Zootaxa 4033 (3): 380–392 ISSN 1175-5326 (print edition) www.mapress.com/zootaxa/ Article ZOOTAXA Copyright © 2015 Magnolia Press ISSN 1175-5334 (online edition) http://dx.doi.org/10.11646/zootaxa.4033.3.4 http://zoobank.org/urn:lsid:zoobank.org:pub:0AE71BF4-A095-473F-9F61-E2766DC77442 Phylogeny of the Rhynchocalamus (Reptilia; ) with a first record from the Sultanate of Oman

JIŘÍ ŠMÍD1, GABRIEL MARTÍNEZ2, JURGEN GEBHART3, JAVIER AZNAR4, JAVIER GÁLLEGO5, BAYRAM GÖÇMEN6, PHILIP DE POUS7,8, KARIN TAMAR9 & SALVADOR CARRANZA8,10 1Department of Zoology, National Museum, Cirkusová 1740, Prague, Czech Republic 2C/ Pedro Antonio de Alarcón nº 34, 5º A, 18002, Granada, Spain 3Welfenstr. 13, 86879 Wiedergeltingen, Germany 4C/ Uruguay nº16, 3ºA, 28016 Madrid, Spain 5Carretera motores, 211, 04720 Aguadulce, Almeria, Spain 6Department of Biology, Zoology Section, Faculty of Science, Ege University, TR 35100 Bornova-Izmir, Turkey 7Faculty of Life Sciences and Engineering, Departament de Producció (Fauna Silvestre), Universitat de Lleida, E-125198, Lleida, Spain 8Institute of Evolutionary Biology (CSIC-Universitat Pompeu Fabra), Passeig Maritim de la Barceloneta 37-49, 08003 Barcelona, Spain 9Department of Zoology, Tel-Aviv University, Tel-Aviv 6997801, Israel 10Corresponding author. E-mail: [email protected]

Abstract

The genus Rhynchocalamus comprises three distributed in Southwest Asia. Little is known about them, most prob- ably because of their secretive fossorial lifestyle. The poor knowledge of the genus is even underscored by the fact that its phylogenetic affinities remained unclear until very recently. The least known of the species, Rhynchocalamus arabicus, is known only from the holotype collected in Aden, Yemen, and it has not been observed since its description in 1933. Here we provide a second record for this species, which represents the first record of this genus for Oman. This extends its range in southern Arabia by more than 1000 km. The observed specimen was determined as R. arabicus on the basis of its similarity in size, color, and scalation with the holotype. Furthermore, we sequenced three mitochondrial (12S, 16S, cytb) and one nuclear (cmos) genes for R. arabicus and for two individuals of R. melanocephalus and one R. satunini and inferred the phylogenetic relationships of all currently recognized species of the genus for the first time. The results of our phylogenetic analyses indicate that Rhynchocalamus is a member of the Western Palearctic clade of and is sis- ter to Lytorhynchus, with which it forms a very well supported clade and shares some morphological characters. As our results show, R. satunini is the basal lineage of the genus and R. melanocephalus is sister to R. arabicus.

Key words: Anatolia, Arabia, Colubrinae, Kukri , Levant, mitochondrial DNA, nuclear DNA, Rhynchocalamus ar- abicus, R. melanocephalus, R. satunini

Introduction

The genus Rhynchocalamus Günther is part of the family Colubridae Oppel and includes only three species of small opisthoglyphous endemic to Southwest Asia—R. arabicus Schmidt, R. melanocephalus Jan and R. satunini Nikolsky. All are characterized by having smooth dorsal scales in 15 rows, small head not distinct from the neck, rather small eyes with round pupils, rostral shield enlarged and wedged between the internasals, a frontal which is approximately half of the length of the parietals, reduced maxillary dentition with 6–8 maxillary teeth, the posterior ones being long (Jan 1862; Schmidt 1933). Our knowledge on the distribution of all three species is still scarce, being probably the result of their secretive fossorial lifestyle and mostly nocturnal activity (Disi et al. 2001; Amr and Disi 2011). The range of R. melanocephalus, the best known of all three species, spans from Sinai, Egypt through the Levant to southern Anatolia (Gasperetti 1988; Avci et al. 2008; Sindaco et al. 2013), R. satunini is

