 (rag1)].

We used the following primer pairs and annealing

temperatures for the amplifications: for 12S, the

primers were 12S268 and 12S916 (Schätti & Utiger,

2001), and the annealing temperature was 65 °C; for

cytb, L14910 and H16064 (Burbrink et al., 2000) and

46 °C; for coi, COIb and COIbdeg (Schätti & Utiger,

2001; Utiger et al., 2002) and 56 °C; for cmos, S77 and

S78 (Lawson et al., 2005) and 53 °C; for nt3, F3 and R4

(Noonan & Chippindale, 2006) and 50 °C; and for rag1,

R13 and R18 (Groth & Barrowclough, 1999) and 58 °C.

The PCRs contained 0.2 µM of each primer, 7.5 µL of

2× QIAGEN Multiplex PCR Kit, 1.5 µL of genomic

DNA (concentration ~30 ng/µL) and double-distilled

H2O to a total volume of 15 µL. The PCR products

were purified with calf intestine alkaline phosphatase

and exonuclease I (New England Biolabs, Ipswich,

MA, USA) and Sanger sequenced along both strands

using the PCR primers in Macrogen Europe (The

Netherlands). Sequences were checked and contigs

assembled in GENEIOUS v.11 (Kearse et al., 2012).

Sampling, Dna extraction anD amplification

of the type SpecimenS of Coluber

rubriCeps thraCius

We sampled ten individuals from the type series of

Coluber rubriceps thracius, the holotype and nine

paratypes (Table 1). The museum samples were

handled in a specialized non-invasive laboratory

designed for work with rare DNA to prevent

contamination (Institute of Vertebrate Biology of

the Academy of Sciences, Studenec, Czech Republic).

DNA was extracted using Invisorb Spin Forensic Kit

(STRATEC). Concentration of samples was measured

on a Qubit 1.0 fluorometer (Thermo Fisher Scientific)

using the Qubit dsDNA HS Assay kit. To obtain DNA

sequences of the type specimens, we modified the

rodent-based DNA mini-barcoding protocol for the

Illumina platform that targets a short cytb fragment

(Galan et al., 2012; Bryja et al., 2014). The mini-

barcode is a fragment of 148 bp, a length short enough

to amplify in samples with fragmented or degraded

DNA (Galan et al. 2012). For the library preparation,

we used a three-step PCR. All PCRs contained 0.4 µM

of each primer, 5 µL (7.5 µL for the third PCR) of

2× QIAGEN Multiplex PCR Kit, 1.0–1.5 µL of DNA

(concentration 0.01–3.00 ng/µL) and double-distilled

H2O to a total volume of 10 µL (for the first and second

PCR) and 15 µL (for the third PCR). The PCRs were

initiated by a denaturation step of 94 °C for 15 min,

followed by 15–25 cycles of denaturation at 94 °C for

30 s, annealing at 45 °C for 45 s and an extension at

72 °C for 30 s, followed by a final extension step at

72 °C for 10 min. All PCRs were prepared in duplicates.

In the first PCR of 25 cycles, we amplified the target

region with short specific primers, L15411_modif

(GAYAAAATYYCHTTYCACCC) and H15553_modif

(GTAGGCRAAYAGGAARTATCA). The product of the

first PCR was then used as a template for the second

PCR of 20 cycles, for which the primers were elongated

at the 5′ end: L15411_modif_F_nextera (TCGTCGG

CAGCGTCAGATGTGTATAAGAGACAGGAYAAAA

TYYCHTTYCACCC) and H15553_modif_R_nextera

(GTCTCGTGGGCTCGGAGATGTGTATAAGAGAC

AGGTAGGCRAAYAGGAARTATCA). The template

for the third PCR of 15 cycles was the product of

the second PCR. Primers for this reaction contained

Illumina adaptors with a unique combination of

barcodes/tags for each sample. After the three-step

amplification, we visualized the products on a 1.5%

agarose gel and measured the gel band intensity with

genoSo ft software (VWR International, Belgium).

The concentration of all samples was measured

with a fluorometer (Qubit 1.0) and purity with a

spectrophotometer (DS-11; DeNovix Inc.). All samples

were then diluted to be of an equimolar concentration

(~100 ng/µL). This library was purified using Agencourt

SPRIselect (Beckman Coulter) and sequenced using

MiSeq Illumina at CEITEC (Masaryk University,

Brno, Czech Republic). Sequences were demultiplexed

based on the unique tags. Primers were cut off and

low-quality reads filtered out as follows: sequences

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658 J. ŠMÍD ET AL.

Table 1. Material used for the genetic analysis

mtDNA nDNA

Taxon Voucher Sample Specimen status 12S cytb coi cmos nt3 rag1 Locality Latitude Longitude

Platyceps

collaris

– DJ7739 MT856882 MT862657 MT862569 MT862592 MT862617 MT862640 Bulgaria,

Sveti Vlas (1)

42.725 27.766

P. collaris – DJ1405 MT856879 MT862654 MT862566 MT862589 MT862614 MT862638 Bulgaria,

Sozopol (2)

42.397 27.703

P. collaris – DJ6132 MT856881 MT862656 MT862568 MT862591 MT862616 – Bulgaria,

Sozopol (3)

42.395 27.689

P. collaris – DJ607 MT856883 MT862658 MT862570 MT862593 MT862618 MT862641 Bulgaria,

Ropotamo (5)

42.305 27.728

P. collaris – DJ1417 MT856880 MT862655 MT862567 MT862590 MT862615 MT862639 Bulgaria,

Sinemorec (7)

42.071 27.963

P. collaris – DJ2478 MT856884 MT862659 MT862571 MT862594 MT862619 MT862642 Israel,

Hermon (22)

33.296 35.762

P. collaris MCCI R-0649 R649 MT856885 – MT862572 MT862595 MT862620 – Israel,

Haifa (23)

32.806 35.007

P. collaris MHNG 2447.74 Schatti1 AY039133 – AY039171 – – – Israel,

Tel Aviv (25)

32.327 34.855

P. collaris MHNG 2447.75 Schatti2 AY039157 – AY039195 – – – Israel,

Tel Aviv (25)

32.327 34.855

P. collaris TAU.R15955 TAU.

R15955

MT856886 – – – – – Israel, Yizre’el

Valley (24)

32.72 35.14

P. collaris TAU.R16921 TAU.

R16921

MT856887 MT862660 – MT862596 MT862621 – Israel, Nezer

Sereni (26)

31.922 34.822

P. collaris TAU.R16927 TAU.

R16927

MT856888 MT862661 – MT862597 MT862622 – Israel,

Shomeron (28)

32.517 35.382

P. collaris HLMD J14 Nagy – AY486922 – AY486946 – – Jordan (27)

P. collaris NMP-P6V 70502 JIR187 MT856889 MT862662 MT862573 MT862598 MT862623 – Syria, 2 km N

of Seydnaya (20)

33.717 36.372

P. collaris NMP-P6V 70503 JIR188 MT856890 MT862663 MT862574 MT862599 MT862624 – Syria, E of

Utaibeh (21)

33.506 36.627

P. collaris – DJ8209 MT856903 MT862685 MT862587 MT862612 MT862636 – Turkey,

Bozdağ-

Ödemiş (8)

38.367 28.103

P. collaris – DJ8198 MT856892 MT862674 MT862576 MT862601 MT862626 MT862644 Turkey,

10 km N

of Saimbeyli (15)

38.061 36.149

P. collaris – DJ8202 MT856896 MT862678 MT862580 MT862605 MT862630 MT862648 Turkey,

Temürağa (16)

38.046 36.586

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PHYLOGEOGRAPHY OF COLLARED DWARF RACER 659

mtDNA nDNA

Taxon Voucher Sample Specimen status 12S cytb coi cmos nt3 rag1 Locality Latitude Longitude

P. collaris – DJ8203 MT856897 MT862679 MT862581 MT862606 MT862631 MT862649 Turkey,

Temürağa (16)

38.046 36.586

P. collaris – DJ8197 MT856891 MT862673 MT862575 MT862600 MT862625 MT862643 Turkey,

Gürümze (14)

38.042 35.811

P. collaris – DJ8206 MT856900 MT862682 MT862584 MT862609 MT862634 MT862652 Turkey,

Suna Plateau

(13)

37.993 35.397

P. collaris – DJ8207 MT856901 MT862683 MT862585 MT862610 Turkey,

Işıklı (9)

37.832 27.799

P. collaris – DJ8200 MT856894 MT862676 MT862578 MT862603 MT862628 MT862646 Turkey,

Ömerli (12)

37.535 34.875

P. collaris – DJ8205 MT856899 MT862681 MT862583 MT862608 MT862633 MT862651 Turkey,

Ceyhan (18)

36.895 35.916

P. collaris – DJ8199 MT856893 MT862675 MT862577 MT862602 MT862627 MT862645 Turkey,

Küplüce (19)

36.757 37.236

P. collaris – DJ8201 MT856895 MT862677 MT862579 MT862604 MT862629 MT862647 Turkey,

Kaldırım (17)

36.674 35.525

P. collaris – DJ8208 MT856902 MT862684 MT862586 MT862611 MT862635 MT862653 Turkey,

Erdemli (11)

36.639 34.340

P. collaris – DJ8204 MT856898 MT862680 MT862582 MT862607 MT862632 MT862650 Turkey,

Bozyazı-

Anamur (10)

36.097 33.042

P. collaris NMP-P6V 75259/1

(DZCHU 435)

CRT01 Paratype of

Coluber

rubriceps

thracius

– MT862664 – – – – Bulgaria,

Arkutino (4)

42.323 27.738

P. collaris NMP-P6V 75259/2

(DZCHU 436)

CRT02 Paratype of

C. r. thracius

– MT862665 – – – – Bulgaria,

Arkutino (4)

42.323 27.738

P. collaris NMP-P6V 75257

(DZCHU 433)

CRT04 Holotype of

C. r. thracius

– MT862666 – – – – Bulgaria,

Arkutino (4)

42.323 27.738

P. collaris NMP-P6V 75259/5

(DZCHU 437)

CRT05 Paratype of

C. r. thracius

– – – – – – Bulgaria,

Arkutino (4)

42.323 27.738

P. collaris NMP-P6V 75259/4

(DZCHU 438)

CRT06 Paratype of

C. r. thracius

– MT862667 – – – – Bulgaria,

Arkutino (4)

42.323 27.738

Table 1. Continued

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660 J. ŠMÍD ET AL.

