Amphibia-Reptilia 30 (2009): 273-282

A preliminary analysis of phylogenetic relationships and biogeography of the dangerously venomous Carpet Vipers, (, Serpentes, ) based on mitochondrial DNA sequences

E. Nicholas Arnold1, Michael D. Robinson2, Salvador Carranza3,*

Abstract. Phylogenetic analysis of 1117 bp of mitochondrial DNA sequences (731 bp of cytochrome b and 386 bp of 16S rRNA) indicate that Echis consists of four main clades: E. ocellatus,andtheE. coloratus, E. pyramidum,andE. carinatus groups. In the E. coloratus group, E. coloratus itself shows substantial genetic divergence from E. omanensis, corroborating their separate status. In the E. pyramidum clade, E. pyramidum from and E. leucogaster from West are genetically very similar, even though samples are separated by 4000 km. South Arabian populations of the E. pyramidum group are much better differentiated from these and two species may be present, from Dhofar, southern probably being referable to E. khosatzkii.IntheE. carinatus group, specimens of E. carinatus sochureki and E. multisquamatus are very similar in their DNA. The phylogeny indicates that the split between the main groups of Echis was followed by separation of African and Arabian members of the E. pyramidum group, and of E. coloratus and E. omanensis. The last disjunction probably took place at the lowlands that run southwest of the North Oman mountains, which are likely to have been intermittently covered by marine incursions; they also separate the E. pyramidum and E. carinatus groups and several sister taxa of other . The E. carinatus group may have spread quite recently from North Oman into its very extensive southwest Asian range, and there appears to have been similar expansion of E. pyramidum (including E. leucogaster) in . Both these events are likely to be associated with the marked climatic changes of the Pleistocene or late Pliocene. Similar dramatic expansions have also recently occurred in three species in Iberia.

Keywords: biogeography, Echis, evolution, mitochondrial DNA, , , venom.

Introduction ineffective in treating bites elsewhere (Warrell and Arnett, 1976; Gillissen et al., 1994). Some- Echis Carpet vipers ( ) are found across the semi- times this is because different species of Echis arid regions of west, northern and east Africa, are likely to have been involved (Gillissen et al., west, south and east Arabia, parts of and 1994), for venom chemistry sometimes varies north to Uzbekistan, and in Pak- greatly between them. Consequently, a good ap- istan, and (fig. 1). They are of- preciation of the taxonomy of the is es- ten abundant and in many areas are a common sential for efficient treatment. cause of fatal snake bite in people (Gasperetti, For a long time, just two species of Echis 1988; Spawls and Branch, 1995). It has been were recognised: E. coloratus (Günther, 1878) shown that raised against venom found in Arabia, , and eastern from a population of Echis in one area may be Egypt, and E. carinatus (Schneider, 1801), which was believed to occur over most of the range of the genus. In E. coloratus, populations 1 - Department of Zoology, The Natural History Museum, London, Cromwell Road, SW7 5BD, London, UK from Israel and west Jordan have recently been 2 - Sultan Qaboos University, Department of Biology, Col- described as a subspecies, E. c. terraesanctae lege of Science, P.O. Box 36, Al-Khod, Muscat, Sul- (Babocsay, 2003) and populations from north- tanate of Oman ern Oman given full species status as E. oma- 3 - Institute of Evolutionary Biology (CSIC-UPF), Passeig Maritim de la Barceloneta 37-49, Barcelona, Spain nensis (Babocsay, 2004), both taxa being recog- *Corresponding author; e-mail: nised on the basis of morphology. The system- [email protected] atics of in its broad sense has

© Koninklijke Brill NV, Leiden, 2009. Also available online - www.brill.nl/amre 274 E.N. Arnold, M.D. Robinson, S. Carranza