380 Accepted by Z. Nagy: 2 Oct. 2015; published: 23 Oct. 2015 distributed from southern Anatolia to Iran and R. arabicus is only known from a single specimen collected in Aden, South Yemen in 1932 and described in 1933 (Schmidt 1933; Gasperetti 1988; Fig. 1). The locality of R. arabicus is isolated by more than 2000 km from the remainder of the genus and the single specimen representing this species makes it one of the rarest snakes in the world, together with other 35 species that are known only from the holotype (Wallach et al. 2014). Virtually nothing is known about its distribution, population status, natural history or threats (Sindaco 2012). The phylogenetic position of the genus Rhynchocalamus remained entirely enigmatic until very recently. A recently published study by Avci et al. (2015) shows that Rhynchocalamus is a member of the Western Palearctic clade of Colubrinae sister to Lytorhynchus. The same study also shows that Muhtarophis barani, formerly part of Rhynchocalamus is an independent genus. Yet the phylogenetic position of the last species of the genus, R. arabicus, still remains unknown. During an expedition to Oman in spring 2013, a specimen morphologically similar to R. arabicus was found in Dhofar, representing the first record of this snake genus for the Sultanate. In this work we present the second-ever reported specimen of R. arabicus, including a morphological comparison with the holotype and a molecular comparison with R. melanocephalus and R. satunini, and reconstruct the phylogeny of the genus within the Palearctic colubrid genera.

Material and methods

Field study. An expedition to Oman was carried out by G.M, J.G., J.A. and J.G. between the 27th of April and the 5th of May 2013 with the objective of surveying the fauna of the Sultanate with an especial emphasis on snakes. Geographic coordinates were obtained with a handheld GPS (datum WGS84) for all the specimens sampled, together with photographs. Tissue samples (tail tips) were preserved in 95% ethanol and deposited in the reptile collection of the Institute of Evolutionary Biology (IBE), Barcelona, Spain. Scale counts and morphological measurements were taken directly in the field. All were released in the same collecting locality after manipulation. Molecular analyses. Material for the phylogenetic analyses included two vouchered samples of R. melanocephalus from Israel (TAUM 15269, Tel Aviv University Zoological Museum, Tel Aviv, Israel) and Syria (ZMHRU 2007–69, Zoological Museum of Harran University, Osmanbey, Sanliurfa, Turkey), one vouchered sample of R. satunini from Iran (CAS 228723, California Academy of Sciences, San Francisco, USA) and an unvouchered sample (code CN4780) of R. arabicus from Dhofar Governorate, Sultanate of Oman (for locality details see Table 1 and Fig. 1). Genomic DNA was extracted from ethanol-preserved tissue samples using the Qiagen DNeasy Blood & Tissue Kit and the following gene fragments were amplified and sequenced: 12S rRNA (12S, 617 bp), 16S rRNA (16S, 523 bp), cytochrome b (cytb, 996 bp) from the mitochondrial DNA (mtDNA) and cmos (459 bp) from the nuclear DNA (nDNA). Primers, primer sources, PCR programs, fragment lengths, numbers of variable and parsimony-informative sites are summarized in Appendix I. Chromatographs were assembled and sequence quality checked in Geneious v.6 (Biomatters, http://www.geneious.com). Heterozygous positions for the nuclear coding gene fragment were identified based on the presence of two peaks of approximately equal height at a single nucleotide site in both strands and were coded according to IUPAC ambiguity codes. DNA sequences were aligned for each gene independently using the online application of MAFFT v.7 (Katoh and Standley 2013) with default parameters (Auto strategy, Gap opening penalty: 1.53, Offset value: 0.0). For the 12S and 16S ribosomal fragment we applied the Q-INS-i strategy, in which information on the secondary structure of the RNA is considered. Poorly aligned positions of the non-protein-coding 12S and 16S genes were eliminated with Gblocks (Castresana 2000) with low stringency option selected (Talavera and Castresana 2007). Alignments of protein- coding genes (cytb, cmos) were trimmed to start on the first codon position; no stop codons were detected when translated into amino acids. To infer the position of Rhynchocalamus within Colubrinae we first carried out a preliminary phylogenetic analysis with the four Rhynchocalamus sequences combined with all colubrines assembled by Pyron et al. (2013) (236 species; data not shown). After we identified the clade Rhynchocalamus belongs to (from here on referred to as the Western Palearctic clade; consisting of the genera Rhynchocalamus, Bamanophis, Dolichophis, , Hemerophis, Hemorrhois, Hierophis, Lytorhynchus, Macroprotodon, Platyceps, Spalerosophis, and “Coluber”

PHYLOGENY OF RHYNCHOCALAMUS Zootaxa 4033 (3) © 2015 Magnolia Press · 381 zebrinus), we narrowed down the dataset to include only these genera in order to further improve the alignment and reduce the computation process. Some sequences of the Western Palearctic clade that were missing in Pyron et al. (2013) were added to complete the dataset, namely: 12S, 16S and cytb sequences of Macroprotodon brevis (Carranza et al. 2004) and 12S and 16S of Dolichophis jugularis (Schätti and Utiger 2001; Nagy et al. 2003a). Four species were used as outgroup (Pyron et al. 2013). GenBank accession numbers of all four genes analyzed for the Western Palearctic clade representatives included in the phylogenetic analyses are listed in Appendix II.

