Molecular Phylogenetics and Evolution Vol. 19, No. 1, April, pp. 94–104, 2001 doi:10.1006/mpev.2001.0912, available online at http://www.idealibrary.com on Evolutionary Relationships among the True Vipers (Reptilia: Viperidae) Inferred from Mitochondrial DNA Sequences Peter Lenk,*,†,1 Svetlana Kalyabina,†,‡ Michael Wink,† and Ulrich Joger* *Zoologische Abteilung, Hessisches Landesmuseum, Friedensplatz 1, D-64283 Darmstadt, Germany; †Institut fu¨ r Pharmazeutische Biologie, Universita¨ t Heidelberg, Im Neuenheimer Feld 364, D-69120 Heidelberg, Germany; and ‡Department of Herpetology, Zoological Institute, Russian Academy of Sciences, Universitetskaya emb.,1, 199034 St. Petersburg, Russia Received April 5, 2000; revised November 7, 2000 iterranean Seas. Taxonomic consequences of these Nucleotide sequences of mitochondrial cyto- findings are discussed. © 2001 Academic Press chrome b and 16S rRNA genes, totaling 946 bp, were used to reconstruct a molecular phylogeny of 42 spe- cies of the subfamily Viperinae representing 12 of the 13 recognized genera. Maximum-parsimony and INTRODUCTION maximum-likelihood were used as methods for phy- logeny reconstruction with and without a posteriori The taxonomy and phylogeny of viperine snakes has weighting. When representatives of the Causinae been controversially discussed in recent years because were taken as outgroup, five major monophyletic morphology-based and molecular trees differ funda- groups were consistently identified: Bitis, Cerastes, mentally in the branching orders proposed (Herrmann Echis, the Atherini (Atheris s.l.), and the Eurasian and Joger, 1997; Herrmann et al., 1999). However, a viperines. Proatheris was affiliated with Atheris, consensus excluding the primitive genera Azemiops and Adenorhinos clustered within Atheris. The Afri- and Causus from the Viperinae was reached and sep- can Bitis consisted of at least three monophyletic arate subfamilies were set up for both of them (Cadle, groups: (i) the B. gabonica group, (ii) the B. caudalis 1988; Groombridge, 1986; Heise et al., 1995; Liem et group, and (iii) the B. cornuta group. B. worthingtoni al., 1971). and B. arietans are not included in any of these Within true vipers, Groombridge (1986) distin- lineages. Eurasian viperines could be unambigu- guished between an African group and a Palaearctic ously devided into four monophyletic groups: (i) group and considered Echis and Cerastes separate en- Pseudocerastes and Eristicophis, (ii) European vi- tities that were tentatively attached to the African pers (Vipera s.str.), (iii) Middle East Macrovipera plus Montivipera (Vipera xanthina group), and (iv) group. Ashe and Marx (1988) and Marx et al. (1988), North African Macrovipera plus Vipera palaestinae however, emphasized characters that united Cerastes and Daboia russelii. These evolutionary lineages are with the Palaearctic Eristicophis and Pseudocerastes, consistent with historical biogeographical patterns. but placed Echis together with the oriental Daboia According to our analyses, the viperines originated russelii and with the Afrotropical genus Bitis. An even in the Oligocene in Africa and successively under- stronger disagreement with biogeographical patterns went a first radiation leading to the five basal was found in these authors’ disruption of Vipera s.l. groups. The radiation might have been driven by the (Vipera, Macrovipera, and Daboia) into a polyphyletic possession of an effective venom apparatus and a assemblage of independent clades. Herrmann and foraging startegy (sit–wait–strike) superior in most Joger (1997) reanalyzed both data sets of Groombridge African biomes and might have been adaptive. The and of Ashe and Marx but found meaningful phyloge- next diversifications led to the Proatheris–Atheris netic signals only in Groombridge’s data. Later, Herr- furcation, the basal Bitis splitting, and the emer- mann et al. (1999) enriched Groombridge’s data set gence of the basal lineages within the Eurasian with cytochrome b amino acid sequence data, confirm- stock. Thereafter, lineages within Echis, Atheris, ing the monophyly of the African and the Palaearctic and Cerastes evolved. The emergence of three group and the affiliation of Echis and Cerastes to the groups within Vipera s.l. might have been forced by African group. Microdermatoglyphic patterns sup- the existence of three land masses during the early ported an association of Echis (and to a lesser degree of Miocene in the area of the Paratethys and the Med- Cerastes) with Atheris (Joger and Courage, 1999). 