Figure 1. Distribution of main taxa of Carpet vipers (Echis). Star indicates distinctive population in north (see below). Maps based on Gasperetti (1988); Spawls and Branch (1995); Babocsay (2004); Geniez et al. (2004); Mazuch (2005); and Trape and Mané (2006). been confused and unstable in recent decades. other in Oman. There are also differences in Within this assemblage, a number of taxa have reproduction, eastern animals apparently being been described on the basis of external fea- viviparous while western ones are egg-laying tures, including E. carinatus leakeyi (Stemm- (Smith, 1943; Minton, 1966; Stemmler, 1971; ler and Sochurek, 1969) and E. c. aliaborri Spawls and Branch, 1995). Among the west- (Drewes and Sacherer, 1974) from . Echis ern populations, the oldest available name is c. sochureki (Stemmler, 1969) was described E. pyramidum (Geoffroy Saint-Hilaire and Ge- from , Iran and North Oman and E. offroy Saint-Hilaire, 1827), which is based on c. astolae (Mertens, 1971) from Astola Island Egyptian material. Morphology indicated that near Pakistan. (Stemmler, 1970) nearly all Echis populations from Egypt to of the savannahs of West Africa was recog- Kenya belonging to the western group could be nised as occurring from Senegal eastwards to at regarded as a single rather variable form, and least western Chad (Wüster et al., 1997), while that E. leucogaster was very similar to these populations assigned to E. leucogaster (Roman, (Arnold, 1980a). On the other hand, E. ocel- 1972) are found in drier areas of West Africa latus appeared to be a separate species distin- and also as isolates in the western Sahara north guished by scale counts, pattern and hemipenial at least to southern Algeria (Wüster et al., 1997). structure. It was also noted that an from A broad study of Echis anatomy, incorporat- Biskra, northern Algeria (Natural History Mu- ing features of the hemipenis and dorsal scale seum, London: BMNH 1907.4.6.55) has a dis- shape (Arnold, 1980a), made it clear that E. car- tinctive hemipenis and may represent yet an- inatus in its broad sense is morphologically dis- other taxon (fig. 1B). junct. The eastern forms (at that time assigned Subsequent to this survey, Echis multisqua- to E. c. carinatus in India and Sri Lanka, E. matus Cherlin, 1981, was described from Turk- c. sochureki and E. c. astolae) are very dif- menistan and Echis was later the subject of an ferent in these aspects from the more western extensive revision (Cherlin, 1990; Cherlin and ones, the two assemblages approaching each Borkin, 1990). In this, a total of twelve species Phylogeny of Echis 275 and a further eight subspecies were recognised, (2000) in Genbank, and with another sequence seven of these taxa being new. For example, an- of E. ocellatus deposited there by W. Wüster. imals from southwest Arabia previously placed The results of these preliminary analyses were in E. pyramidum were assigned to E. khosatzkii used to assess aspects of the systematics and Cherlin, 1990 and E. varia borkini Cherlin, biogeography of Echis. 1990. Of Cherlin’s new forms, E. multisqua- matus was shown to be close to E. carinatus sochureki in its external morphology (Auffen- Material and methods berg and Rehman, 1991). But many of Cherlin’s A total of 17 specimens of the genus Echis were included other radical taxonomic changes have never in this study, among them representatives of at least seven been formally reassessed, and the systematics of nominal species covering much of the geographical distrib- the Echis carinatus complex has remained un- ution of the genus. One cerastes (Linnaeus, 1758) stable. Species-level distinction between E. car- and one C. (Linnaeus, 1758) were used as outgroups. Specimen data are given in table 1. inatus and the more western forms has gener- ally been accepted (see for instance Gasperetti, Extraction of DNA and PCR amplification 1988; Schätti and Gasperetti, 1994), but many authors use conflicting taxonomic arrangements The DNeasy tissue kit, Qiagen, was used to extract genomic DNA from the samples, following the manufacturers’ in- elsewhere in the genus, especially in Africa. structions. Primers used in both amplification and sequenc- Thus, Largen and Rasmussen (1993), Spawls ing were L 14846F1 (5-CAA CAT CTC AGC ATG ATG and Branch (1995), and Mazuch (2005) all vary AAA CTT CG 3 ; Lenk et al., 2001) and S2 (5 -TGG GAT TGA TCG TAG GAT GGC GTA-3) for the cytochrome b in the names they use for some populations, (cytb) gene, and L2510 (5 -CGC CTG TTT ATC AAA AAC and the last two publications do not incorporate AT-3 ) and H3062 (5-CCG GTT TGA ACT CAG ATC A- some of Cherlin’s changes in the regions they 3) for 16S rRNA (Lenk et al., 2001). The two gene frag- cover. Regrettably, confusion in the systemat- ments were amplified using PCR procedures described by Carranza et al. (1999, 2001) and processed with an ABI Echis ics of has probably been greatest in the 377 automated sequencer following the manufacturer’s pro- medical and toxinological literature (Wüster et tocols. al., 1997). For example, less than 50% of exper- imental venoms and animals involved in bites Phylogenetic analyses that were mentioned could be confidently as- DNA sequences were aligned using ClustalX (Thompson et signed to the currently more widely accepted al., 1997) with default parameters (gap opening = 10; gap species (Wüster and McCarthy, 1996). extension = 0.2). The cytb alignment included no gaps and Because of these problems, the relationships no stop codons were observed when the sequences were translated into amino acids using the vertebrate mitochon- of some of the more critical populations of drial code, suggesting that all the cytb sequences analyzed Echis are surveyed here using mitochondrial were functional. Only five gaps had to be postulated to un- DNA sequences. So far, their use in the genus ambiguously align all the 16S rRNA sequences; therefore has been limited, although individuals assigned all the positions were included in the phylogenetic analy- ses. to six taxa have been investigated using frag- Topological incongruence among partitions was tested ments of cytochrome b (cytb) and 16S rRNA using the incongruence length difference (ILD) test (Michke- (Lenk et al., 2001). In the course of a contin- vich and Farris, 1981; Farris et al., 1994). In this, 10 000 uing broader molecular study on the Arabian heuristic searches were carried out after removing all invari- able characters from the dataset (Cunningham, 1997). To fauna, tissue samples were collected by test for incongruence among data sets we also used a recip- the present authors from three taxa of Echis in rocal 70% bootstrap proportion (Mason-Gamer and Kel- Oman during 2005. The DNA sequences pro- logg, 1996) or a 95% Bayesian posterior probability thresh- duced from these were combined with other old. Topological conflicts were considered significant if two different relationships for the same set of taxa were both new ones from Mauritania, Niger and with those supported with bootstrap values > 70%, or posterior proba- of Lenk et al. (2001) and Malhotra and Thorpe bility values > 95%. 276 E.N. Arnold, M.D. Robinson, S. Carranza

