Recent advances in phylogeny and taxonomy of Near and Middle Eastern Vipers – an update

Th e number of recognized viper species in the Near and Middle East has been raised signifi cantly in the last 25 years (Table 1). While some smaller genera remained more or less stable, the genus Vipera has been subdivided into four genera on the basis of molecular genetic data. Of these genera, Daboia contains the former Vipera palaestinae and D. russelii, Macrovipera the species M. lebetina, M. schweizeri and an undescribed, basal species from Iran, and Montivipera the former Vipera xanthina and V. raddei complexes. While the genetic diversity in the M. raddei complex is fairly low, it is high in the M. xanthina complex. Th is may give reason to synonymize several taxa in the M. raddei complex, while new taxa can be described in the Turkish M. xanthina complex. Th e number of known species in the Middle Eastern Saw-scaled vipers (genus Echis) must be raised from 2 to 6. Th ese species belong to 3 diff erent species complexes (an Asian, an African and an Arabian complex). A particularly high diversity of Echis is found in southern Arabia. Antivenom producers should pay particular attention to new species in the medically important genera Echis and Macrovipera.


Introduction
Th e taxonomy and phylogeny of Near and Middle Eastern vipers have been controversially discussed in the past.Until the eighties of the 20 th century Palaearctic vipers, except Eristicophis, Echis and Cerastes, were subsumed under the genus Vipera (e.g.Schwarz 1936, Marx & Rabb 1965).Obst (1983) revalidated the genus Daboia for the large Asiatic taxa.Based on immunological comparisons of blood serum albumin Herrmann et al. (1992) resurrected the genus Macrovipera for lebetina and mauritanica and restricted the name Daboia to russelii.In 1999 Nilson et al. introduced the subgenus Montivipera for species of the 'xanthina complex' and 'raddei complex' which was raised to full rank by Joger (2005).
Th e number of recognized species in the region rose from 25 (Joger 1984) to 31 (David & Ineich 1999).Th e purpose of this contribution is to summarize the latest development, partly on the basis of own molecular phylogenetic analyses.
Molecular methods have transformed taxonomy and phylogenetics.First molecular analyses of Herrmann et al. (1999) and Lenk et al. (2001) found Pseudocerastes and Eristicophis, Vipera s. str., Daboia, Macrovipera and Montivipera to be monophyletic groups.Echis and Cerastes were found monophyletic by Joger & Courage (1999) but not by Lenk et al. (2001).Internal relationships and species concepts especially within Montivipera and Macrovipera, but also within Echis, have been debated controversially (e.g.Schätti et al. 1991, Cherlin1990).To elucidate the confusing taxonomy we used large samples and diff erent molecular markers.
Th ese marginal taxa are not typical inhabitants of the Middle East, and therefore will not be focused on in the present paper.