mtDNA nDNA

Taxon Voucher Sample Specimen status 12S cytb coi cmos nt3 rag1 Locality Latitude Longitude

P. collaris NMP-P6V 75258

(DZCHU 441)

CRT12 Paratype of

C. r. thracius

– MT862669 – – – – Bulgaria,

Arkutino (4)

42.323 27.738

P. collaris NMP-P6V 75259/3

(DZCHU 439)

CRT13 Paratype of

C. r. thracius

– MT862670 – – – – Bulgaria,

Arkutino (4)

42.323 27.738

P. collaris NMP-P6V 75262

(DZCHU 440)

CRT14 Paratype of

C. r. thracius

– MT862671 – – – – Bulgaria,

Arkutino (4)

42.323 27.738

P. collaris NMP-P6V 75260

(DZCHU 442)

CRT15 Paratype of

C. r. thracius

– MT862672 – – – – Bulgaria,

Arkutino (4)

42.323 27.738

P. collaris NMP-P6V 75261

(DZCHU 443)

CRT11 Paratype of

C. r. thracius

– MT862668 – – – – Bulgaria,

Ahtopol (6)

42.098 27.917

Platyceps

najadum

MCCI R-1399 R1399 MT856904 MT862686 MT862588 MT862613 MT862637 – Armenia,

Khosrov

Reserve

39.968 44.951

Telescopus

dhara

– CN10774 MK372075 MK373064 – MK373173 MK373244 MK373213 Oman, Wadi

Ayoun

17.253 53.894

Telescopus

fallax

ZMHRU 2012_103 2012_103 MK372088 MK373073 – MK373186 MK373253 MK373222 Turkey,

Şanlıurfa

37.212 37.969

Telescopus

obtusus

TMHC841 TMHC841 MK372104 MK373090 – MK373202 MK373269 MK373237 Somalia,

9 km SE of

Boorama

9.86 43.244

Telescopus

pulcher

NMP-P6V 75609 TMHC843 MK372105 MK373092 – MK373204 MK373271 MK373239 Somalia,

15 km SE of

Sheikh

9.825 45.29

The sample column refers to codes shown in the Supporting Information (Figs S1–S4). Numbers in parentheses in the locality column show locality numbers as given in Figures 1 and 2. Collection

acronyms are as follows: DZCHU, Department of Zoology, Charles University, Prague, Czech Republic; HLMD, Hessisches Landesmuseum, Darmstadt, Germany; MCCI, Museo Civico di Storia

Naturale di Carmagnola, Torino, Italy; MHNG, Muséum d’histoire naturelle, Genève, Switzerland; NMP-P6V, National Museum in Prague, Czech Republic; TAU.R, Steinhardt Museum of Natural

History, Tel Aviv, Israel; TMHC, Tomas Mazuch herpetological collection (private), Dříteč, Czech Republic; ZMHRU, Zoology Museum of Harran University, Sanliurfa, Turkey. GenBank accessions of

sequences newly produced for this study are those starting with ‘MT’.

Table 1. Continued

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PHYLOGEOGRAPHY OF COLLARED DWARF RACER 661

containing more than two expected sequencing errors

were eliminated; then we used the dada2 denoising

algorithm (https://benjjneb.github.io/dada2/) for

correction of sequencing errors in the quality-filtered

dataset. Only variants with a minimum coverage of

four sequences per variant and those that occurred in

both duplicates were used for a BLAST analysis.

phylogenetic analySeS

The six genetic markers were aligned independently

with MAFFT v.7 (Katoh et al., 2019) using the default

settings. No stop codons were found in the alignments

of the protein-coding genes (cytb, coi, cmos, nt3 and

rag1), indicating that no pseudogenes were amplified.

The final alignments had the following lengths:

12S, 633 bp; cytb, 1072 bp; coi, 594 bp; cmos, 567 bp;

nt3, 486 bp; and rag1, 1009 bp. We concatenated all

markers in an alignment of a total length of 4361 bp.

Platyceps najadum and four species of Telescopus

Wagler, 1830 were used as outgroup taxa based on

published evidence (Table 1; Zheng & Wiens, 2016).

Phylogenetic analyses were conducted by two

means: maximum likelihood (ML) and Bayesian

inference (BI). For the ML analysis, we used raxml

v.7.3 (Stamatakis, 2006). The concatenated alignment

was partitioned by gene, and the GTRGAMMA model

was used for all partitions. A heuristic search included

100 random addition replicates and 1000 thorough

bootstrap pseudo-replicates. Identical haplotypes

were retained in the analysis to assess intraspecific

variability. The Bayesian analysis ran in BEAST v.2.5.2

(Bouckaert et al., 2014). The dataset was partitioned

by gene, and heterozygous positions in the nuclear

markers were coded using International Union of Pure

and Applied Chemistry (IUPAC) ambiguity codes. The

best evolutionary substitution models were estimated

using the reversible-jump algorithm (RB; Bouckaert

et al., 2013) with four exponentially distributed rate

categories (+Γ; mean = 1) and the shape parameter

estimated. The RB algorithm estimates substitution

models alongside the phylogeny without the need for

a priori selection. The parameter for the proportion

of invariable sites (+I) was not estimated given

its correlation with the +Γ parameter, resulting in

inadequate models when both +Γ and +I are included

(Sullivan et al., 1999; Mayrose et al., 2005). Given that

the dataset focused on intraspecific diversification,

we did not assume the substitution rate to vary

substantially throughout the tree and, as a result, we

used a strict clock model with lognormally distributed

priors (mean = 1, SD = 1.25) for all partitions and a

coalescent constant population tree prior. The analysis

ran three times for 5 × 107 Markov chain Monte

Carlo generations sampled every 25 000 generations,

producing a set of 2000 posterior trees. We discarded

10% of trees as burn-in after determining that the

effective sample size (ESS) values of all parameters

exceeded the recommended value of 200 using

TRACER v.1.6 (Rambaut et al., 2014), and ensuring

that convergence and stationarity had been reached

by assuring that parameter values in the independent

runs plateaued at similar values. Tree files from the

three runs were combined using logcombiner, and

a maximum clade credibility tree was identified using

treeannotator (both programs are from the BEAST

package). Nodes were considered supported when they

received a Bayesian posterior probability (pp) ≥ 0.95

and ML bootstrap ≥ 70. All phylogenetic analyses ran

through the CIPRES Science Gateway (Miller et al.,

2010). To inspect the influence of the fast-evolving

mitochondrial markers on the tree topology visually,

we ran the same analyses as described above (both

ML and BI) with a concatenated dataset of the three

mitochondrial genes. Together with the haplotype

networks (see two paragraphs below), this was meant

to assess the extent to which the variation in the

nuclear markers contributes to the topology of the tree.

We also constructed a phylogenetic network

among the P. collaris samples using Spl i tS tree

v.4.15.1 (Huson & Bryant, 2006), with the Neighbor-

Net algorithm (Bryant & Moulton, 2004). Phylogenetic

networks are useful in that they can help to visualize

reticulate relationships among samples and detect

hybrid individuals. We used the alignment of all

six markers concatenated as an input. The nuclear

loci were phased as described below, and the

mitochondrial markers were duplicated to match

the phased nuclear samples. Outgroups were not

included in this analysis, nor were the short cytb

sequences of the C. r. thracius type specimens. We

used 1000 bootstrap replicates to assess support for

the network groups. We tested individual nuclear loci

for recombination using the φ statistic implemented

in SplitStree (Bruen et al., 2006).

Intraspecific relationships at the level of individual

genes were assessed by reconstructing haplotype

networks. Heterozygous positions present in the

alignments of the three nuclear loci were resolved by

phasing. We used SeqphaSe (Flot, 2010) to convert

input files and PHASE (Stephens et al., 2001) to

reconstruct the gametic phases, with the probability

threshold set to 0.7 (Harrigan et al., 2008). Networks

were constructed with the TCS algorithm (Clement

et al., 2000) as implemented in POPART (Leigh &

Bryant, 2015). For the cytb gene network, we used the

148-bp-long alignment that included sequences of the

C. r. thracius types.

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662 J. ŠMÍD ET AL.

Uncorrected genetic distances (p-distances) for

the mitochondrial genes were calculated in MEGA7

(Kumar et al., 2016).

moDelling the potential SpecieS DiStribution

in the preSent anD in the paSt

We compiled a dataset of distribution records by

searching published sources, museum catalogues, public

biodiversity databases and from our own fieldwork. The

final set totalled 539 georeferenced records that covered

the known range of the species densely. However, the

records varied considerably in their geographical

accuracy (0.005–55 km), and the less accurate ones

might affect model performance. We therefore selected

only records with an accuracy of ≤ 2 km. For this reason,

the outlying localities in central Anatolia and by the

Syrian border could not be included. Also, we removed

duplicates and included only records collected after

1980 (Supporting Information, Table S1).

As a background for modelling in present conditions,

we selected an area within 200 km of the species

range, as presented by Roll et al. (2017), from which

we cropped countries where the species has never

been recorded: Cyprus, Egypt, Iran, Romania, Saudi

Arabia and the Greek Aegean Sea islands. To avoid

model bias caused by high densities of records in

certain regions, we thinned the dataset using the

spThin R package (Aiello-Lammens et al., 2015). We

tested three minimum distance radii to separate

any two records: 5, 20 and 50 km. They all produced

comparable models, and we show results only for the

5 km model. The thinning ran ten times, and each run

produced a different dataset of 90 records. These were

used as independent random replicates. We did not

develop independent models for the two clades found

within P. collaris because their genetic differentiation

is shallow, especially when compared with the genetic

differentiation between P. collaris and its sister species,

P. najadum (see Results below); thus, we assumed

their ecological niches to be similar.

As input variables, we used the 19 BioClim

variables (CHELSA; Karger et al., 2017), elevation

slope and land cover (GlobCover 2009 2.3; European

Space Agency; http://due.esrin.esa.int/page_globcover.

php) and tested them for collinearity using Pearson’s

r coefficient in enmtoo lS (Warren et al., 2010).

Based on the correlation matrix, we selected only

the uncorrelated (r < 0.75) and more biologically

meaningful variables. The final set of variables was

as follows: elevation, slope, land cover, Bio2 (mean

diurnal temperature range), Bio4 (temperature

seasonality), Bio8 (mean temperature of wettest

quarter), Bio9 (mean temperature of driest quarter),

Bio16 (precipitation of wettest quarter) and Bio17

(precipitation of driest quarter).

