ABlackwell Publishing mitogenomic Ltd study on the phylogenetic position of snakes DESIRÉE A. DOUGLAS, AXEL JANKE & ULFUR ARNASON Accepted: 16 August 2006 Douglas, D. A., Janke, A. & Arnason, U. (2006). A mitogenomic study on the phylogenetic doi:10.1111/j.1463-6409.2006.00257.x position of snakes. — Zoologica Scripta, 35, 545–558. Phylogenetic relationships of squamates (lizards, amphisbaenians and snakes) have received considerable attention, although no consensus has been reached concerning some basal diver- gences. This paper focuses on the Serpentes (snakes), whose phylogenetic position within the Squamata remains uncertain despite a number of morphological and molecular studies. Some mitogenomic studies have suggested a sister-group relationship between snakes and varanid lizards, while other studies have identified snakes and lizards as sister groups. However, recent studies using nuclear data have presented a different scenario, with snakes being more closely related to anguimorph and iguanian lizards. In this mitogenomic study we have examined the above hypotheses with the inclusion of amphisbaenians, one gekkotan and one acrodont lizard, taxa not represented in previous mitogenomic studies. To this end we have also extended the representation of snakes by sequencing five additional snake genomes: two scolecophidians (Ramphotyphlops australis and Typhlops mirus) two henophidians (Eunectes notaeus and Boa constrictor) and one caenophidian (Elaphe guttata). The phylogenetic analysis recovered snakes and amphisbaenians as sister groups, thereby differing from previous hypotheses. In addition to a discussion on previous morphological and molecular studies in light of the results presented here, the current study also provides some details regarding features of the new snake mitochondrial genomes described. Desirée Douglas, ,Division of Evolutionary Molecular Systematics, Department of Cell and Organism Biology, University of Lund, Sölvegatan 29, 22362 Lund, Sweden. E-mail: [email protected] Axel Janke, Division of Evolutionary Molecular Systematics, Department of Cell and Organism Biology, University of Lund, Sölvegatan 29, 22362 Lund, Sweden. E-mail: [email protected] Ulfur Arnason, Division of Evolutionary Molecular Systematics, Department of Cell and Organism Biology, University of Lund, Sölvegatan 29, 22362 Lund, Sweden. E-mail: [email protected] Introduction Caldwell, 2000; Lee et al. 1999; Caldwell and dal Sasso 2004). The position of Serpentes (snakes) within the Squamata has On the grounds that these fossils possess hindlimbs, and are been debated since the 19th century (e.g. Cope 1869). The therefore taken to be primitive, they have been placed as the traditional view is that snakes arose from within the lizards, sister group to all living snakes (Caldwell & Lee 1997; Lee & although Underwood (1970) suggested that lizards and snakes Caldwell 2000; Scanlon & Lee 2000). could have separate origins. While the traditional view has An alternative hypothesis is that snakes evolved from gained almost universal acceptance, opinion has differed as to terrestrial lizards (Greene 1997; Greene & Cundall 2000; which group of lizards is most closely related to snakes. One Rieppel et al. 2003), more specifically those with a nocturnal hypothesis posits that the closest living relatives of snakes are and/or burrowing habit resembling that of blind snakes varanid (monitor) lizards, which belong to the infraorder (scolecophidians), which are traditionally thought to be the Anguimorpha. Some morphological studies have, with the most basal of living snakes (Bellairs & Underwood 1951; inclusion of fossil data, placed snakes within the Mosasauroidea Underwood 1970; Pough et al. 2005). Some authors have argued — a group of large, marine, extinct varanoids that lived during that fossil snakes also possessed more advanced characters the Cretaceous period. This grouping is supported by skeletal (e.g. in the skull), and that this justifies a more derived placement characters common to both mosasauroids and fossil snakes than that of scolecophidians, which would mean that fossil purported to have been marine (Caldwell & Lee 1997, Lee & snakes do not have any bearing on snake origins (Zaher © 2006 The Authors. Journal compilation © 2006 The Norwegian Academy of Science and Letters • Zoologica Scripta, 35, 6, November 2006, pp545–558 545 Phylogenetic position of snakes • D. A. Douglas et al. 1998; Coates & Ruta 2000; Tchernov et al. 2000; Greene & new snake genomes that include representatives from all Cundall 2000; Zaher & Rieppel 2002; Rieppel et al. 2003). three major lineages of snakes: two scolecophidians ( Jan’s Previous molecular studies based on mitochondrial (mt) blind snake, Typhlops mirus and the southern blind