Near and Middle Eastern vipers
Phylogenetic analysis of concatenated mtDNA (Fig. 1) using Bayesian inference (Metropolis-coupled Markov-Chain Monte-Carlo) produced a fully resolved bifurcating topology that strongly supports the monophyly of the genera Pseudocerastes and Eristicophis, Daboia, Macrovipera and Montivipera.Our data confi rm the major topology of Lenk et al. (2001), except the basal position of Vipera s.str.All major branches are supported by robust posterior probabilities, except the monophyly of Macrovipera xanthina.
Th e deeply forked group of desert snakes, Pseudocerastes (Fig. 4) and Eristicophis, is the most basal group, followed by the Afro-Asiatic cluster of D. russelii, D. palaestinae (Fig. 5) and D. mauritanica.Both groups are characterised by a high number of apomorphic states.Th e Pseudocerastes have hornlike scale structures on top of their supraocular scales, a convergence to Cerastes (see below).Th ere are three nominal species: P. fi eldi (northern part of Arabian peninsula and Sinai), P. persicus (Oman, UAE, Iran, Afghanistan, Pakistan) and a newly described species from western Iran, P. urarachnoides Bostanchi et al. (2006).Eristicophis bears a horseshoe-shaped scale on top of its snout and has a prehensile tail.Its single species, E. macmahoni, is restricted to the deserts of Baloutchistan shared by Iran, Afghanistan and Pakistan.Daboia has a disjunct distribution, with one or two North African species (mauritanica, deserti) formery included in Macrovipera, the Levantine D. palaestinae (formerly Vipera palaestinae) and the South Asian Daboia russelii, which reaches the Middle East only peripherally in Pakistan (Joger 1984, Lenk et al. 2001).Daboia is characterized by a raised numbers of body scales.
Montivipera has been the subject of controversial interpretations of species concepts (Schätti et al 1991, 1992and Nilson & Andrén 1992).Following Nilson's and Andrén's species concept, populations of the 'xanthina complex' were geographically isolated along the "Anatolian Diagonal".Th ese vicariance events resulted in the speciation of isolated populations.Consequently, they separated the eastern populations from the west Anatolian xanthina and evaluated three new species albizona, bulgardaghica and wagneri (Nilson & Andrén 1984, 1985a, 1990).In 1991, Schätti et al. present a well founded but oppositional study about the morphological variation of the 'xanthina complex'.In their opinion, the new species are conspecifi c and refl ect merely diff erences between distant and polymorphic populations of xanthina.(Hall 1999) and concatenated into a single amino acid alignment with 2566 positions.Phylogenetic relations were inferred using MrBayes 3.1.2(Ronquist & Huelsenbeck 2001) under the best fi t model (GTR+I+G) selected using MrModeltest 2.2 (Nylander 2004).Two independent runs with one cold and three heated chains (MC3) were run for three million generations sampling every 100 generations and discarding the fi rst 25% of the trees as burnin.Convergence was estimated in Tracer v1.4.1 (Rambaut & Drummond 2007).A more detailed description of material and methods will be published elsewhere (in prep).2) M. bornmuelleri (Levant), wagneri (East Anatolia) and bulgardaghica (Taurus).Note that these haplogroups are concordant with the disjunct geographic distribution.Haplotypes of bulgardaghica are nested within albizona and do not support the validity of albizona.Within the West Anatolian xanthina (s.str.) cluster, there is substantial well supported phylogenetic structure suggesting the presence of taxonomically unrecognised genetic diversity.
Th e genus Macrovipera (Fig. 3) has its main distribution area in Asia.It is found in semi-deserts and steppe habitat of the Levantine countries (Jordan, Syria, Turkey, Iraq, Iran and Azerbaijan) northeastwards to Middle Asia (Turkmenistan, Uzbekistan, Tadzhikistan, Kirgistan and Afghanistan) (Joger 1984, Bruno 1985).Together with Echis, Macrovipera is responsible for the majority of serious, often-lethal clinical problems in western Asia (e.g.Fatehi-Hassanabad and Fatehi 2004).Nevertheless, the taxonomic status of some taxa is still debated.Joger (1984) accepted only the subspecies lebetina and obtusa (Fig. 7), of which the latter includes the synonyms euphratica and   turanica.Nilson and Andrén (1988) raised the isolated island population M. l. schweizeri of the Greece Cyklades to species status and described a new subspecies transmediterranea from Algeria and Tunisia.Central Asian populations were treated as valid subspecies cernovi by Chikin and Szczerbak (1992).In our analysis haplotypes of M. lebetina segregate into four major lineages which support the validity of the allopatric subspecies lebetina, obtusa, turanica and cernovi.