We used the maximum entropy approach

implemented in maxent v.3.3 (Phillips et al., 2006) to

develop the model and to assess the importance of each

input variable. maxent ran with the following settings

(other than default): random seed, replicated run

type = cross validate and maximum iterations = 5000.

We ran ten replicates for each of the ten thinned

datasets, thus producing 100 models. In each run, 10%

of records were randomly selected as test points.

The area under the receiver-operating characteristic

curve (AUC) value of each model was taken as a

measure of the model accuracy. Models with AUC > 0.8

were considered to perform well (Araújo et al., 2005).

To test whether the predictive models performed better

than random, we generated 100 null models based on

100 sets of 90 records randomly distributed in the

background of the model. The probabilistic models

were converted to binary maps of potential presence/

absence using two thresholds, a ten-percentile training

presence threshold and maximum training sensitivity

plus specificity threshold (Jiménez-Valverde & Lobo,

2007; Worth et al., 2014). The resulting areas of the

species potential presence were largely overlapping for

the two thresholds, and we show results for the latter.

All spatial analyses for the current conditions were

done at the scale of 30 arc s.

Besides modelling the potential distribution of

P. collaris in current climatic and topographical

conditions, we projected the model to four past periods,

which were: the Last Glacial Maximum (LGM;

~21 kya), the Last Interglacial (LIG; ~130 kya), the

mid-Pleistocene (~787 kya) and the mid-Pliocene

(~3.2 Mya). We only used the BioClim variables for

the past projections, and Bio2 was excluded because

it was not available for all the past periods. Past

climatic data were downloaded collectively from

the PaleoClim database (Brown et al., 2018), with

the following original sources: Karger et al. (2017)

for the LGM; Otto-Bliesner et al. (2006) for the LIG;

Brown et al. (2018) for the mid-Pleistocene; and Hill

(2015) for the mid-Pliocene. The background onto

which the models were projected had to be expanded

to account for the different continental outlines in the

past. For this reason, we converted the background that

was used for the current conditions into an envelope,

i.e. a rectangle defined by minima and maxima of the

x- and y-coordinates of the input. The past projections

were all done at a resolution of 2.5 arc min.

RESULTS

Dna amplification

For the fresh material of P. collaris (25 samples) we

produced 133 new DNA sequences of the six markers

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PHYLOGEOGRAPHY OF COLLARED DWARF RACER 663

analysed, resulting in a genetic data matrix with few

missing data (89% gene sampling).

For the type material of C. r. thracius, we were able

to obtain the targeted 148-bp-long cytb fragment for

nine of the ten specimens. DNA concentrations of

these specimens ranged between 0.01 and 3.42 ng/µL,

and average number of reads generated for them

was 5735 (range 2113–10 302; Table 2). Artefactual

variants and contaminations were identified based

on a BLAST analysis and discarded manually.

One specimen was removed from further analysis

(CRT05) because the reads showed a mix of various

contaminations (predominantly Homo). There were

several synonymous substitutions along the 148 bp

fragment, indicating that the amino acid content of

this gene fragment was similar across all samples.

phylogenetic analySeS

The samples of P. collaris form two well-differentiated

clades in all analyses. One comprises all samples from

Bulgaria (including the types of C. r. thracius), western

Turkey and most samples from southern Turkey

(ML bootstrap = 98/BI pp = 1.00; support values are

given in this order hereafter), and we term it here the

Balkan–Anatolian clade. The other clade comprises

samples from Israel, Jordan and Syria, and we term

it the Levantine clade, although this clade has only

moderate support in the ML analysis (Fig. 1; support

65/1.00; for original ML and BI trees, see Supporting

Information, Figs S1, S2, respectively). Relationships

within both clades remain unresolved owing to low

branch support. The position of sample DJ8199, from

a locality in extreme southern Turkey (locality 19 in

Fig. 1), differs in the two analyses. It was reconstructed

and supported as sister to the Bulgarian and other

Turkish samples in the ML analysis (support 74), but

the BI analysis recovers it as sister to the Levantine

clade (support 0.94). Both support values are on the

edge of interpretability, but the haplotype networks

constructed for each marker independently show

the sample to be closer to the Levantine clade in its

mitochondrial DNA, whereas the nuclear markers are

more similar to the Balkan–Anatolian clade (networks

in Fig. 1). When only mitochondrial markers are

analysed, topologies of the ML and BI trees remain

similar to those of the complete dataset, including

the varying position of sample DJ8199 (Supporting

Information, Figs S3, S4). The haplotype networks

clearly differentiate the two clades described above,

but the nuclear ones show a certain degree of allele

sharing.

The phylogenetic network analysis resulted in

similar groups to those recovered in the ML and BI

analyses (Fig. 2). The reciprocal monophyly of the

Balkan–Anatolian and Levantine clade is strongly

supported (bootstrap 99.9). Sample DJ8199, whose

phylogenetic position varied in the ML and BI analysis,

is clustered at the base of the Levantine clade. No signs

of recombination are detected for the three nuclear loci

(P-values ranging between 0.68 and 1.00).

Genetic p-distances between the Balkan–Anatolian

and Levantine clades are (±SD): 1.3 ± 0.2% in 12S,

2.2 ± 0.2% in cytb and 1.6 ± 0.2% in coi. Genetic

diversity within the Balkan–Anatolian clade is 0.2% in

12S, 0.6% in cytb and 0.3% in coi; within the Levantine

clade it is 0.3% in 12S, 0.7% in cytb and 0.4% in coi.

Genetic distances of P. collaris to its sister species

P. najadum are 10.6 ± 0.3% in 12S, 11.2 ± 0.3% in cytb

10.8 ± 0.2% in coi.

Table 2. Type specimens of Coluber rubriceps thracius included in the genetic analysis

Voucher Sample DNA concentration (ng/µL) BLAST results Number of reads after filtering

First coverage Second coverage

NMP-P6V 75259/1 CRT01 0.038 Platyceps collaris 2113 3988

NMP-P6V 75259/2 CRT02 1.08 P. collaris 3022 6351

NMP-P6V 75257 CRT04 2.61 P. collaris 6405 6817

NMP-P6V 75259/5 CRT05 0.038 Contamination 4208 6372

NMP-P6V 75259/4 CRT06 0.128 P. collaris 7015 8626

NMP-P6V 75261 CRT11 < 0.01 P. collaris 6272 5512

NMP-P6V 75258 CRT12 3.42 P. collaris 4546 6947

NMP-P6V 75259/3 CRT13 < 0.01 P. collaris 3349 5910

NMP-P6V 75262 CRT14 2.07 P. collaris 3018 5019

NMP-P6V 75260 CRT15 < 0.01 P. collaris 8910 10 302

The DNA concentration was measured using a Qubit 1.0 fluorometer (Qubit High Sensitivity Kit). Results of the BLAST search showed that one of

the specimens was contaminated by human DNA. The columns headed ‘Number of reads after filtering’ show the total number of sequences obtained

from the two independent duplicates.

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664 J. ŠMÍD ET AL.

SpecieS DiStribution moDelling

Species distribution models of all replicates

performed well, with AUC values ranging between

0.8779 and 0.8905 (mean = 0.8864). The standard

deviations of the models are low (0.0385–0.0457;

mean = 0.0429), indicating model stability

regardless of the input data. Models based on real

data performed significantly better than null models

(AUC range = 0.649–0.762; mean = 0.705). The main

environmental predictors explaining the distribution

of P. collaris are Bio4 (47.1% contribution averaged

over the ten random thinning replicates, range

45.9–49.3%), Bio2 (mean contribution 22.4%,

range 20.5–23.6%) and Bio17 (mean contribution

10.6%, range 10.0–10.9%; for the contribution of all

variables and their response curves, see Supporting

Information, Fig. S5). The predicted distribution

of suitable habitat spans from south-eastern

Bulgaria and north-western Turkey in a narrow

band continuously along the Turkish Mediterranean

coast all the way to the coast of Syria and further

across Lebanon to Israel and Jordan. The suitable

habitat is rarely found > 200 km inland from the

Mediterranean coast, and especially in southern

Turkey it is formed by a considerably narrow coastal

zone (Figs 1, 3).

Figure 1. Phylogenetic tree resulting from the Bayesian inference (BI) analysis of three mitochondrial and three nuclear

genes concatenated. Nodes were considered supported when Bayesian posterior probability was ≥ 0.95 and maximum

likelihood (ML) bootstrap values ≥ 70. Lengths of branches connecting the split between Platyceps collaris and Platyceps

najadum and the crown nodes of those species are not proportional to the rest of the tree and the scale, which is indicated

by their partial transparency. The two clades, the Balkan–Anatolian and the Levantine, are highlighted in the tree with the

red and green shading, respectively. Four species of Telescopus used to root the tree are not shown. Each tree tip is connected

by a dashed line with the locality of its sample, which is marked by a number (for details, see Table 1). Type localities are

marked with stars: P. collaris collaris in green, P. collaris rubriceps in white and Coluber rubriceps thracius in pink. The

potential current distribution of P. collaris based on the species distribution model with the maximum training sensitivity plus

specificity threshold applied is shown in blue. Haplotype networks reconstructed for the six markers are on the right. Circles

are colour coded according to the clade assignment, and their size is proportional to the number of individuals. Lines represent

mutational steps. The network for the cytb gene was constructed using the 148-bp-long fragment that was available for the

types of Coluber rubriceps thracius. The position of sample DJ8199 from locality 19, whose phylogenetic placement differed in

the maximum likelihood and Bayesian inference analyses, is marked with the locality number in each network.

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PHYLOGEOGRAPHY OF COLLARED DWARF RACER 665

Projections of the suitable conditions into the past

show that certain parts of the range of P. collaris have

been stable since the mid-Pliocene. The overall extent

of suitable habitat was smallest in the mid-Pleistocene,

when it was restricted to the Levant and western

Anatolia, with a thin coastal strip that might have

been a corridor connecting these two refugia. During

the climatic optimum of the LIG, this coastal strip was

much broader and most probably allowed population

connectivity. During the LGM, western Anatolia had

the most suitable conditions, whereas the southern

Levant was mostly unsuitable. Since the LGM, the

western and southern Anatolian part of the suitable

area contracted noticeably, whereas it expanded

considerably into the southern Levant (Fig. 3).

DISCUSSION

In this study, we applied a multidisciplinary approach

to disentangle the phylogeographical structure, genetic

diversity and distribution dynamics of a racer snake,

Platyceps collaris. We used a multilocus genetic dataset

generated by Sanger sequencing in combination

with a newly developed method for amplifying and

Illumina sequencing short DNA barcodes for museum

specimens, which often suffer from DNA fragmentation

and degradation. The main advantage of this approach

is that it allows separation of individual reads of each

sequence and exclusion of those that are obviously

contaminated, typically by human DNA (Galan et al.,

2012), which also happened with one of our samples.