FIGURE 1. Distribution of the genus Rhynchocalamus in the Middle East. Numbered localities indicate the origin of material used for the phylogenetic analyses. Spatial data for R. melanocephalus and R. satunini combined from various sources (IUCN - www.iucnredlist.org; Darevsky 1970; Baha El Din 2006; Amr and Disi 2011; Sindaco et al. 2013).

Models of nucleotide evolution for the dataset including the Western Palearctic snakes were assessed using PartitionFinder v.1.1.1 (Lanfear et al. 2012) with the following settings: branch lengths linked, only models available in BEAST evaluated, AIC model selection criterion applied, all partition schemes analyzed. Each gene was set as an independent partition, this partition scheme was also the one preferred by PartitionFinder with GTR+I+G model of evolution selected for all genes. For the phylogenetic analyses all genes were concatenated to a single alignment with a final length of 2541 bp. Maximum likelihood (ML) analysis was performed in RAxML

382 · Zootaxa 4033 (3) © 2015 Magnolia Press ŠMÍD ET AL. v.7.0.3 (Stamatakis 2006) using the raxmlGUI v.1.2 graphical interface (Silvestro and Michalak 2012), with parameters estimated independently for each partition, GTRGAMMA model of nucleotide evolution, and 100 random addition replicates. Reliability of the tree was assessed by bootstrap analysis with 1000 pseudoreplications (Felsenstein 1985). The software BEAST v.1.7.5 (Drummond and Rambaut 2007) was used for Bayesian analysis (BA). Three individual runs of 5x107 generations were carried out, sampling at intervals of 104 generations. Models and prior specifications applied were as follows (otherwise by default): model of sequence evolution for each partition GTR+I+G as selected by PartitionFinder (see above); Yule tree prior for the phylogenetic reconstruction; random starting tree; base substitution prior Uniform (0,100); alpha prior Uniform (0,10). Partitions and clock models were unlinked. Posterior trace plots and effective sample sizes (ESS) of the runs were monitored in Tracer v.1.5 (Rambaut and Drummond 2007) to ensure convergence. The results of the individual runs were combined in LogCombiner discarding 10% of the samples and the maximum clade credibility ultrametric tree was produced with TreeAnnotator (both provided with the BEAST package).

TABLE 1. Specimens of Rhynchocalamus included in the phylogenetic analyses, with respective taxonomic identification, voucher reference, corresponding geographical distribution data (latitude, longitude and country), locality code as shown in Fig. 1, and GenBank accession numbers.

Species Voucher Latitude Longitude Country Locality 12S 16S cytb cmos reference code R. arabicus - 17.253 53.887 Oman 4 KT878842 KT878847 KT878854 KT878851 R. ZMHRU 35.063 35.899 Syria 1 KT878840 KT878844 KT878852 KT878848 melanocephalus 2007-69 R. TAUM 32.452 35.423 Israel 2 KT878841 KT878845 KT878853 KT878849 melanocephalus 15269 R. satunini CAS 34.584 49.891 Iran 3 KT878843 KT878846 KT878855 KT878850 228723