Table 1. Details of material and sequences used in the present study. The question mark indicates that the cytb and 16S rRNA sequences of the specimen of E. ocellatus by Lenk et al. (2001) are probably the result of a misidentification of an E. leucogaster specimen as an E. ocellatus (see fig. 2).

Taxa Locality Accession numbers References cytb/16S rRNA

Cerastes cerastes Erfoud () AJ275703/AJ275755 Lenk et al. (2001) Djebil () AJ275705/AJ275757 Lenk et al. (2001) Wadi Rishrash (Egypt) AJ275708/AJ275760 Lenk et al. (2001) Echis omanensis-1 Jebel Akhdar (north Oman) EU642590/EU642581 E3026.8 Echis omanensis-2 North of Tanuf (north Oman) EU642587/EU642578 E3026.9 Echis omanensis-3 Wadi Bani Habib (north Oman) EU642588/EU642579 E3026.10 Echis omanensis-4 Wadi Bani Khalid (north Oman) EU642589/EU642580 E23116.1 Echis carinatus subsp. (Pakistan) AJ275706/AJ275758 Lenk et al. (2001) Echis multisquamatus (Turkmenistan) AJ275702/AJ275763 Lenk et al. (2001) Echis carinatus sochureki Manah (north Oman) EU642591/EU642582 E3026.2 Echis ocellatus-1 West Africa AF292568/ Wüster, unpublished Echis ocellatus-2 West Africa AF191579/ Malhotra and Thorpe (2000) Echis ocellatus-3 10 km N. of Tapoua (Niger) EU642592/FJ808740 SPM0050 Echis sp. () AJ275707/AJ275759 Lenk et al. (2001) Echis khozatskii NW. of Ayun pools (Dhofar, Oman) EU642584/EU642575 E3026.15 (Egypt) AJ275709/AJ275761 Lenk et al. (2001) Echis ocellatus? (Mali) AJ275710/AJ275762 Lenk et al. (2001) -2 Ayoûn el’Atrous (Mauritania) EU642585/EU642576 E3026.3 Echis leucogaster-3 Ayoûn el’Atrous (Mauritania) EU642586/EU642577 E3026.4