The biggest potential of this method is, in our view, that

it allows an alignment-based placement of important

specimens (e.g. types, extinct populations) within the

phylogenetic context to resolve outstanding taxonomic

or conservation questions.

intraSpecific genetic DiverSification

Platyceps collaris is endemic to the Eastern

Mediterranean, a region that experienced intense

Figure 2. Phylogenetic network from SplitStree, showing the reticulate relationships within the two clades of Platyceps

collaris, the Balkan–Anatolian clade (red) and the Levantine clade (green). Numbers in circles are locality numbers shown

in Figure 1 and detailed in Table 1. Bootstrap support values for major nodes ≥ 70 are indicated. The specimen depicted is

from Ropotamo, Bulgaria.

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666 J. ŠMÍD ET AL.

phylogeographical research on squamates in the past

decade that has often resulted in uncovering hidden

genetic diversity or even cryptic species (Moravec

et al., 2011; Bellati et al., 2015; Jandzik et al., 2018;

Kotsakiozi et al., 2018; Jablonski & Sadek, 2019; Šmíd

et al., 2019). The results of all phylogenetic analyses

conducted in the present study show a clear genetic

differentiation of P. collaris into two geographically

well-delineated clades. One, the Balkan–Anatolian

clade, occupies the north-western part of the species

range. It spans from the Black Sea coast in south-

eastern Bulgaria along the Mediterranean coast of

Turkey to the Antakya region at the Turkish–Syrian

border. The second, the Levantine clade, covers the

southern part of the species range from extreme

southern Turkey through Syria to Israel and Jordan.

The two clades are clearly differentiated in their

mitochondrial DNA, but they share some alleles in

the cmos and nt3 nuclear loci. As the networks show,

these shared nuclear alleles are either ancestral or

shared across multiple samples from both clades,

which indicates retention of ancestral polymorphism

rather than them being a result of hybridization. The

boundary between the two clades corresponds well with

the geographical position of the Nur Mountain range in

Hatay Province of south-central Turkey (Fig. 1), which

runs parallel to the Gulf of İskenderun and separates

the Anatolian part of the Eastern Mediterranean from

the Levant. These mountains are part of the so-called

Anatolian Diagonal, a natural and probably the most

important biogeographical barrier in the region, which

has repeatedly been proved effective in separating

closely related taxa (Davis, 1971; Tamar et al., 2015;

Kornilios, 2017; Jandzik et al., 2018; Jablonski et al.,

2019; Jablonski & Sadek, 2019).

Genetic diversity within the Balkan–Anatolian and

Levantine clades is of a comparable magnitude. All

mitochondrial markers show the presence of several

Figure 3. Species distribution models for Platyceps collaris in current conditions (A) and in conditions during the Last

Glacial Maximum (B), the Last Interglacial (C), mid-Pleistocene (D) and mid-Pliocene (E). Black points in A indicate the

records that were used for the species distribution modelling. The bottom right panel shows reconstructions of global

temperature in the last 4 Myr relative to the peak Holocene temperature (Hansen & Sato, 2012), with grey arrows and

dashed lines highlighting the temperature in the time periods used in the present study.

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PHYLOGEOGRAPHY OF COLLARED DWARF RACER 667

substitutions within both clades, and the nuclear loci

also show a considerable degree of variance, despite

their generally slower substitution rates compared

with the mitochondrial genes. Relationships within

these two clades could not be resolved, which was

most probably because the phylogenetic relationships

at this shallow evolutionary depth do not necessarily

form a bifurcating tree but rather a reticulate pattern,

which becomes obvious when they are visualized using

the phylogenetic network (Fig. 2).

DiStribution anD hiStorical range DynamicS

The distribution of P. collaris is well documented

in the literature thanks to the unique appearance

of the snake, although misidentifications with the

sister species, P. najadum, can happen (e.g. Berger-

Dell’Mour, 1986). Although the range of the species

spans a relatively large territory, from south-eastern

Bulgaria to southern Israel and Jordan, the species

distribution modelling analysis shows that the

predicted suitable habitat is found only in a narrow

stretch of land along the Mediterranean coast (Fig. 1).

The potential distribution reaches further inland in

the Levant, even beyond the Dead Sea Rift in Jordan,

but there it is restricted to the Mediterranean ecozone

(Disi, 1996; Disi et al., 2001). The character of the

environmental variables that contribute most to the

potential distribution of the species suggests that

P. collaris prefers areas of low climatic seasonality,

without fluctuating temperatures (Supporting

Information, Fig. S5). For example, the probability of

presence of the species decreases significantly with

increasing elevation. This means that in south-western

Turkey, where the mountains come close to the sea, the

suitable habitat is present only along the immediate

vicinity of the coast.

The Levantine part of the species range corresponds

well to the distribution of the Eastern Mediterranean

conifer–broadleaf forest ecoregion (Dinerstein et al.,

2017), which covers the eastern Mediterranean coastal

zone (Supporting Information, Fig. S6). The ecoregion

forms a narrow coastal strip in southern Turkey, and

it reaches eastwards along the entire length of the

Turkish–Syrian border all the way to the isolated

P. collaris populations. However, these populations lie

well away from the suitable habitat of the species. It

might be that the environmental variables used in the

modelling process did not capture all dimensions of

the biotic requirements of the species and thus failed

to identify these localities to have a suitable habitat.

It is nonetheless interesting that there have not been

more observations of P. collaris in the broadleaf forest

ecoregion along the Turkish–Syrian border. These

eastern localities might represent microclimatic

pockets where the species survives in isolation from the

Mediterranean populations, a hypothesis that could be

addressed easily if the specimens were sequenced and

placed in a phylogenetic framework. More enigmatic in

this respect is the record from central Anatolia, which

is > 200 km away from other records of the species

(İğci et al., 2015). Given that a voucher of the specimen

is catalogued in a collection [Zoology Museum of

Adıyaman University, Turkey (ZMADYU) 2013/57], we

recommend collecting a tissue sample and genotyping

it in order to assess its phylogenetic affinities.

The models of suitable habitat distributions in the

past show that the climatic conditions of the Eastern

Mediterranean have been stable since the Late

Tertiary and suitable for the occurrence of P. collaris.

Despite some oscillations, western and southern

Anatolia and the northern Levant have remained

suitable for the species for the past 3 Myr (Fig. 3).

This contrasts with south-eastern Bulgaria and the

southern Levant, which lacked suitable habitats in

the LGM and mid-Pleistocene. Western Anatolia, one

of the current hotspots for the species distribution,

had a larger extent of suitable habitats in the LGM

and LIG, whereas further in the past its suitability

corresponded more or less to that of the current

conditions. Together with the northern Levant, these

areas might have provided refugia where populations

of P. collaris survived climatically challenging times,

such as the LGM, and from where they subsequently

colonized the rest of the current range. Here, it is of

importance to note that the historical oscillations of

suitable habitats did not necessarily have to lead to

population expansions and contractions for reasons

associated with factors such as dispersal limitations,

competitive exclusion and predation (Peterson, 2011).

However, the genetic evidence that confirmed the

presence of the two distinct clades provides credibility

to the presence of the two refugia. It is therefore likely

that the Anatolia refugium served as a source for the

colonization of the south-eastern Balkans after the

LGM, whereas the south of the Levant was repopulated

from the north, from the Syrian coastal region, as has

been suggested for the genus Lacerta Linnaeus, 1758

(Ahmadzadeh et al., 2013).

SubSpecific taxonomy of platyCeps Collaris

The genetic differentiation between the two P. collaris

clades is, to some degree, mirrored in the morphological

traits. Specimens from Bulgaria (including the types of

Coluber rubriceps thracius) and south-western Turkey

have been shown to have a lower number of ventral

(185–203) and subcaudal scales (79–105) compared

with specimens from the Levant (ventrals, 189–220;

subcaudals, 89–128; Rehák, 1985, 1986; Rehák & Obst,

1993; Schätti et al., 2001). However, the morphological

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668 J. ŠMÍD ET AL.

variability seems to be more of a cline, with specimens

from southern Turkey being intermediate in these

traits. Unfortunately, all authors who have collected

morphological data for P. collaris compared populations

and did not provide values for individual specimens.

For example, Baran (1976) gave only trait means and

variations for specimens from across the species range,

Rehák (1986) compared populations from Bulgaria and

Israel, and Rehák & Obst (1993) compared populations

from Bulgaria with those from Turkey, Israel and Jordan.

The most detailed comparison was provided by Schätti

et al. (2001), who compared the following five geographical

groups: Bulgaria, western Anatolia, southern Anatolia,

Lebanon and north-western Syria. However, as our

results show, southern Anatolia is occupied by both

clades, and the groupings by Schätti et al. (2001) might

have meant that both were represented in this category,

thus obfuscating the potential difference between the

clades. Therefore, future morphological comparison of

ideally genotyped specimens from southern Anatolia

will be necessary to clarify this issue.

Based on the genetic results obtained in the present

study, we are able to draw some conclusions regarding

the subspecific taxonomy of P. collaris. Considering the

phylogeographical structure found within the species,

the parapatry of the two clades and their complete

segregation of mitochondrial haplotypes, the two

clades can be considered distinct subspecies. Given

that the type locality of P. collaris in Beirut falls within

the range of the Levantine clade, this clade retains

the nominotypical name Platyceps collaris collaris

(Müller, 1878).

In the case of the Balkan–Anatolian clade, the name

Zamenis dahlii rubriceps Venzmer, 1919 has priority

over the name Coluber rubriceps thracius Rehák, 1985.

By sequencing the holotype and eight paratypes of

C. r. thracius, we show that they are genetically identical

with all other specimens of P. collaris from Bulgaria

included in the analysis and also with two specimens

from western and southern Turkey. Although we

could not analyse the type material of Zamenis dahlii

rubriceps directly in the present study, we had samples

collected not far away from its type locality that have

proved to represent the Balkan–Anatolian clade. In fact,

the type locality is surrounded by samples of that clade,

and we believe that it is reasonable to assume that, if

genotyped, it would cluster with the Balkan–Anatolian

clade. Therefore, following the principle of priority, we

suggest that this clade should bear the name Platyceps

collaris rubriceps (Venzmer, 1919) comb. nov., and the

name Coluber rubriceps thracius Rehák, 1985 should

be its junior synonym.