1 To whom correspondence should be addressed. E-mail: However, immunological data contradict this view, [email protected]. as they position Echis and Cerastes closer to the Palae- 94 1055-7903/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved. RELATIONSHIPS AMONG TRUE VIPERS 95 arctic Macrovipera and the Oriental Daboia (Herr- dye cy5) used were L 14846, H 15556/7, H 15149, L mann and Joger, 1995, 1997). A preliminary analysis of 15162, L 2510, and H 3062. partial cytochrome b gene sequences was ambiguous, For amplification, total DNA was used as a template depending on the particular tree reconstruction algo- plus 25 pmol of the above-mentioned primers, 1.5 mM rithm used, and even positioned Cerastes as a basal MgCl2, 0.1 mM each dNTP, 5 ␮l amplification buffer, taxon in the viperine tree (Herrmann et al., 1999). and 1.5 U Taq DNA Polymerase (Amersham Pharma- In this study we have analyzed nucleotide sequences cia Biotech) in a total volume of 50 ␮l. After an initial of the mitochondrial cytochrome b and 16S rRNA genes denaturation step (4 min at 94°C) 30 cycles of 30 s at and used them in a combined data set. The aim of this 95°C, 30 s at 45°C, and 90 s at 72°C were performed on study was to further elucidate phylogenetic relation- a Biometra thermocycler. After 30 cycles the reaction ships in the viperine complex. temperature was maintained at 72°C for 4 min and We use the term “Eurasian vipers” for palaearctic than lowered to 4°C for further storage. The quality of genera (Eristicophis, Pseudocerastes, Vipera and Mac- the resulting PCR products was controlled by electro- rovipera) plus Daboia. The term “Vipera s.l.” is applied phoresis on a 1.5% agarose gel (Qualex Gold Agarose; to Vipera, Macrovipera, and Daboia. The latter two FMC Bioproducts). Depending on the strength of the were taxonomically separated from Vipera by Herr- band, 1 to 6 ␮l of the PCR product was used to perform mann et al. (1992). The unit “Atherini” includes the direct cycle sequencing of the PCR products employing genera Atheris, Proatheris, Montatheris (not studied the “ThermoSequenase flourescent labeled primer cy- here), and Adenorhinos (see Fig. 1). cle sequencing kit with 7-deaza-dGTP” (Amersham Pharmacia Biotech) according to the protocol of the manufacturer. Cycle sequencing was performed at MATERIAL AND METHODS 94°C for 3 min, followed by 25 cycles of 94°C for 30 s and 50°C for 90 s. The sequencing products were DNA was extracted from blood collected by caudal loaded on longranger acrylamide gel (FMC Bioprod- vein puncture as described in Joger and Lenk (1995) ucts) without further purification and run on an ALF from living specimens or tissue samples removed from EXPRESS II sequencer (Amersham Pharmacia Bio- freshly dissected or ethanol-preserved animals. The tech) for automatic fluorescence detection of the nucle- dataset includes 42 species representing 12 of the 13 otide sequence. Both heavy and light strands were recognized viperine genera with the exception of Mon- sequenced in such a way that large overlapping seg- tatheris, a monotypic genus of East Africa that has ments (80%) were obtained. All sequence outputs were been recently separated from Atheris (Broadley, 1996). compared with the electrophoretograms and aligned The selection of outgroup species was done on the manually using the human sequence (Accession No. strength of taxonomical considerations. We regarded J01415) as reference (Anderson et al., 1981). 16S rRNA the genus Causus as the most important candidate, as sequences were aligned with the aid of the secondary it is classified in a separate subfamily and lacks apo- structure model of the human sequence (Gutell and morphic states of viperines in several characters Fox, 1988) to optimize site homology in the alignment. (venom apparatus, topology of arteries, shape of pupils, Sequences were deposited at GenBank/EMBL (Acces- scalation, reproduction), but simultaneously shares sion Nos. AJ275679–AJ275784; see Table 1). with viperines some synapomorphic states in other Because the resolution of the 16S rRNA data set characters (discussed in Underwood, 1999) which give alone was limited due maybe to the low number of reason to assume a sister group relationship. informative characters, we combined the sequences Additional analyses were performed with other out- from the cytochrome b and 16S rRNA genes into one groups (i.e., the viperid Azemiops feae and the colubrid data set. We used MEGA version 1.01 (Kumar et al., Dinodon semicarinatus as more distantly related alter- 1993) to calculate base compositional frequencies for natives). Table 1 shows all specimens used. both genes. Sequence divergences were calculated with Tissue samples were stored in 95% ethanol or PAUP* 4b3a (Swofford, 1998). To test the amount of EDTA–buffer (10% EDTA, 0.5% sodium fluoride, 0.5% phylogenetic information in the data set, the g1 value thymol, 1% Tris, pH 7.0; Arctander, 1988) at Ϫ20°C. (Hillis and Huelsenbeck, 1992) derived from 10,000 Total genomic DNA was prepared following standard random trees was determined with PAUP* 4b3a. proteinase K and phenol chloroform protocols (Sam- Many philosophies and algorithms concerning tech- brook et al., 1989). Portions of the cytochrome b and niques for extracting maximum information from DNA 16S rRNA gene were amplified using the polymerase sequence data exist. Systematic analyses should princi- chain reaction. Primers listed in Table 2 were used in pally avoid assumptions about evolutionary processes in varying combinations to obtain the desired cytochrome their methods (Mindell and Thacker, 1996). However, b fragment of all species.