Results

The new record of Rhynchocalamus arabicus from Oman. On the 29th of April 2013 at 21:10 a black snake was observed and caught resting between stones in Wadi Ayoun (17.253° N, 53.887° E), Dhofar Governorate, Sultanate of Oman (Fig. 1). The temperature was 25ºC, windless and with no moonlight. The specimen was identified as R. arabicus on the basis of the following morphological characters when compared with photographs of the holotype (FMNH 18219, Field Museum of Natural History, Chicago, USA) and its detailed description in the literature (Schmidt 1933; Gasperetti 1988; Egan 2007): jet-black coloration of the head and body (with scales “faintly and narrowly outlined with light” ex. Schmidt 1933) (Fig. 2); body size and aspect (289 mm in the new specimen and 278 mm in the holotype) with elongate, slender body and head only slightly distinct from neck; 15 rows of dorsal scales at midbody; round pupil; large rostral shield wedged between the internasals; nasal undivided; one loreal scale; one preocular, one postocular, and two temporal scales on each side; 6–6 upper labials (Fig. 3). The specimen, however, differed from the holotype in several other morphological characters: the number of subcaudal scales (71 in the new specimen vs. 81 in the holotype); different head shape with the Omani specimen having longer snout, longer frontal shield, and larger dorsal scale between the parietals (Fig. 3). We consider these differences to be intraspecific variability similar in magnitude to that reported in R. melanocephalus, in which, for instance, the number of subcaudals varies between 47 and 66 (Franzen and Bischoff 1995; Avci et al. 2008). We find the general habitus of the snake and the corresponding numbers and arrangements of head and body scales to be similar enough to consider the specimens conspecific. The correct generic assignment to the genus Rhynchocalamus and the differences from the two other species are further confirmed by genetic data (see below). Phylogenetic comparison. Both ML and BA analyses resulted in trees with similar topologies but that differed in six nodes (the position of Eirenis barani, Hierophis spinalis, Spalerosophis diadema, Hemerophis socotrae, “Coluber” zebrinus, and Bamanophis dorri). We therefore present in Fig. 4 the BA tree with the ML bootstrap values of the well-supported nodes added but we also provide the ML tree in Appendix III for comparison. Contrary to Pyron et al. (2013), who recovered relationships among most of the Western Palearctic

PHYLOGENY OF RHYNCHOCALAMUS Zootaxa 4033 (3) © 2015 Magnolia Press · 383 genera basically resolved and strongly supported, our analyses differed in the position of some genera, regarding mostly the monotypic ones or those represented in the analyses by a single species (Hemerophis, “Coluber” zebrinus, Lytorhynchus). Both BA and ML analyses invariably recovered the genus Rhynchocalamus as monophyletic (Bayesian pp 1/ML bootstrap 100) and sister to Lytorhynchus (1/97). The sister group to these two genera was not resolved due to low support (see Fig. 4 and Appendix III). The uncorrected genetic distances (p- distance, complete deletion) between R. arabicus and its sister taxon, R. melanocephalus, are 5.8 ± 0.9% for the 12S (after Gblocks), 4.0 ± 0.8% for the 16S (after Gblocks), and 10.3 ± 0.9% for the cytb. Genetic distances between R. melanocephalus and R. satunini are 7.2 ± 1.1% for the 12S, 3.6 ± 0.8% for the 16S, and 12.4 ± 1.0% for the cytb. The level of genetic variability between the two samples of R. melanocephalus from Israel and Syria is very low, they share identical haplotypes in the 16S and differ only by 0.7 ± 0.3% in the 12S and by 0.6 ± 0.2% in the cytb.

FIGURE 2. Comparison of body habitus (left) and head coloration (right) of all known Rhynchocalamus species. A, B—R. arabicus from Wadi Ayoun, Dhofar Governorate, Sultanate of Oman. Photos by Javier Aznar Gonzalez de Rueda; C, D—R. melanocephalus from Tartus, Syria. Photos by Bayram Göçmen; E, F—R. satunini from Artuklu, Mardin Province, Turkey. Photos by Bayram Göçmen.

384 · Zootaxa 4033 (3) © 2015 Magnolia Press ŠMÍD ET AL. FIGURE 3. Dorsal and lateral view of the head of the holotype of R. arabicus (FMNH 18219; left) and its comparison with the new specimen reported herein (right). The original black coloration of the holotype has faded due to fixation. Left photo by Kathleen Kelly and right photo by Javier Aznar Gonzalez de Rueda.

Discussion

In this work we present the second-ever reported specimen of Rhynchocalamus arabicus, which represents the first record of this species and genus for the Sultanate of Oman. This finding constitutes more than a 1000 km range extension of the genus Rhynchocalamus in southern Arabia. The addition of a new genus of snake to the reptile fauna of Oman is surprising considering the century-long herpetological surveys conducted in the region (e.g. Arnold 1980; Schätti and Desvoignes 1999; van der Kooij 2001). The recent observations of rare nocturnal snakes in the southern Arabian Peninsula (Gardner et al. 2009; Šmíd 2010) strengthens the importance of conducting new surveys even in relatively well studied places as Dhofar, as well as in less explored regions. Additional fieldwork in the Dhofar region will be needed to collect more data on the distribution, population status, and natural history of R. arabicus.