Genetic methods have enabled a boom in species

descriptions that is unprecedented since the times

of Linnaeus. Despite this, subspecific taxonomy

shows the opposite trend in squamates (Torstrom

et al., 2014). However, recognition of subspecies

can serve as a useful formalization that reflects

character differentiation (genetic, morphological and

geographical) within species and should be adhered

to (Kindler & Fritz, 2018), especially when there is

congruence between more lines of evidence. In our case,

these congruent lines are the genetic differentiation

between the Levantine clade (P. c. collaris) and the

Balkan–Anatolian clade (P. c. rubriceps), which is

probably a result of different Quaternary refugia used

by the clades. However, morphological differentiation

between the clades needs to be verified.

ACKNOWLEDGEMENTS

We thank Roberto Sindaco (Carmagnola Museum,

Italy) and Shai Meiri (Tel Aviv Museum, Israel)

for providing tissue samples and Jana Poláková

(Comenius University in Bratislava, Slovakia) for

her laboratory work. Dagmar Čížková (Institute of

Vertebrate Biology, Czech Republic), Jakub Kreisinger

(Charles University, Czech Republic) and Boris Tichý

(Central European Institute of Technology [CEITEC],

Czech Republic) kindly helped with the study design

and the analysis of museum samples. The project

was supported and permits issued by the Ministry of

Environment and Water of Bulgaria (permit numbers

656/08.12.2015 and 767/24.01.2019) and TÜBITAK

(The Scientific and Technical Research Council of

Turkey; permit numbers TBAG-104T294 and TBAG-

108T162). The work of J.Š. was supported by the

Czech Science Foundation (GACR, project number

18-15286Y), that of J.Š. and J.M. by the Ministry of

Culture of the Czech Republic (DKRVO 2019–2023/6.

VII.b, 00023272), the work of D.V. by the Charles

University (SVV 260571/2020) and the work of D.J. by

the Slovak Research and Development Agency (under

contracts no. APVV-15-0147 and APVV-19-0076).

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SUPPORTING INFORMATION

Additional Supporting Information may be found in the online version of this article at the publisher’s web-site:

Figure S1. Phylogenetic tree of Platyceps collaris resulting from the maximum likelihood analysis of all genes

concatenated. Bootstrap support values ≥ 60 are shown above branches. Type specimens of Coluber rubriceps

thracius are in red.

Figure S2. Phylogenetic tree of Platyceps collaris resulting from the Bayesian inference analysis of all genes

concatenated. Posterior probability values ≥ 0.90 are shown above branches. Type specimens of Coluber rubriceps

thracius are in red.

Figure S3. Phylogenetic tree of Platyceps collaris resulting from the maximum likelihood analysis of the three

mitochondrial genes concatenated. Bootstrap support values ≥ 60 are shown above branches. Type specimens of

Coluber rubriceps thracius are in red.

Figure S4. Phylogenetic tree of Platyceps collaris resulting from the Bayesian inference analysis of the three

mitochondrial genes concatenated. Posterior probability values ≥ 0.90 are shown above branches. Type specimens

of Coluber rubriceps thracius are in red.

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672 J. ŠMÍD ET AL.

Figure S5. Response curves of the environmental variables used to develop the distribution model, showing how

each variable affected the prediction and its relative contribution to the model (averaged over ten runs). Each plot

shows the probability of presence of the species (y-axis) as a function of the environmental predictor value (x-axis).

Red lines show mean responses of ten replicate runs; standard deviations are in blue. The key in the lower right

refers to the categorical land cover variable in the lower left.

Figure S6. Geographical distribution of the Eastern Mediterranean conifer–broadleaf forest ecoregion (Dinerstein

et al., 2017).

Table S1. Locality details of records used for the species distribution modelling and their original sources.

Coordinates from Bulgaria have been truncated to two decimal places for conservation purposes.

Downloaded from https://academic.oup.com/zoolinnean/article/193/2/655/6048377 by Department of Plant Physiology, Faculty of Science, Charles University user on 30 September 2021

Phylogeography of the collared dwarf racer

Supplementary Materials – 1

Supplementary Figures and Table for

Quaternary range dynamics and taxonomy of the Mediterranean collared dwarf racer,

Platyceps collaris (Squamata: Colubridae)

Šmíd et al.

Phylogeography of the collared dwarf racer

Supplementary Materials – 2

Figure S1. Phylogenetic tree of Platyceps collaris resulting from the ML analysis of all genes

concatenated. Bootstrap support values ≥ 60 are shown above branches. Type specimens of

Coluber rubriceps thracius are in red.

0.02

Platyceps_collaris_Turkey_8205

Platyceps_collaris_Israel_TAUR16921

Platyceps_collaris_Turkey_8202

Platyceps_collaris_Syria_JIR187

Platyceps_collaris_Israel_R649

Platyceps_collaris_Bulgaria_7739

Coluber_rubriceps_thracicus_CRT02

Coluber_rubriceps_thracicus_CRT06

Coluber_rubriceps_thracicus_CRT13

Platyceps_collaris_Bulgaria_6132

Platyceps_collaris_Turkey_8206

Platyceps_collaris_Jordan_Nagy

Platyceps_collaris_Israel_TAUR15955

Coluber_rubriceps_thracicus_CRT11

Platyceps_collaris_Turkey_8208

Platyceps_collaris_Israel_Schatti2

Coluber_rubriceps_thracicus_CRT01

Coluber_rubriceps_thracicus_CRT12

Platyceps_collaris_Turkey_8203

Telescopus_obtusus_TMHC841

Platyceps_collaris_Bulgaria_DJ607

Coluber_rubriceps_thracicus_CRT04

Platyceps_collaris_Turkey_8207

Platyceps_collaris_Israel_Schatti1

Telescopus_dhara_CN10774

Platyceps_collaris_Turkey_8204

Platyceps_collaris_Turkey_8201

Coluber_rubriceps_thracicus_CRT15

Telescopus_pulcher_TMHC843

Platyceps_collaris_Syria_JIR188

Platyceps_collaris_Turkey_8209

Platyceps_collaris_Israel_2478

Platyceps_collaris_Turkey_8198

Telescopus_fallax_syriacus_2012_103

Platyceps_collaris_Bulgaria_1417

Platyceps_collaris_Bulgaria_1405

Platyceps_najadum_R1399

Platyceps_collaris_Turkey_8197

Coluber_rubriceps_thracicus_CRT14

Platyceps_collaris_Israel_TAUR16927

Platyceps_collaris_Turkey_8200

Platyceps_collaris_Turkey_8199

100

Phylogeography of the collared dwarf racer

Supplementary Materials – 3

Figure S2. Phylogenetic tree of Platyceps collaris resulting from the BI analysis of all genes

concatenated. Posterior probability values ≥ 0.90 are shown above branches. Type specimens

of Coluber rubriceps thracius are in red.

0.02

Platyceps_collaris_Turkey_8203

Platyceps_collaris_Turkey_8209

Coluber_rubriceps_thracicus_CRT12

Telescopus_obtusus_TMHC841

Platyceps_najadum_R1399

Platyceps_collaris_Turkey_8205

Platyceps_collaris_Turkey_8202

Platyceps_collaris_Israel_Schatti2

Platyceps_collaris_Israel_R649

Platyceps_collaris_Israel_Schatti1

Coluber_rubriceps_thracicus_CRT06

Platyceps_collaris_Israel_TAUR16921

Coluber_rubriceps_thracicus_CRT04

Platyceps_collaris_Turkey_8207

Telescopus_fallax_syriacus_2012_103

Coluber_rubriceps_thracicus_CRT15

Platyceps_collaris_Turkey_8206

Platyceps_collaris_Syria_JIR187

Platyceps_collaris_Bulgaria_1417

Platyceps_collaris_Bulgaria_7739

Platyceps_collaris_Turkey_8208

Platyceps_collaris_Turkey_8201

Platyceps_collaris_Bulgaria_6132

Coluber_rubriceps_thracicus_CRT11

Coluber_rubriceps_thracicus_CRT02

Platyceps_collaris_Turkey_8199

Coluber_rubriceps_thracicus_CRT14

Platyceps_collaris_Bulgaria_1405

Platyceps_collaris_Turkey_8197

Platyceps_collaris_Turkey_8204

Platyceps_collaris_Bulgaria_DJ607

Coluber_rubriceps_thracicus_CRT13

Platyceps_collaris_Israel_TAUR15955

Platyceps_collaris_Israel_2478

Telescopus_dhara_CN10774

Telescopus_pulcher_TMHC843

Platyceps_collaris_Turkey_8198

Platyceps_collaris_Turkey_8200

Platyceps_collaris_Israel_TAUR16927

Coluber_rubriceps_thracicus_CRT01

Platyceps_collaris_Jordan_Nagy

Platyceps_collaris_Syria_JIR188

0.94

Phylogeography of the collared dwarf racer

Supplementary Materials – 4

Figure S3. Phylogenetic tree of Platyceps collaris resulting from the ML analysis of the three

mitochondrial genes concatenated. Bootstrap support values ≥ 60 are shown above branches.

Type specimens of Coluber rubriceps thracius are in red.

0.05

Platyceps_collaris_Turkey_8206

Platyceps_collaris_Israel_TAUR16921

Platyceps_collaris_Israel_Schatti2

Platyceps_collaris_Bulgaria_7739

Platyceps_collaris_Turkey_8199

Platyceps_collaris_Bulgaria_6132

Platyceps_collaris_Turkey_8204

Coluber_rubriceps_thracicus_CRT14

Platyceps_collaris_Israel_R649

Telescopus_obtusus_TMHC841

Platyceps_collaris_Israel_TAUR16927

Platyceps_collaris_Bulgaria_1405

Platyceps_collaris_Turkey_8209

Platyceps_collaris_Turkey_8208

Platyceps_collaris_Syria_JIR187

Platyceps_collaris_Bulgaria_1417

Platyceps_collaris_Turkey_8203

Coluber_rubriceps_thracicus_CRT13

Platyceps_collaris_Turkey_8201

Telescopus_pulcher_TMHC843

Telescopus_dhara_CN10774

Coluber_rubriceps_thracicus_CRT02

Coluber_rubriceps_thracicus_CRT01

Platyceps_collaris_Turkey_8200

Platyceps_collaris_Jordan_Nagy

Platyceps_najadum_R1399

Platyceps_collaris_Turkey_8202

Platyceps_collaris_Turkey_8205

Coluber_rubriceps_thracicus_CRT11

Platyceps_collaris_Bulgaria_DJ607

Coluber_rubriceps_thracicus_CRT15

Platyceps_collaris_Israel_Schatti1

Coluber_rubriceps_thracicus_CRT12

Platyceps_collaris_Syria_JIR188

Coluber_rubriceps_thracicus_CRT06

Coluber_rubriceps_thracicus_CRT04

Platyceps_collaris_Turkey_8207

Platyceps_collaris_Turkey_8197

Platyceps_collaris_Israel_2478

Telescopus_fallax_syriacus_2012_103

Platyceps_collaris_Turkey_8198

Platyceps_collaris_Israel_TAUR15955

100

Phylogeography of the collared dwarf racer

Supplementary Materials – 5

Figure S4. Phylogenetic tree of Platyceps collaris resulting from the BI analysis of the three

mitochondrial genes concatenated. Posterior probability values ≥ 0.90 are shown above

branches. Type specimens of Coluber rubriceps thracius are in red.