snake, gene data (Forstner et al. 1995; Rest et al. 2003) have placed Ramphotyphlops australis), two henophidians (the yellow varanid lizards as the sister group of snakes, in support of the anaconda, Eunectes notaeus and the Columbian red-tailed snake−mosasauroid hypothesis. Other molecular studies boa, Boa constrictor imperator), and one caenophidian (the corn based on one or two nuclear genes (c-mos and RAG-1) have snake, Elaphe guttata) (see Table 1). This was done to increase all recovered a group containing snakes, anguimorphs and the taxon sampling across Serpentes for which, prior to this iguanians (Saint et al. 1998; Harris 2003; Vidal & Hedges study, only two mt genomes — Leptotyphlops dulcis and Dinodon 2004; Townsend et al. 2004). In addition, a study by Vidal & semicarinatus — had been sequenced. Hedges (2005), based on nine nuclear genes, also produced Six additional alethinophidian (i.e. all snakes with the the same result, with snakes as the sister group to anguimorphs exception of scolecophidians) snake genomes were recently and iguanids. In comparison, two recent studies based on all described (Dong & Kumazawa 2005). This included another mt genes (Kumazawa 2004; and Dong & Kumazawa 2005) Boa constrictor. However, for the purposes of this study it was have placed snakes as the sister group of lizards. However, no important to increase the sampling of the most basal group — amphisbaenians, gekkotans or acrodonts were included in the Scolecophidia — as it was apparent from this, and previous, these studies. studies (Kumazawa 2004; Dong & Kumazawa 2005) that In this study we aimed to test the above hypotheses on the snake mt genes have a much faster rate of evolution than position of snakes using heavy-strand protein-coding mt those of other squamates. Although the mt genomes of some genes for phylogenetic analysis. The sampling included five lizard families have not been sequenced, the current study Table 1 The names and GenBank accession Taxon (scientific name) Common name Accession numbers of mt sequences of all species used Snakes Elaphe guttata guttata* Corn snake AM 236349 in this study. Dinodon semicarinatus Ryukyu odd-tooth snake NC 001945 Boa constrictor imperator* Boa constrictor AM 236348 Eunectes notaeus*Yellow anaconda AM 236347 Ramphotyphlops australis* Southern blind snake AM 236346 Typhlops mirus*Jan’s blind snake AM 236345 Leptotyphlops dulcis Texas blind snake NC 005961 Lizards Varanus komodoensis Komodo dragon AB080275 and AB080276 Abronia graminea Green arboreal alligator lizard NC 005958 Shinisaurus crocodilurus Crocodile lizard NC 005959 Cordylus warreni Warren’s spiny-tail lizard NC 005962 Eumeces egregius Mole skink NC 000888 Sceloporus occidentalis Western fence lizard NC 005960 Iguana iguana Common iguana NC 002793 Pogona vitticeps Central bearded dragon NC 006922 Teratoscincus keyserlingii Giant frog-eyed gecko NC 007008 Amphisbaenians Bipes biporus Five-toed worm lizard NC 006287 Amphisbaena schmidti Schmidt’s worm lizard NC 006284 Diplometopon zarudnyi Zarudnyi’s worm lizard NC 006283 Rhineura floridana Florida worm lizard NC 006282 Shpenodontidans Sphenodon punctatus Tuatara NC 004815 Crocodilians Caiman crocodylus Spectacled caiman NC 002744 Alligator mississippiensis American alligator NC 001922 Birds Gallus gallus Chicken NC 001323 Struthio camelus Ostrich NC 002785 Turtles Chelonia mydas Green turtle NC 000886 Chrysemys picta Painted turtle NC 002073 Mammals Mus musculus House mouse NC 005089 Didelphis virginiana North American opossum NC 001610 Amphibians Ranodon sibiricus Siberian salamander NC 004021 Xenopus laevis African clawed frog NC 001573 *Taxa sequenced in this study. 546 Zoologica Scripta, 35, 6, November 2006, pp545–558 • © 2006 The Authors. Journal compilation © 2006 The Norwegian Academy of Science and Letters D. A. Douglas et al. • Phylogenetic position of snakes allows examination of recent hypotheses on snake origin as it includes taxa purported to be their closest living sister groups, anguimorphs and iguanians. We also aimed to investigate mitogenomic features in the new genomes sequenced. The mt genomes of snakes are interesting in that they contain gene duplications and rearrangements. Kumazawa (2004) reported a novel position of the tRNA-Gln gene in the mt genome of the Texas blind snake (L. dulcis) and the absence of the origin of light strand replication (OL) that could be characteristic of all scole- cophidian genomes. Duplicated control regions have been reported in all alethinophidian snakes (Kumazawa et al. 1998; Dong & Kumazawa 2005). Gene rearrangements and duplication events have been used as potential phylogenetic markers in previous analyses (e.g. Macey et al. 1997, 2000, 2004) and are discussed in this study. Materials and