PHYLOGENY OF RHYNCHOCALAMUS Zootaxa 4033 (3) © 2015 Magnolia Press · 385 Oligodon cinereus 1/100 Oligodon arnensis Oligodon sublineatus Oligodon taeniolatus “Coluber” zebrinus Bamanophis dorri Macroprotodon abubakeri 1/98 0.99/100 1/99 Macroprotodon cucullatus Macroprotodon brevis Lytorhynchus diadema 1/97 Rhynchocalamus satunini 1/100 Rhynchocalamus arabicus 0.95/- 0.98/77 1/100 Rhynchocalamus melanocephalus-1 Rhynchocalamus melanocephalus-2 Spalerosophis diadema

1/98 Hemorrhois algirus

1/100 Hemorrhois hippocrepis 1/97 Hemorrhois ravergieri 0.99/- Hemorrhois nummifer 0.99/74 Platyceps rogersi Platyceps rhodorachis 1/94 Platyceps karelini

0.99/- Platyceps florulentus Platyceps collaris -/74 Platyceps najadum Hemerophis socotrae

1/100 Dolichophis caspius Dolichophis jugularis Hierophis spinalis 1/94 1/100 Hierophis gemonensis Hierophis viridiflavus 0.99/- 1/100 Eirenis modestus Eirenis aurolineatus Eirenis decemlineatus 0.99/77 Eirenis levantinus Eirenis medus 0.06 subs./site 0.99/82 0.99/88 Eirenis thospitis

0.99/76 Eirenis lineomaculatus Eirenis coronelloides Eirenis punctatolineatus Eirenis barani 0.99/- 1/100 Eirenis collaris Eirenis eiselti

FIGURE 4. Maximum clade credibility tree obtained from BEAST based on the concatenated dataset of three mitochondrial and one nuclear gene (2541 bp). Values at the nodes indicate Bayesian posterior probability/ ML bootstrap support; only values above 0.95 and 70, respectively, are shown. The numbers of R. melanocephalus samples correspond to the locality numbers in Fig. 1 and Table 1.

The phylogeny of Rhynchocalamus. Previous molecular phylogenetic studies invariably recovered a monophyletic group within Colubrinae, termed here the Western Palearctic clade, although the support of this clade varied from none (Lawson et al. 2005) through moderate (Pyron et al. 2011) to very strong (Nagy et al. 2003b,

386 · Zootaxa 4033 (3) © 2015 Magnolia Press ŠMÍD ET AL. 2004; Pyron et al. 2013), which was probably a result of different taxa included and markers targeted. Our results clearly indicate that the genus Rhynchocalamus is a member of this clade and is a sister lineage to Lytorhynchus. Contrary to the tree presented by Avci et al. (2015), in which the clade of Rhynchocalamus + Lytorhynchus is sister to all other Western Palearctic genera, we were not able to deduce further phylogenetic relationships of this clade with the other genera due to low support at deeper levels of the phylogeny (Fig. 4; see also Appendix III). The Bayesian posterior probabilities were high at most deeper nodes, however, posterior probability as a measure of phylogenetic reliability has been shown to be excessively high compared with ML bootstrap values (Cummings et al. 2003; Erixon et al. 2003), particularly in analyses of concatenated data sets as carried out here (Suzuki et al. 2002). We therefore interpret the tree branches as unresolved unless they are supported by both high pp (≥ 0.95) and ML bootstrap (≥ 70) (Zander 2004). The well-supported sister taxa Lytorhynchus and Rhynchocalamus share several morphological characters as reduced dentition with 6–9 and 6–8 maxillary teeth, respectively, or enlarged wedge-shaped rostral shield. These similarities can thus be understood as shared synapomorphies of this monophyletic group. Although there is only one out of six currently recognized Lytorhynchus species included in the analyses, we do not expect that including more species of the genus would affect the tree topology greatly since all the species are very similar in their morphology (Leviton and Anderson 1970), and we therefore anticipate them to cluster with L. diadema (the type species of the genus Lytorhynchus). The status of R. satunini has been discussed repeatedly. Some authors have raised it to species level (Reed and Marx 1959; Avci et al. 2015) whereas others have considered it conspecific with R. melanocephalus (Franzen and Bischoff 1995; Avci et al. 2007). According to our analyses, the two subspecies are separated by high genetic distances (see results) that correspond with those reported for other Western Palearctic colubrine genera (Fig. 4) (e.g. Nagy et al. 2003a; Carranza et al. 2004). Additionally, there are several morphological differences reported between the two species: R. satunini differs from R. melanocephalus in having a greater number of upper labials (7 versus 6), greater number of lower labials (8 versus 7) and in not having the top of the head and nape uniformly black (Fig. 2; Reed and Marx 1959; Darevsky 1970; Franzen and Bischoff 1995). These results suggest that, as already proposed by Reed and Marx (1959) and Avci et al. (2015), R. satunini and R. melanocephalus should be considered as different species. As can be seen in Fig. 1, the distribution of all species of Rhynchocalamus is extremely patchy and limited. The range of R. melanocephalus is restricted to the Levant and Sinai and resembles the distribution pattern of other Levantine snakes (e.g. Eirenis lineomaculatus, E. rothii, Micrelaps muelleri, Daboia palaestinae; Sindaco et al. 2013), while R. satunini is confined to the Irano-Anatolian biodiversity hotspot (Mittermeier et al. 2004). Although the boundary between the two species seems to be well documented (e.g. Sindaco et al. 2013), further field research in southern Turkey and northwestern Syria is necessary for a precise delineation of their ranges and possible contact zones. The distribution of the south Arabian R. arabicus is even more enigmatic. The only two known localities are separated by a distance of more than a 1000 km. We assume that this can be attributed to the poor knowledge of the reptile fauna of central and eastern Yemen and that the distribution is, in fact, more continuous. Moreover, this species is separated by a 2000 km hiatus from its nearest congeners. Whether this gap in the genus distribution, spanning literally across the whole of the Arabian Peninsula, is caused by a lack of collecting data from the Arabian interior or by real absence of these snakes remains to be investigated. Recent field surveys in western Saudi Arabia provided evidence that the distribution of reptile taxa known previously from Egypt, Jordan, or Yemen is more continuous along the Hijaz and Asir Mountains (Šmíd et al. 2013; Badiane et al. 2014; Metallinou et al. 2015) and can indicate that Rhynchocalamus may also be present there. The collection of additional specimens and tissue samples of all Rhynchocalamus species from the southern Arabian Peninsula and other areas such as southeastern Anatolia and more intense fieldworks in the eastern Arabian mountains will be crucial to clarify the distribution and evolutionary history of the genus.