0.02

Coluber_rubriceps_thracicus_CRT13

Platyceps_collaris_Bulgaria_1417

Coluber_rubriceps_thracicus_CRT04

Platyceps_najadum_R1399

Platyceps_collaris_Turkey_8200

Platyceps_collaris_Turkey_8202

Platyceps_collaris_Israel_Schatti2

Platyceps_collaris_Israel_Schatti1

Platyceps_collaris_Turkey_8198

Platyceps_collaris_Turkey_8207

Platyceps_collaris_Israel_TAUR15955

Coluber_rubriceps_thracicus_CRT06

Platyceps_collaris_Turkey_8203

Telescopus_fallax_syriacus_2012_103

Platyceps_collaris_Bulgaria_6132

Platyceps_collaris_Bulgaria_DJ607

Platyceps_collaris_Turkey_8197

Telescopus_pulcher_TMHC843

Coluber_rubriceps_thracicus_CRT01

Platyceps_collaris_Turkey_8199

Coluber_rubriceps_thracicus_CRT12

Platyceps_collaris_Bulgaria_1405

Platyceps_collaris_Turkey_8206

Coluber_rubriceps_thracicus_CRT14

Platyceps_collaris_Turkey_8205

Platyceps_collaris_Turkey_8209

Coluber_rubriceps_thracicus_CRT15

Platyceps_collaris_Turkey_8201

Platyceps_collaris_Syria_JIR187

Platyceps_collaris_Bulgaria_7739

Platyceps_collaris_Israel_2478

Telescopus_dhara_CN10774

Coluber_rubriceps_thracicus_CRT11

Platyceps_collaris_Israel_R649

Telescopus_obtusus_TMHC841

Platyceps_collaris_Turkey_8208

Platyceps_collaris_Israel_TAUR16921

Coluber_rubriceps_thracicus_CRT02

Platyceps_collaris_Israel_TAUR16927

Platyceps_collaris_Jordan_Nagy

Platyceps_collaris_Syria_JIR188

Platyceps_collaris_Turkey_8204

0.93

Phylogeography of the collared dwarf racer

Supplementary Materials – 6

Figure S5. Response curves of the environmental variables used to develop the distribution

model showing how each variable affected the prediction and its relative contribution to the

model (averaged over 10 runs). Each plot shows the probability of the species’ presence (y axis)

as a function of the environmental predictor value (x axis). Red lines show mean responses of

ten replicate runs, standard deviations are in blue. The legend in the lower right refers to the

categorical land cover variable in the lower left.

Phylogeography of the collared dwarf racer

Supplementary Materials – 7

Figure S6. Geographic distribution of the Eastern Mediterranean conifer-broadleaf forest

ecoregion (Dinerstein et al., 2017).

Phylogeography of the collared dwarf racer

Supplementary Materials – 8

Table S1. Locality details of records used for the SDM and their original sources. Coordinates

from Bulgaria have been truncated to two decimal places for conservation purposes.

Country

Locality

Latitude

Longitude

Source

Bulgaria

Rezovo

27.99

smartbirds.org

Bulgaria

Sinemorec

42.03

27.95

This study

Bulgaria

42.04

27.99

This study

Bulgaria

Sinemorets

42.05

27.98

smartbirds.org

Bulgaria

Sinemorets

42.06

27.92

This study

Bulgaria

Ahtopol

42.06

27.95

smartbirds.org

Bulgaria

Brodilovo

42.11

27.83

smartbirds.org

Bulgaria

Tsarevo

42.17

27.83

smartbirds.org

Bulgaria

42.18

27.75

This study

Bulgaria

Velika

42.19

27.75

smartbirds.org

Bulgaria

42.19

27.8

This study

Bulgaria

Primorsko

42.28

27.74

smartbirds.org

Bulgaria

42.28

27.75

This study

Bulgaria

Primorsko

42.29

27.74

iNaturalist

Bulgaria

42.3

27.69

This study

Bulgaria

Ropotamo

42.3

27.72

Balcanica.info

Bulgaria

Primorsko

42.3

27.74

smartbirds.org

Bulgaria

42.31

27.72

This study

Bulgaria

Primorsko

42.31

27.77

smartbirds.org

Bulgaria

Arkutino

42.33

27.72

Rehák (1985)

Bulgaria

Sozopol

42.37

27.69

smartbirds.org

Bulgaria

Sozopol

42.39

27.68

This study

Bulgaria

Sozopol

42.39

27.69

This study

Bulgaria

Sveti vlas

42.71

27.75

This study

Bulgaria

Elenite

42.71

27.81

Panner (2009)

Bulgaria

Sveti Vlas

42.72

27.75

smartbirds.org

Bulgaria

Sveti Vlas

42.72

27.76

This study

Bulgaria

42.73

27.71

This study

Israel

31.61406

34.92282

GBIF

Israel

31.67055

34.57677

GBIF

Israel

31.7424

35.10922

GBIF

Israel

31.74498

35.00052

GBIF

Israel

31.75104

34.87807

GBIF

Israel

31.7621

34.98781

GBIF

Israel

31.76345

34.80309

GBIF

Israel

Jerusalem

31.79367

35.21999

GBIF

Israel

Mt. Scopus

31.79447

35.2439

Schatti et al. (2001)

Israel

31.79454

34.97507

GBIF

Israel

31.91965

34.86479

GBIF

Israel

Southern Coastal Plain, Nezer Sereni

31.922

34.822

This study

Israel

31.93234

34.89118

GBIF

Israel

Miqwe Yisrael

32.0292

34.7814

Schatti et al. (2001)

Israel

32.15777

34.88299

GBIF

Israel

Haifa

32.5241

35.1407

GBIF

Israel

32.58816

35.00289

GBIF

Israel

32.61228

35.5153

GBIF

Israel

32.62401

35.51427

GBIF

Israel

32.66846

35.03896

GBIF

Israel

Mt. Tabor

32.683

35.4

Schatti et al. (2001)

Israel

Yizre’el (Jezreel) Valley, Qiryat Tiv'on

32.72

35.14

This study

Israel

32.83671

35.8023

iNaturalist

Israel

HaZafon

32.89259

35.09821

GBIF

Israel

32.90455

35.74612

GBIF

Israel

32.90545

35.74613

GBIF

Israel

32.91079

35.7622

GBIF

Israel

32.91084

35.74937

GBIF

Israel

32.91266

35.74617

GBIF

Israel

32.93816

35.79532

iNaturalist

Israel

32.97109

35.54115

GBIF

Israel

33.03372

35.30589

GBIF

Israel

HaZafon

33.17679

35.63402

GBIF

Israel

Golan

33.18039

35.77118

GBIF

Israel

HaZafon

33.241

35.65568

GBIF

Israel

33.24576

35.64843

GBIF

Israel

33.24844

35.65381

GBIF

Israel

33.24897

35.74609

GBIF

Israel

33.28952

35.75171

GBIF

Israel

33.29223

35.75173

GBIF

Israel

Golan

33.29272

35.75448

GBIF

Israel

33.29328

35.75557

iNaturalist

Israel

Hermon

33.29599

35.76239

This study

Israel

33.30293

35.77864

GBIF

Israel

33.30927

35.77116

GBIF

Jordan

Dilāghah

30.13333

35.4

Amr & Disi (2011)

Jordan

Ma‘an

30.2

35.73333

Disi et al. (2001)

Jordan

Ayl

30.21667

35.53333

Amr & Disi (2011)

Jordan

Petra

30.3333

35.4333

Disi et al. (2001)

Jordan

Ash Shawbak

30.52

35.53833

Amr & Disi (2011)

Phylogeography of the collared dwarf racer

Supplementary Materials – 9

Country

Locality

Latitude

Longitude

Source

Jordan

Ash Shawbak

30.5333

35.5667

Disi et al. (2001)

Jordan

Aṭ Ṭafīla

30.83333

35.6

Amr & Disi (2011)

Jordan

Al Mazār

30.97722

35.85889

Amr & Disi (2011)

Jordan

Al Karak

31.1667

35.75

Disi et al. (2001)

Jordan

Al Karak

31.18472

35.70472

Amr & Disi (2011)

Jordan

Rākīn

31.22389

35.70667

Amr & Disi (2011)

Jordan

Batīr

31.26417

35.70472

Amr & Disi (2011)

Jordan

‘Ammān

31.96667

35.98333

Amr & Disi (2011)

Jordan

Al Jubayhah

32.01667

35.86667

Disi et al. (2001)

Jordan

Şuwayliḩ

32.025

35.83806

Amr & Disi (2011)

Jordan

Yājūz

32.03333

35.91667

Amr & Disi (2011)

Jordan

Salīhī

32.12278

35.83111

Amr & Disi (2011)

Jordan

Dayr ‘Allā

32.19778

35.62111

Amr & Disi (2011)

Jordan

Ibbin

32.36667

35.81667

Disi et al. (2001)

Jordan

Dayr Abu Sa’id

32.5

35.68333

Disi et al. (2001)

Syria

E of Utaibeh, Lake shore

33.50606

36.6274

This study

Syria

2 km N of Seydnaya

33.7173

36.3717

This study

Turkey

Bozyazı-Anamur/Mersin

36.09666

33.04152

Aydın Adnan Menderes University

Turkey

Yılan adası, Kalkan - Antalya

36.21502

29.35472

Dokuz Eylül University

Turkey

Yilan Adasi, Kalkan

36.21517

29.35585

Schatti et al. (2001)

Turkey

Erdemli/Mersin

36.63865

34.34009

Aydın Adnan Menderes University

Turkey

Kaldırım Village-Yumurtalık/Adana

36.67375

35.52499

Aydın Adnan Menderes University

Turkey

Waldgebiet bei Aydolun

36.752

31.774

Kucharzewski (2016)