Cerastes and Echis
Th ese two genera have been united in a monophyletic group based on the shared character of serrated lateral scales which they use for producing a warning sound like a rattle-snake (Groombridge 1986).It is considered an adaptation to desert conditions.However, as the harmless colubrid Dasypeltis and some species of African bushvipers (genus Atheris) possess the same type of serrated scales, a convergent development is possible (Joger & Courage 1999).A new phylogenetic analysis (Pook et al., in press) confi rms the monophyly of Echis and Cerastes.
Cerastes, the Horned vipers, do not always bear horns on top of their supraorbital scales.Th ere is a hornless species, Cerastes vipera, which is found in sand dune areas in North Africa, Egypt and Israel/Palestine.Cerastes cerastes, a less specialized species, maybe horned or hornless (Figs 8,9) and occupies, in a number of subspecies, desert and semi-desert areas in North Africa and western Arabia (Werner et al. 1991, Werner andSivan 1992).Th e typical desert snake of most of Arabia and Khuzistan province (Iran) is Cerastes gasperettii Leviton & Anderson, 1967(Gasperetti 1980), the sister species of C. cerastes, but which nearly always bears horns.
Echis, the Saw-scaled vipers, are found in a very large area from West Africa to India, including most of the Middle East countries.Th eir systematics and taxonomy have been a discussed for decades.Klemmer (1963) recognized only two species: Echis carinatus in most of the range and E. coloratus in Arabia.Joger (1984Joger ( , 1987) added E. pyramidum for southwestern Arabia.Cherlin (1990) described a number of new species and subspecies and increased the total number of Echis species signifi cantly.A new species within the E. coloratus group was described by Babocsay (2004).Pook et al. (in press), using molecular genetic methods, have now clarifi ed the complicated situation.On the basis of their results and additional data, we recognize the following six species in the Near and Middle East: Echis  (1990).As we did not fi nd a close phylogenetic relationship between Echis populations from Yemen and Ethiopia, we consider borkini a separate species.
Th ere is a strong zoogeographical division in Arabian Echis, E. sochureki and E. omanensis being found in the eastern part of the peninsula only, whereas southwestern Arabia (including Dhofar province, Oman) is inhabited by E. coloratus, E. khosatzkii and E. cf.borkini (see also Joger 1987).
As Echis bites frequently cause death and successful bite treatment depends on choosing a species-specifi c antivenom (if available), it is of great importance to know which species of Echis occur in which area.Th ere is still need for additional research in this genus.It is also time for an eff ort to study the venom of species like E. khosatzkii and E. cf borkini, and start production of antivenom against their bites.

Figure 1 .
Figure1.Bayesian 50% majority-rule consensus tree of 176 specimens with posterior probability values showing the phylogenetic relations among west palearctic vipers.Th e genealogy was inferred from three protein coding mitochondrial genes (Cytb, COI, ND5).Sequences were aligned separately using Clus-talW(Th ompson et al. 1994) implemented in Bioedit 7.0.9(Hall 1999) and concatenated into a single amino acid alignment with 2566 positions.Phylogenetic relations were inferred using MrBayes 3.1.2(Ronquist& Huelsenbeck 2001)  under the best fi t model (GTR+I+G) selected using MrModeltest 2.2(Nylander 2004).Two independent runs with one cold and three heated chains (MC3) were run for three million generations sampling every 100 generations and discarding the fi rst 25% of the trees as burnin.Convergence was estimated in Tracer v1.4.1(Rambaut & Drummond 2007).A more detailed description of material and methods will be published elsewhere (in prep).
from 70 xanthina complex individuals with 39 haplotypes support the monophyly of two diverging haplogroups with (1) M. xanthina from West Anatolia and (

Figure 2 .
Figure 2. Approximate distribution of Montivipera in Asia Minor, Iran, Levantine and adjacent regions.Geographic origin of sampling locations are indicated by open circles.Nominal taxa are given with corresponding terrae typicae.

Figure 3 .
Figure 3. Approximate distribution of Macrovipera in the Middle East.Geographic origin of sampling locations are indicated by open circles.Nominal taxa are given with corresponding terrae typicae.

Table 1 .
Progress in taxonomical knowledge about Near and Middle East vipers during the last 25 years.Numbers of species in each genus occurring in the area of the Near and Middle East.Th e genus Vipera has been subdivided into 4 genera.Asterisks (*) indicate genera that only occur at the northern margin of the area.transcaucasiana) the latter two probably in the extreme north of Iran.Mountain areas of northern Iran, Afghanistan and Pakistan are also populated by species of the genus Gloydius (formerly Agkistrodon), the only pitvipers (Crotalinae) in the area (see Joger