Acknowledgements

Special thanks are due to Saleh Al Saadi, Mohammed Al Shariani, Thuraya Alsariri, Ali Alkiyumi, and the other members of the Nature Conservation Department of the Ministry of Environment and Climate, Sultanate of Oman

PHYLOGENY OF RHYNCHOCALAMUS Zootaxa 4033 (3) © 2015 Magnolia Press · 387 for their help and support and for issuing all the necessary permits. The expedition was made with the Permit nº 14/ 2013 of the General Director of Nature Conservation of the Ministry of Environment & Climate Affairs of the Sultanate of Oman. We would like to thank Octavio Jiménez Robles and Juan Ramón Fernández Cardenete for help with the morphological analysis and Dr. Mehmet Zulfu Yildiz (Adıyaman University, Adıyaman, Turkey) and Mr. Musa Geçit (Mardin, Turkey) for field assitance. To Alan Resetar and Kathleen Kelly of the Fieldmuseum of Chicago, for taking the photographs of the holotype of R. arabicus. To Drew Gardner and all the people who helped us with the identification of the Omani specimen. To Jens Vindum of the California Academy of Sciences and to Erez Maza and Shai Meiri from the Steinhardt Museum of Natural History and National Research Center in Tel Aviv for providing tissue samples of R. satunini and R. melanocephalus, respectively. This work was supported by the project “Field study for the conservation of in Oman” funded by the Ministry of Environment and Climate Affairs (Ref: 22412027), and grant CGL2012-36970 from the Ministerio de Economía y Competitividad, Spain (co-funded by FEDER). SC is member of the Grup de Recerca Emergent of the Generalitat de Catalunya: 2014SGR1532. The work of JS was supported by Ministry of Culture of the Czech Republic (DKRVO 2015/15, National Museum, 00023272). PdP is funded by a FI-DGR grant from the Generalitat de Catalunya, Spain (2014FI_B2 00197). KT is supported by the Steinhardt Museum of Natural History and National Research Center, Tel Aviv, Israel.