Three methods were employed for phylogenetic analyses (Huelsenbeck and Ronquist, 2001); only values equal or of the two separate gene fragments and for those in which above 95% were considered to indicate sufficient support they were combined. These were: maximum-likelihood (Wilcox et al., 2002). (ML), maximum-parsimony (MP) and Bayesian analysis. Modeltest (Posada and Crandall, 1998) was used to select the most appropriate model of sequence evolution for the ML and Bayesian analyses under the Akaike Information Results Criterion. This was the GTR model taking into account gamma distribution and the number of invariable sites for each independent partition (cytb and 16S) and for the com- The two gene partitions used (cytb and 16S) bined data set (cytb + 16S). were shown to be congruent using the ILD- Both ML and MP analyses were performed in test (P = 0.90), and independent analyses of PAUP*4.0b10 (Swofford, 1998) and included searches in- volving tree bisection and reconnection (TBR) branch swap- the two gene partitions confirmed there were ping with 10 and 100 step-wise additions of taxa, respec- no topological conflicts (Mason-Gamer and tively. In the MP analyses, gaps were included as a fifth state and transitions and transversions were given the same Kellogg, 1996). Therefore, both mitochondrial weight. Bayesian analyses were performed on MRBAYES fragments were combined for further analyses. v.3.1.2 (Huelsenbeck and Ronquist, 2001) and each parti- Of the 1117 bp of the combined data set (731 tion had its own model of sequence evolution and model parameters (see above). Four incrementally heated Markov of cytb and 386 of 16S), 327 were variable and chains with the default heating values were used. All analy- 276 parsimony-informative. × 6 ses started with randomly generated trees and ran for 2 10 The results of the phylogenetic analyses are generations in two independent runs with samplings at in- tervals of 100 generations that produced 20 000 trees. After shown in figure 2. ML, MP and Bayesian phy- verifying that stationarity had been reached, both in terms logenetic analyses all produced trees with iden- of likelihood scores and parameter estimation, the first 5000 tical topologies in which four well-supported trees were discarded in both independent runs of the cytb and 16S rRNA and the combined analyses and a major- main units are discernable. They are the E. ity rule consensus tree was generated from the remaining ocellatus, E. pyramidum, E. coloratus, and E. 15 000 post-burnin trees. The frequency of any particular clade among the individual trees contributing to the con- carinatus groups which have uncorrected ge- sensus tree represents the posterior probability of that clade netic divergences of about 12-18% from each Phylogeny of Echis 277

Figure 2. Relationships of Carpet vipers (Echis) based on 731 base pairs of cytochrome b and 386 of 16S rRNA mitochondrial genes. Two other viperines, Cerastes cerastes and C. vipera are used as outgroups. Maximum likelihood (ML), maximum parsimony (MP) and Bayesian analyses all gave identical topologies. Figures close to nodes are: ML bootstrap value/MP bootstrap value/Bayesian posterior probability value (only values equal or higher than 0.95 are indicated with an asterisk “*”). The question mark indicates that the cytb and 16S rRNA sequences of the specimen of E. ocellatus by Lenk et al. 2001 (see table 1) are probably the result of misidentification of an E. leucogaster. other in the cytb fragment investigated (see ta- (1%) and E. leucogaster (5%) and very diver- ble 2). In the E. pyramidum group, there is a gent from the other E. ocellatus included in this quite deep divergence between African and Ara- study (14-16%); it may consequently actually bian representatives (11-13%). In North Africa, also be an E. leucogaster. Overall divergence a specimen of E. pyramidum from Egypt is very in these four widely separated North African similar to two assigned to E. leucogaster from snakes is only about 2-5%. Mauritania (5%). A further snake from Mali, The two south Arabian members of the E. identified in GenBank as E. ocellatus (Lenk pyramidum group, from Yemen and Dhofar in et al., 2001), is also close to E. pyramidum south Oman, are sisters but show a divergence 278 E.N. Arnold, M.D. Robinson, S. Carranza

Table 2. Uncorrected genetic distances for the cytochrome b gene fragment used in this study.