Turkey

Küplüce Village-Kilis

36.75719

37.23555

Aydın Adnan Menderes University

Turkey

İztuzu Kuzeyi - Dalyan - Muğla

36.78493

28.62834

Dokuz Eylül University

Turkey

Kızılburun Mevkii - Köyceğiz - Muğla

36.80226

28.52348

Dokuz Eylül University

Turkey

Çandır-Köyceğiz

36.82751

28.60819

Dokuz Eylül University

Turkey

Ekincik- Köyceğiz

36.83261

28.55059

Dokuz Eylül University

Turkey

Toroslar, Mersin

36.83932

34.61363

Dokuz Eylül University

Turkey

Ceyhan (Botaş)/Adana

36.89531

35.91594

Aydın Adnan Menderes University

Turkey

Kavak arası Köyceğiz

36.89906

28.72029

Dokuz Eylül University

Turkey

Toparlar Kuzeyi - Köyceğiz - Muğla

36.98732

28.6671

Dokuz Eylül University

Turkey

Gökova - Muğla

37.05563

28.36512

Dokuz Eylül University

Turkey

Beşkonak - Antalya

37.1403

31.19879

Dokuz Eylül University

Turkey

Ömerli Village-Pozantı/Adana

37.53545

34.87516

Aydın Adnan Menderes University

Turkey

Karina und Umgebung

37.632

27.11756

GBIF

Turkey

Işıklı Village/Aydın

37.832

27.7987

Aydın Adnan Menderes University

Turkey

Suna Plateau-Yahyalı/Kayseri

37.99278

35.39736

Aydın Adnan Menderes University

Turkey

Obruk şelalesi - Saimbeyli - Adana

38.001

36.09318

Dokuz Eylül University

Turkey

Gürümze Village-Feke/Adana

38.04202

35.81172

Aydın Adnan Menderes University

Turkey

Temürağa Village-Göksun/Kahramanmaraş

38.0458

36.58581

Aydın Adnan Menderes University

Turkey

10 km North of Saimbeyli/Adana

38.06065

36.14885

Aydın Adnan Menderes University

Turkey

Gümüldür, İzmir

38.0775

27.01762

Dokuz Eylül University

Turkey

Bozdağ-Ödemiş/İzmir

38.36708

28.10343

Aydın Adnan Menderes University

Turkey

Menemen, İzmir

38.60313

27.07201

Dokuz Eylül University

Turkey

Tahtakuşlar - Kaşdağ

39.59337

26.85549

Dokuz Eylül University

Turkey

Yeniköy - Tekirdağ

40.64698

26.99938

Dokuz Eylül University

Turkey

Orhaniye village, Kocaeli

40.85767

30.0669

Afsar et al. (2013)

West Bank

31.62698

35.12515

GBIF

West Bank

31.78608

35.3019

GBIF

Phylogeography of the collared dwarf racer

Supplementary Materials – 10

References for Supplementary Figures and Table S1.

Afsar, M., Cicek, K., Dincaslan, Y. E., Ayaz, D., & Tok, C. V. (2013). New record localities

of five snake species in Turkey. Herpetozoa, 25, 179–183.

Amr, Z. S., & Disi, A. M. (2011). Systematics, distribution and ecology of the snakes of

Jordan. Vertebrate Zoology, 61, 179–266.

Dinerstein, E., Olson, D., Joshi, A., Vynne, C., Burgess, N.D., Wikramanayake, E., Hahn, N.,

Palminteri, S., Hedao, P., Noss, R., Hansen, M., Locke, H., Ellis, E.C., Jones, B.,

Barber, C.V., Hayes, R., Kormos, C., Martin, V., Crist, E., Sechrest, W., Price, L.,

Baillie, J.E.M., Weeden, D., Suckling, K., Davis, C., Sizer, N., Moore, R., Thau, D.,

Birch, T., Potapov, P., Turubanova, S., Tyukavina, A., de Souza, N., Pintea, L., Brito,

J.C., Llewellyn, O.A., Miller, A.G., Patzelt, A., Ghazanfar, S.A., Timberlake, J.,

Klöser, H., Shennan-Farpón, Y., Kindt, R., Lillesø, J.-P.B., van Breugel, P., Graudal,

L., Voge, M., Al-Shammari, K.F., Saleem, M., 2017. An ecoregion-based approach to

protecting half the terrestrial realm. Bioscience 67, 534–545.

Disi, A. M., Modrý, D., Necas, P., & Rifai, L. (2001). Amphibians and reptiles of the

Hashemite Kingdom of Jordan. Frankfurt am Main: Edition Chimaira.

Kucharzewski, Ch. (2016). Weiterer Beitrag zur Herpetologie der Südwest-Türkei. Sauria,

38, 37–56.

Panner, T. (2009). Bemerkungen zur Herpetofauna des Emine-Balkan mit neuem nördlichsten

Verbreitungsnachweis für Platyceps collaris in Bulgarien. Elaphe, 17, 48–49.

Rehák, I. (1985). Coluber rubriceps thracius ssp. n. from Bulgaria (Reptilia: Squamata:

Colubridae). Věstník Československé společnosti zoologické, 49, 276–282.

Schätti, B., Baran, I., & Maunoir, P. (2001). Taxonomie, Morphologie und Verbreitung der

Masken-Schlanknatter Coluber (s. l.) collaris (Müller, 1878). Revue Suisse de

Zoologie, 108, 11–30.

A new, rare, small-ranged, and endangered mountain snake of the genus Elaphe from the Southern Levant

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Full-text available

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Apr 2023

Zlatozar Nikolaev Boev

This study presents for the first time summarized data on 759 species/taxa (628 species at least) of six classes of Quaternary vertebrates of Bulgaria: Chondrichthyes (1); Actinopterygii (34); Amphibia (18); Reptilia (33); Aves (299); and Mammalia (374). The richest fauna has been recorded in the Late Pleistocene (285 species), followed by the Calabrian (255). Bulgaria has lost 32.3% of its former total Quaternary vertebrate fauna. The number of the lost taxa is as follows: species (245), genera (80), families (16), orders (5), of them three mammalian (Perissodactyla, Proboscidea, and Primates), and two avian (Otidiformes and Pteroclidiformes). Extinct families are: one amphibian (Palaeobatrachidae); two reptilian (Varanidae and Elapidae); three avian (Gruidae, Otididae, and Pteroclididae), and ten mammalian (Dipodidae, Eomyidae, Hystricidae, Ochotonidae, Hyaenidae, Phocidae, Equidae, Rhinocerotidae, Elephantidae, and Cercopithecidae). After the small mammals (mainly Cricetidae; 52 taxa), the composition of bovids (27 taxa) and canids (13 taxa) impoverished in a higher extent. The biggest number of recorded vertebrate families is found in the Meghalayan (79), followed by the Greenlandian (63) and the Late Pleistocene (62). At order and family levels, the most varied was the vertebrate fauna in the Meghalayan (39 orders, 79 families). In the Calabrian, the number of genera was a three times greater than in the Northgrippian, which indicates more diversified paleoenvironment. One genus, 25 species, and one subspecies have been described as new to the science from the Quaternary localities in Bulgaria.

Biogeography of Angolan rodents: The first glimpse based on phylogenetic evidence

Article

Full-text available

Oct 2021
DIVERS DISTRIB

Aim Assessment of phylogenetic diversity and biogeographical affinities of the rodent fauna from one of the most neglected areas in Africa. Location Angola (with biogeographical implications in adjacent areas). Methods Inference of mitochondrial phylogenies for rodent genera occurring in Angola, delimitation of molecular operational taxonomic units (MOTUs), assessment and comparative analysis of their geographical distribution. Results We provide the first genetic evidence for the presence of 44 rodent taxa from 19 genera and 5 families in Angola, including twelve MOTUs endemic to Angola, and 12 candidate new species, pending integrative taxonomic revisions. The endemic MOTUs were found almost exclusively in the Angolan Miombo Woodlands and in Angolan montane forest−grassland mosaic. Main conclusions The highly diverse Angolan rodent fauna is mostly composed of and shows affinities with taxa originating from three major biogeographical regions of sub‐Saharan Africa (Zambezian, South African and Congolian). It is also composed of a unique fauna comprising palaeoendemics from the Angolan Highlands. The species richness and the endemism detected in the Angolan montane forest−grassland mosaic and in the Angolan escarpment forests suggest that these are relevant for conservation, but more studies including other biological groups are needed to fundament this.

Morphological and genetic differentiation in the anguid lizard Pseudopus apodus supports the existence of an endemic subspecies in the Levant

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Apr 2021

The Levant represents one of the most important reptile diversity hotspots and centers of endemism in the Western Palearctic. The region harbored numerous taxa in glacial refugia during the Pleistocene climatic oscillations. Due to the hostile arid conditions in the warmer periods they were not always able to spread or come into contact with populations from more distant regions. One large and conspicuous member of the Levantine herpetofauna is the legless anguid lizard Pseudopus apodus. This species is distributed from the Balkans to Central Asia with a portion of its range running along the eastern Mediterranean coast. Mitochondrial and nuclear DNA sequences, microsatellite genotypes, and morphology show that populations in this region differ from the two named subspecies and presumably had a long independent evolutionary history during the Quaternary. Here we describe the Levantine population as a new subspecies and present biogeographic scenarios for its origin and diversification. The new subspecies is genetically highly diverse, and it forms a sister lineage to Pseudopus from the remaining parts of the range according to mtDNA. It is the largest-bodied of the three subspecies, but occupies the smallest range.

Coluber rubriceps thracius ssp. n. from Bulgaria (Reptilia: Squamata: Colubridae)

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Full-text available

Jan 1985

Ivan Rehak

Coluber rubriceps thracius ssp. n. from Bulgaria is described on the basis of differences in its external morphology from the Asian specimens. Characteristic of the new subspecies are a relatively small number both of ventrals and subcaudals and a relatively shorter tail, in addition to other minute differences in a larger number of praeoculars and a smaller number of upper labials. As to sexual dimorphism, the tail of the male is relatively longer, and the number of ventrals smaller than in the female. A new faunistic record (Akhtopol) was added to its area of distribution so far known.