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PHYLOGENY OF RHYNCHOCALAMUS Zootaxa 4033 (3) © 2015 Magnolia Press · 389 Šmíd, J., Moravec, J., Kratochvíl, L., Gvoždík, V., Nasher, A.K., Busais, S.M., Wilms, T., Shobrak, M.Y. & Carranza, S. (2013) Two newly recognized species of Hemidactylus (Squamata, Gekkonidae) from the Arabian Peninsula and Sinai, Egypt. ZooKeys, 355, 79–107. http://dx.doi.org/10.3897/zookeys.355.6190 Stamatakis, A. (2006) RAxML-VI-HPC: Maximum likelihood-based phylogenetic analyses with thousands of taxa and mixed models. Bioinformatics, 22, 2688–2690. http://dx.doi.org/10.1093/bioinformatics/btl446 Suzuki, Y., Glazko, G.V. & Nei, M. (2002) Overcredibility of molecular phylogenies obtained by Bayesian phylogenetics. Proceedings of the National Academy of Sciences, 99, 16138–16143. http://dx.doi.org/10.1073/pnas.212646199 Talavera, G. & Castresana, J. (2007) Improvement of phylogenies after removing divergent and ambiguously aligned blocks from protein sequence alignments. Systematic Biology, 56, 564–577. http://dx.doi.org/10.1080/10635150701472164 van der Kooij, J. (2001) The herpetofauna of the sultanate of Oman. Part 4: the terrestrial snakes. Pod@rcis, 2, 54–64. Wallach, V., Williams, K.L. & Boundy, J. (2014) Snakes of the World. A catalogue of living and extinct species. Boca Ratón, Florida (USA): CRC Press, Taylor & Francis, 1227 pp Zander, R.H. (2004) Minimal values for reliability of bootstrap and jackknife proportions, decay index, and Bayesian posterior probability. Phyloinformatics, 2, 1–13.

APPENDIX I. Information on the sequenced gene fragments, primers, primer sequences and source references, PCR programs, fragment lengths (for the two ribosomal mtDNA genes, 12S and 16S, the length is shown both before and after Gblocks, respectively), models of sequence evolution selected by PartitionFinder, and number of variable and parsimony-informative (Pi) sites (for the Gblocks fragments in the case of the 12S and 16S genes). Gene Primers Primer sequence Primer source 12S 12S268 GTGCCAGCGACCGCGGTTACACG Schätti and Utiger 2001 12S916 GTACGCTTACCATGTTACGACTTGCCCTG 16S 16Sa CGCCTGTTTATCAAAAACAT Palumbi et al., 1991 16Sb CCGGTCTGAACTCAGATCACGT cytb L14910 GACCTGTGATMTGAAAACCAYCGTTGT Burbrink et al. 2000 H16064 CTTTGGTTTACAAGAACAATGCTTTA cmos Cmos-FUF TTTGGTTCKGTCTACAAGGCTAC Gamble et al. 2008 Cmos-FUR AGGGAACATCCAAAGTCTCCAAT continued. Gene Primers PCR cycles program Length (bp) Model Variable sites Pi sites 12S 12S268 94° 1', 65° 1', 72° 1' 617/577 GTR+I+G 213 153 12S916 35 cycles 16S 16Sa 94° 30'', 48° 45'', 72° 1' 523/509 GTR+I+G 141 83 16Sb 35 cycles cytb L14910 94° 40'', 46° 30'', 72° 1' 996 GTR+I+G 479 401 H16064 40 cycles cmos Cmos-FUF 94° 30'', 53° 45'', 72° 1'30'' 459 GTR+I+G 54 22 Cmos-FUR 35 cycles

References to Appendix I not cited in the text

Burbrink, F.T., Lawson, R. & Slowinski, J.B. (2000) Mitochondrial DNA phylogeography of the polytypic North American rat snake (Elaphe obsoleta): a critique of the subspecies concept. Evolution, 54, 2107–2118. http://dx.doi.org/10.1554/0014-3820(2000)054[2107:MDPOTP]2.0.CO;2 Gamble, T., Bauer, A.M., Greenbaum, E. & Jackman, T.R. (2008) Evidence for Gondwanan vicariance in an ancient clade of gecko lizards. Journal of Biogeography, 35, 88–104. http://dx.doi.org/10.1111/j.1365-2699.2007.01770.x

390 · Zootaxa 4033 (3) © 2015 Magnolia Press ŠMÍD ET AL. Palumbi, S.R., Martin, A.P., Romano, S.L., McMillan, W.O.D., Stice, L. & Grabowski, G. (1991) The simple fool’s guide to PCR. University of Hawaii, Honolulu, HI, 94 pp.