Species 1

1 E. sp. Ð2 2 E. khosatzkii 0.10 Ð 3 3 E. ocellatus? 0.12 0.12 Ð 4 4 E. pyramidum 0.12 0.11 0.01 Ð 5 5 E. leucogaster-2 0.13 0.10 0.05 0.05 Ð 6 6 E. leucogaster-3 0.13 0.10 0.05 0.05 0.00 Ð 7 7 E. omanensis-2 0.17 0.15 0.15 0.14 0.16 0.16 Ð 8 8 E. omanensis-3 0.17 0.15 0.15 0.14 0.16 0.16 0.00 Ð 9 9 E. omanensis-4 0.16 0.14 0.15 0.14 0.16 0.16 0.01 0.01 Ð 10 10 E. omanensis-1 0.16 0.14 0.15 0.14 0.16 0.16 0.01 0.01 0.01 Ð 11 11 E. coloratus 0.17 0.15 0.16 0.16 0.16 0.16 0.10 0.10 0.09 0.09 Ð 12 12 E. multisquamatus 0.18 0.16 0.16 0.16 0.16 0.16 0.16 0.16 0.16 0.16 0.16 Ð 13 13 E. carinatus 0.18 0.16 0.15 0.15 0.15 0.15 0.15 0.15 0.15 0.15 0.16 0.02 Ð 14 sochureki 14 E. carinatus subsp. 0.18 0.16 0.15 0.15 0.16 0.16 0.16 0.16 0.16 0.16 0.15 0.03 0.03 Ð 15 15 E. ocellatus-1 0.18 0.16 0.16 0.15 0.16 0.16 0.17 0.17 0.17 0.17 0.17 0.16 0.16 0.16 Ð 16 16 E. ocellatus-2 0.18 0.16 0.16 0.15 0.16 0.16 0.17 0.17 0.17 0.17 0.17 0.17 0.16 0.16 0.00 Ð 17 17 E. ocellatus-3 0.15 0.15 0.14 0.13 0.12 0.12 0.17 0.17 0.17 0.18 0.17 0.18 0.16 0.18 0.06 0.06 Ð 18 18 Cerastes cerastes 0.19 0.18 0.18 0.18 0.19 0.19 0.18 0.18 0.18 0.18 0.18 0.21 0.20 0.21 0.20 0.20 0.18 Ð 19 19 Cerastes vipera 0.20 0.18 0.21 0.21 0.20 0.20 0.18 0.18 0.18 0.18 0.17 0.20 0.19 0.20 0.21 0.21 0.21 0.13 Ð of about 10%. In the E. coloratus clade, there is grounds, E. p. aliaborri should be synonymised a deep dichotomy between the western E. col- (Mazuch, 2005). oratus itself and E. omanensis of North Oman, The strong divergence of the two Arabian with a divergence of about 9-10%. In the E. car- populations of the E. pyramidum group studied inatus group, animals assigned to E. multisqua- here, from African ones and from each other, matus from Turkmenistan and E. c. sochureki indicates they represent at least one separate from North Oman are very similar (about 2%) species. The name E. khosatzkii (Cherlin, 1990) and a further specimen, apparently from Pak- may be available for the population in Dhofar, istan shows a divergence of 3% from these. southern Oman (fig. 1B). The locality of the holotype of E. khosatskii, is ‘’ (in fact, somewhere on an itinerary from Mukallah Discussion on the coast to the Hadhramaut valley and back to the sea at Ash Shihr, fide Anderson, 1896). Systematics The paratype comes from Ghayl Ba Wazir in the same general area of eastern Yemen, and a sim- The great divergence between samples of E. ilar specimen has been reported 170 km to the ocellatus and E. pyramidum considered to be east at Sayhut (Schätti and Desvoignes, 1999). valid here indicates that they are not closely These specimens come from 300-500 km west related as has been previously suggested (Lenk of the Dhofar ones. They share with these a dis- et al., 2001). As noted, the specimen assigned tinctive range of dorsal patterns and, compared to E. ocellatus on which this relationship was with most other Arabian animals of the E. pyra- based is more likely to be assignable to the E. midum group from further west, relatively high pyramidum group (see fig. 2). It remains to be ventral and subcaudal scale counts (East Yemen seen if DNA indicates this is also true for the and Dhofar: 165-179 ventral scales and 39-46 generally similar Kenyan E. pyramidum leakeyi, subcaudal scales in males (n = 6), and respec- with which, it is suggested on morphological tively 183-189, and 36-39 in females (n = 4). Phylogeny of Echis 279