Ecological Niches and Geographic Distributions (MPB-49)

Book

Full-text available

Nov 2011

This book provides a first synthetic view of an emerging area of ecology and biogeography, linking individual- and population-level processes to geographic distributions and biodiversity patterns. Problems in evolutionary ecology, macroecology, and biogeography are illuminated by this integrative view. The book focuses on correlative approaches known as ecological niche modeling, species distribution modeling, or habitat suitability modeling, which use associations between known occurrences of species and environmental variables to identify environmental conditions under which populations can be maintained. The spatial distribution of environments suitable for the species can then be estimated: a potential distribution for the species. This approach has broad applicability to ecology, evolution, biogeography, and conservation biology, as well as to understanding the geographic potential of invasive species and infectious diseases, and the biological implications of climate change. The book lays out conceptual foundations and general principles for understanding and interpreting species distributions with respect to geography and environment. Focus is on development of niche models. While serving as a guide for students and researchers, the book also provides a theoretical framework to support future progress in the field.

Taxonomic Evaluation of Coluber rubriceps (Venzmer, 1919) From Bulgaria

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Full-text available

Oct 1986

Ivan Rehak

An examination has been made of the external morphology of C. rubriceps from the southern part of the Bulgarian Black Sea coast, and of C. rubriceps from Israel. Several notes on bionomics are given.

MEGA7: Molecular Evolutionary Genetics Analysis Version 7.0 for Bigger Datasets

Article

Full-text available

Jul 2016

We present the latest version of the Molecular Evolutionary Genetics Analysis (MEGA) software, which contains many sophisticated methods and tools for phylogenomics and phylomedicine. In this major upgrade, MEGA has been optimized for use on 64-bit computing systems for analyzing bigger datasets. Researchers can now explore and analyze tens of thousands of sequences in MEGA. The new version also provides an advanced wizard for building timetrees and includes a new functionality to automatically predict gene duplication events in gene family trees. The 64-bit MEGA is made available in two interfaces: graphical and command line. The graphical user interface (GUI) is a native Microsoft Windows application that can also be used on Mac OSX. The command line MEGA is available as native applications for Windows, Linux, and Mac OSX. They are intended for use in high-throughput and scripted analysis. Both versions are available from www.megasoftware.net free of charge.

The biogeography of Elaphe sauromates (Pallas, 1814), with a description of a new rat snake species

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May 2019

Background: The rat snake genus Elaphe once comprised several dozens of species distributed in temperate through tropical zones of the New and Old World. Based on molecular-genetic analyses in early 2000s, the genus was split into several separate genera, leaving only 15 Palearctic and Oriental species as its members. One of the three species also occurring in Europe is Elaphe sauromates, a robust snake from the Balkans, Anatolia, Caucasus, Ponto-Caspian steppes, and Levant that has been suspected to be composed of two or more genetically diverse populations. Here, we studied the genetic structure and morphological variation of E. sauromates, aiming to better understand its inter-population relationships and biogeography, and subsequently revise its taxonomy. Methods: We reconstructed the phylogeography and analyzed the genetic structure of E. sauromates populations originating from most of its geographic range using both mitochondrial (COI, ND4) and nuclear (C-MOS, MC1R, PRLR, RAG1) DNA gene fragments. We employed Maximum likelihood and Bayesian inference methods for the phylogenetic tree reconstructions, supplemented with species delimitation methods, analysis of haplotype networks, and calculation of uncorrected p-distances. Morphological variation in 15 metric and 18 meristic characters was studied using parametric univariate tests as well as multivariate general linearized models. In total, we analyzed sequences originating from 63 specimens and morphological data from 95 specimens of E. sauromates sensu lato. Results: The molecular phylogeny identiﬁed two clearly divergent sister lineages within E. sauromates, with both forming a lineage sister to E. quatuorlineata. The genetic distance between them (5.80–8.24% in mtDNA) is similar to the distances among several other species of the genus Elaphe. Both lineages are also moderately morphologically differentiated and, while none of the characters are exclusively diagnostic, their combination can be used for conﬁdent lineage identiﬁcation. Here, following the criteria of genetic and evolutionary species concepts, we describe the lineage from eastern Anatolia and parts of the Lesser and Great Caucasus as a new species E. urartica sp. nov. Discussion: Elaphe urartica sp. nov. represents a cryptic species whose ancestors presumably diverged from their common ancestor with E. sauromates around the Miocene-Pliocene boundary. The intraspeciﬁc genetic structure indicates that the recent diversity of both species has been predominantly shaped by Pleistocene climatic oscillations, with glacial refugia mainly located in the Balkans, Crimea, and/or Anatolia in E. sauromates and Anatolia and/or the Caucasus in E. urartica sp. nov.

PaleoClim, high spatial resolution paleoclimate surfaces for global land areas

Article

Full-text available

Nov 2018

High-resolution, easily accessible paleoclimate data are essential for environmental, evolutionary, and ecological studies. The availability of bioclimatic layers derived from climatic simulations representing conditions of the Late Pleistocene and Holocene has revolutionized the study of species responses to Late Quaternary climate change. Yet, integrative studies of the impacts of climate change in the Early Pleistocene and Pliocene – periods in which recent speciation events are known to concentrate – have been hindered by the limited availability of downloadable, user-friendly climatic descriptors. Here we present PaleoClim, a free database of downscaled paleoclimate outputs at 2.5-minute resolution (~5 km at equator) that includes surface temperature and precipitation estimates from snapshot-style climate model simulations using HadCM3, a version of the UK Met Office Hadley Centre General Circulation Model. As of now, the database contains climatic data for three key time periods spanning from 3.3 to 0.787 million years ago: the Marine Isotope Stage 19 (MIS19) in the Pleistocene (~787 ka), the mid-Pliocene Warm Period (~3.264–3.025 Ma), and MIS M2 in the Late Pliocene (~3.3 Ma).

Phylogeography and taxonomy of the barred grass snake (Natrix helvetica), with a discussion of the subspecies category in zoology

Article

Full-text available

Nov 2018

Using 12 microsatellite loci, mitochondrial DNA sequences and previously published morphological evidence, we examined the phylogeographic and taxonomic structure of Natrix helvetica. Our results support tentatively the recognition of five subspecies: (1) N. h. helvetica (Lacepède, 1789) from Western Europe, (2) N. h. cetti Gené, 1839 from Sardinia, (3) N. h. corsa (Hecht, 1930) from Corsica, (4) N. h. lanzai Kramer, 1970 from the Po drainage and peninsular Italy, except for parts of Calabria and perhaps Apulia, and (5) N. h. sicula (Cuvier, 1829) from Sicily and parts of Calabria. The status of the subspecies from Corsica and Sardinia warrants further research. Grass snakes from the two islands are genetically deeply divergent from their continental and Sicilian conspecifics. However, the mitochondrial haplotypes of N. h. cetti and N. h. corsa are not reciprocally monophyletic, and our analyses of microsatellite data did not unambiguously support their distinctness. Natrix helvetica lanzai comprises three mitochondrial lineages and could represent more than one taxon. Grass snakes of the northern mitochondrial lineage of N. h. lanzai (mainly from the Po drainage system) were previously assigned to N. h. helvetica. We propose that the recognition of subspecies within N. helvetica reflects its genetic differentiation best. Our subspecies concept resembles the definition of Evolutionarily Significant Units (ESUs) in that subspecies should be ideally confirmed by two independent genetic lines of evidence (mtDNA, nuclear genomic markers). In contrast to species, subspecies are still fully capable of extensive gene flow and may disappear in the evolutionary process as a consequence of secondary contact. Thus, subspecies represent an early stage of (incomplete) speciation. Using the subspecies category facilitates communication within and beyond science (legislation, conservation), a clear advantage compared to the cumbersome concept of ESUs.

The species identity and biogeography of Blanus (Amphisbaenia: Blanidae) in Lebanon

Article

Jul 2019

The genus Blanus Wagler, 1830 represents limbless, burrowing reptiles of the family Blanidae with disjunct circum-Mediterranean distribution. The recently described species Blanus alexandri Sindaco, Kornilios, Sacchi & Lymberakis, 2014 is known from south-eastern Turkey with a presumed occurrence in the Levant and Iraq. We provide here records from Lebanon and confirm the affiliation of Lebanese populations to this species by mitochondrial and nuclear DNA. Blanus alexandri comprises at least seven deeply evolved phylogenetic clades with up to 11% of uncorrected p-distances in their mitochondrial DNA. This suggests a probably older than the Miocene origin of some of these clades. Populations from Lebanon form a different clade with a genetic diversity that is close to populations from southern Turkey.

Ancient diversification, biogeography, and the role of climatic niche evolution in the Old World cat snakes (Colubridae, Telescopus)

Article

Jan 2019

Genome‐wide markers untangle the green‐lizard radiation in the Aegean Sea and support a rare biogeographical pattern

Article

Jan 2019

Aim The Aegean Sea constitutes a major biogeographic barrier between the European and Asian continents and several models of diversification in the Aegean have been documented. Here, we test three of those models for the Aegean green‐lizards (Lacerta trilineata–pamphylica group): Vicariance versus Overland Dispersal versus Island Stepping‐stone Dispersal. We investigate these hypotheses and complement our knowledge on the impact of the Aegean Barrier on east Mediterranean taxa. Location Aegean Sea, east Mediterranean. Taxon Lacerta lizards. Methods We analysed ddRAD loci (double‐digest restriction‐site‐associated DNA) to estimate species‐trees under coalescent models and maximum likelihood trees using concatenation. We performed hierarchical population structure analyses and inferred ancestral distribution‐areas. We also sequenced the complete cytochrome b gene and produced a time‐calibrated mtDNA gene‐tree to conduct a critical comparison with previous studies. Results Aegean green‐lizards diverged into four main groups in parallel during the Late Pliocene with distributions to the East and West of the Aegean. The Eastern group includes Lacerta pamphylica and East Aegean L. trilineata, while the Western group contains the Central Cyclades populations and the remaining populations of the Balkan Peninsula. The Aegean green‐lizards’ ancestor occurred in Anatolia, while the West lineage ancestor occurred in the Central Cyclades islands, revealing a dispersal between the two regions. The radiations of all major green‐lizard groups, including trilineata+pamphylica, occurred in parallel in the Late Pliocene. Main Conclusions In contrast to previously suggested biogeographical hypotheses for the group, based on mtDNA, the Island Stepping‐stone Dispersal scenario is strongly supported. Green lizards offer a rare paradigm of diversification in the Aegean, where populations largely expanded their geographical distribution and crossed the Aegean Barrier using the central Aegean islands as stepping stones.

Quaternary range dynamics and taxonomy of the Mediterranean collared dwarf racer, Platyceps collaris (Squamata: Colubridae)

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