APPENDIX II. GenBank accession numbers for all the Western Palearctic colubrine snakes included in the phylogenetic analyses, with the only exception of the newly sequenced members of the genus Rhynchocalamus for which data is presented in Table 1. Species 12S 16S cytb cmos Bamanophis dorri – AY188081 AY188040 AY188001 “Coluber” zebrinus – AY188084 AY188043 AY188004 Dolichophis caspius AY039135 AY376768 AY039173 AY376797 Dolichophis jugularis AY039152 AY376769 AY486917 AY486941 Eirenis aurolineatus – AY376778 AY376749 AY376807 Eirenis barani – AY376785 AY376764 AY376822 Eirenis collaris – AY376795 AY376766 AY376824 Eirenis coronelloides – AY376787 AY376758 AY376816 Eirenis decemlineatus – AY376789 AY376760 AY376818 Eirenis eiselti – AY376776 AY376747 AY376805 Eirenis levantinus – AY376794 AY376765 AY376823 Eirenis lineomaculatus – AY376791 AY376762 AY376820 Eirenis medus AY647226 AY376796 AY376767 AY376825 Eirenis modestus AY039143 AY376792 AY486933 AY486957 Eirenis punctatolineatus AY647227 AY376781 AY376755 AY376813 Eirenis thospitis – AY376790 AY376761 AY376819 Hemerophis socotrae AY039132 AY188083 AY188042 AY188003 Hemorrhois algirus AY039149 – AY486911 AY486935 Hemorrhois hippocrepis DQ451992 – DQ451987 AY486940 Hemorrhois nummifer AY039163 AY376771 AY039201 AY376800 Hemorrhois ravergieri AY039131 – AY486920 AY486944 Hierophis gemonensis AY039145 AY376770 AY039183 AY376799 Hierophis spinalis AY541508 AY376773 AY486924 AY486948 Hierophis viridiflavus AY541505 AY376774 AY486925 AY486949 Lytorhynchus diadema AY647229 AY188064 AY188025 AY187986 Macroprotodon abubakeri AY643297 AY643338 AY643383 – Macroprotodon brevis AY643291 AY643332 AY643374 - Macroprotodon cucullatus AY643290 AY188065 AY188026 AY187987 Oligodon arnensis KC347327 KC347365 KC347481 KC347405 Oligodon cinereus HM591503 HM591508 AF471033 AF471101 Oligodon sublineatus KC347329 KC347367 KC347483 KC347407 Oligodon taeniolatus KC347330 KC347368 KC347484 KC347408 Platyceps collaris AY039157 – AY486922 AY486946 Platyceps florulentus AY039130 – AY486915 AY486939 Platyceps karelini AY647232 – AY486918 AY486942 Platyceps najadum AY039128 – AY486912 AY486936 Platyceps rhodorachis AY039154 – AY486921 AY486945 Platyceps rogersi AY039127 AY188082 AY188041 AY188002 Spalerosophis diadema AY039144 HQ658450 AF471049 AF471155

PHYLOGENY OF RHYNCHOCALAMUS Zootaxa 4033 (3) © 2015 Magnolia Press · 391 APPENDIX III. Maximum likelihood tree inferred using the concatenated dataset of three mitochondrial and one nuclear gene (2541 bp; see Material and Methods). Values by the nodes indicate bootstrap support; only values above 70 are shown. The numbers of R. melanocephalus samples correspond to the locality numbers in Fig. 1 and Table 1.

Oligodon taeniolatus 100 Oligodon arnensis Oligodon sublineatus Oligodon cinereus Macroprotodon abubakeri 98 99 Macroprotodon brevis Macroprotodon cucullatus 100 Bamanophis dorri “Coluber” zebrinus Lytorhynchus diadema 97 Rhynchocalamus satunini 100 Rhynchocalamus arabicus 77 -1 100 Rhynchocalamus melanocephalus Rhynchocalamus melanocephalus -2 Hemerophis socotrae 97 Hemorrhois ravergieri Hemorrhois nummifer 100 98 Hemorrhois algirus Hemorrhois hippocrepis Spalerosophis diadema Platyceps rhodorachis 74 Platyceps rogersi 94 Platyceps karelini Platyceps florulentus Platyceps najadum 76 Platyceps collaris

100 Dolichophis jugularis Dolichophis caspius 94 Hierophis spinalis Hierophis viridiflavus 100 Hierophis gemonensis Eirenis modestus 100 Eirenis aurolineatus 77 Eirenis decemlineatus Eirenis levantinus Eirenis thospitis 82 88 Eirenis medus 76 Eirenis lineomaculatus 0.09 subs./site Eirenis coronelloides Eirenis punctatolineatus Eirenis barani

100 Eirenis eiselti Eirenis collaris

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