Western Yemen and adjoining southwest Saudi Historical biogeography Arabia: usually 145-169 ventral scales and 29- Echis belongs to the , a group for = 37 subcaudal scales in males (n 5) and re- which an African origin has been postulated = spectively 158-170 and 29-38 in females (n (Lenk et al., 2001). , the sister group 6) Ð E.N. Arnold, unpublished data; Gasperetti, of the Viperinae is found in Africa and, of the 1988; Schätti and Gasperetti, 1994). However, five units that comprise the subfamily, , adults from eastern Yemen have narrow heads, and Cerastes also occur there with the whereas they are broad in the ones from Dho- last two extending into Arabia, an area that is far (Arnold, 1980a). The other genetically dis- geologically part of that continent. The only unit tinct southern Arabian member of the E. pyra- of the Viperinae to occur predominantly outside midum group investigated here does not have Africa is made up of Vipera, Pseudocerastes a precise locality within Yemen (Lenk et al., and and mainly occurs in 2001), but its genetic divergence makes it un- and . Echis itself has three of its main units likely to be conspecific with the Dhofar popu- entirely in the African-Arabian region and the lation. It could conceivably be a representative fourth, the mainly Asian E. carinatus group, is of the populations in western Yemen for which also represented there in North Oman (fig. 1D). Given the overall distribution pattern of taxa the name E. borkini (Cherlin, 1990) (type local- within the Viperinae, and the topology of the ity Lahej near Aden) may be available (E. sp. phylogenetic tree from figure 2 in which the E. in fig. 1B). More DNA sampling of the E. pyra- coloratus group is basal to all the other three midum group in Yemen and Oman is required groups, it is most parsimonious to assume an before these problems of nomenclature can be African-Arabian origin for Echis. This contrasts resolved. However, it is quite possible that at with an Asian genesis in the Irano-Turanian least two species of Echis could occur in south- region suggested by Cherlin (1990). But that west Arabia, for some other reptile taxa have hypothesis did not take the distribution of the two or more closely related allopatric or parap- relations of Echis into account and the putative atric species there distributed from west to east. phylogeny of the genus on which it was based These include spiny-tailed agamids (Uromastyx (Cherlin, 1990, fig. 6) is different from the one yemenensis and U. benti Ð Wilms and Schmitz, presented here. 2007) and semaphore geckos (Pristurus carteri The DNA phylogeny (fig. 2) suggests that group Ð Arnold, 1986a). In other cases more dis- Echis spread and diversified relatively rapidly tantly related congenerics occur allopatrically in to produce its main groups, while disjunction this region, for example in Hemidactylus and of the E. pyramidum group around the Red Ptyodactylus geckos (Carranza and Arnold, un- Sea, and the separation of E. coloratus and E. omanensis within Arabia, occurred rather published data). later. The Red Sea is a site of disjunction for The strong genetic divergence between Echis many other reptile clades, including ground coloratus and the recently distinguished E. oma- geckos (Stenodactylus), sand skinks (Scincus), nensis corroborates the species status of these lacertids of the Acanthodactylus scutellatus and forms. In contrast, similarity in DNA sequence Mesalina brevirostris-M. rubropunctata groups, confirms an assessment from morphology (Auf- sand vipers (Cerastes) and awl-headed snakes fenberg and Rehman, 1991) that Echis mul- (Lytorhynchus) (see Arnold, 1980b, 1986b, tisquamatus is conspecific with E. carinatus 1987). Similarly, the disjunction between E. sochureki. The genetic similarity of E. multi- coloratus and E. omanensis in eastern Arabia, squamatus and E. c. sochureki has already been is matched by many other groups including noted by Lenk et al. (2001). Bunopus, Hemidactylus and Pristurus geckos 280 E.N. Arnold, M.D. Robinson, S. Carranza

(Arnold, 1980a; Arnold and Carranza, unpub- nonicus and Pseudocerastes (Arnold and Gal- lished DNA data), sand skinks (Scincus m. lagher, 1977). mitranus and S. mitranus muscatensis ÐCar- Echis carinatus may have extended its range ranza et al., 2008), and lacertids (Acanthodacty- comparatively recently and rapidly in southwest lus schmidti and A. blandfordii Ð Harris et al., Asia, as the North Oman and Turkemenistan 1998). Different degrees of genetic divergence animals investigated here are genetically simi- between western and eastern representatives of lar, even though they are separated by at least these units suggest that there was not a sin- 1200 km. It remains to see if spread into In- gle vicariance event but a series of temporary dia was also comparatively recent. Such rapid interruptions that affected different taxonomic expansion by snake species, once the edge groups at different times. This possibility gains of a new inhabitable region is colonised, has some support from topography. The highland also occurred elsewhere. For example, DNA regions of Dhofar and North Oman are sep- phylogenies show that the Montpellier snake arated by a lowland area that stretches from (Malpolon monspessulanus), Horseshoe whip the Arabian Gulf southeast to the Arabian Sea. snake (Hemorrhois hippocrepis) and Western A fault-line running through this area and exten- false smooth snake (Macroprotodon brevis) sive inland sabkhas (salt flats) suggest there may have only reached the Iberian peninsula from have been an intermittent shallow sea separat- north Africa recently, but have spread exten- ing the more elevated regions on either side of it sively there (Carranza et al., 2004; Carranza and (Gasperetti, 1988). As already noted, a number Arnold, 2006). of taxa, perhaps including Echis, have different Limited genetic divergence (table 2) suggests species distributed along the southern coastal ar- that quite rapid range extension has also oc- eas of southwest Arabia, indicating another re- curred in Echis pyramidum (including E. leuco- gion where some vicariance took place. If this gaster) in North Africa. This may have hap- really applies to Echis, genetic distances (ta- pened after restriction by climatic change al- ble 2) suggest separation of the two forms con- though, if so, the core area where E. pyramidum cerned might have been around the time when survived is not identifiable. There also appears the ancestor of E. coloratus and E. omanensis to have been some restriction after expansion, as speciated. the range of E. pyramidum is very fragmented. Parsimony suggests E. carinatus may have These events are likely to have occurred during originated by vicariance in North Oman and the well-substantiated climatic fluctuations that subsequently expanded eastwards, although the characterised the Pleistocene and late Pliocene topology of the phylogeny (fig. 2) indicates (Sarnthein, 1978; Schuster et al., 2006). Move- this may have involved more than one invasion, ment of E. carinatus in southwest Asia may or alternatively an invasion and a later back- also have been around the same general pe- colonisation. Such movement between North riod. Oman and Iran and neighbouring areas may have taken place in other reptile groups. Hemi- Acknowledgements. We are grateful to Ali Alkiyumi dactylus persicus and Acanthodactylus blan- and Salim Al-Saadi and the Department of Diwan affairs, fordii appear to have moved from Oman into Sultanate of Oman, for permission to collect tissue sam- Iran (Arnold and Carranza, unpublished data) ples in Oman in 2005 (Permit number: 08/2005). E.O.Z. Wade, J.M. Padial and J. Brito provided tissue samples, and this may also have happened in Asacccus J. Roca essential technical assistance, and D. Donaire ac- geckos (Arnold and Gardener, 1994). Move- tive help in the field. E.N. Arnold also thanks Sultan Qa- ments in the opposite direction are likely to have boos Bin Said for permitting him to join the Oman Flora and Fauna Survey 1977, during which the first specimens of occurred in the ancestor of Bunopus hajarensis Echis from Dhofar were collected; and also M.D. Gallagher and B. spatalurus, and in Ablepharus cf. pan- for organising this and many other collecting excursions. Phylogeny of Echis 281

B.C. Groombridge and C.J. MacCarthy provided advice Carranza, S., Arnold, E.N., Mateo, J.A., López-Jurado, L.F. on morphological aspects of this study. Work was funded (2001): Parallel gigantism and complex colonization by grants from the Natural Environment Research Council patterns in the Cape Verde scincid lizards Mabuya and (NERC) NER/B/S/2000/00714 and NER/A/S/2001/00511 Macroscincus (Reptilia: Scincidae) revealed by mito- to E.N. Arnold and by project CGL2005-06876/BOS from chondrial DNA sequences. Proc. R. Soc. Lond. Ser. B. the Ministerio de Educación y Ciencia, Spain and Grant 268: 1595-1603. 2005SGR00045 (Grup de Recerca Emergent) from the Gen- Carranza, S., Arnold, E.N., Wade, E., Fahd, S. (2004): eralitat de Catalunya. 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