Molecular Phylogenetics and Evolution Vol. 12, No. 3, August, pp. 250–272, 1999 Article ID mpev.1999.0615, available online at http://www.idealibrary.com on Molecular Phylogenetics, tRNA Evolution, and Historical Biogeography in Anguid Lizards and Related Taxonomic Families J. Robert Macey,*,1 James A. Schulte II,* Allan Larson,* Boris S. Tuniyev,† Nikolai Orlov,‡ and Theodore J. Papenfuss§ *Department of Biology, Box 1137, Washington University, St. Louis, Missouri 63130; †Caucasian State Biosphere Reserve, Sochi, Russia; ‡Zoological Institute, Russian Academy of Sciences, St. Petersburg, Russia; and §Museum of Vertebrate Zoology, University of California, Berkeley, California 94720 Received December 24, 1997; revised October 15, 1998 persed to Asia via the Bering land bridge with subse- Phylogenetic relationships among lizards of the fami- quent colonization of Europe and Morocco, requires a lies Anguidae, Anniellidae, Xenosauridae, and Shinisau- phylogenetic tree seven steps longer than the most ridae are investigated using 2001 aligned bases of parsimonious hypothesis. North African, European, mitochondrial DNA sequence from the genes encoding and West Asian anguines were isolated from others by ND1 (subunit one of NADH dehydrogenase), tRNAIle, the rapid uplift of Tibet in the late Oligocene to tRNAGln, tRNAMet, ND2, tRNATrp, tRNAAla, tRNAAsn, Miocene. Phylogenetic analysis of evolutionary tRNACys, tRNATyr, and COI (subunit I of cytochrome c changes in the gene encoding tRNACys suggests gradual oxidase), plus the origin for light-strand replication reduction of dihydrouridine (D) stems by successive Asn Cys (OL) between the tRNA and the tRNA genes. The deletion of bases in some lineages. This evolutionary aligned sequences contain 1013 phylogenetically infor- pattern contrasts with the one observed for parallel mative characters. A well-resolved phylogenetic hy- elimination of the D-stem in mitochondrial tRNAs of pothesis is obtained. Because monophyly of the family eight other reptile groups, in which replication slip- Xenosauridae (Shinisaurus and Xenosaurus) is statisti- page produces direct repeats. An unusual, enlarged cally rejected, we recommend placing Shinisaurus in a T␺C (T) stem is inferred for tRNACys in most separate family, the Shinisauridae. The family Annielli- species. ௠ 1999 Academic Press dae and the anguid subfamilies Gerrhonotinae and Key Words: Reptilia; Sauria; Anguimorpha; Anguidae; Anguinae each form monophyletic groups receiving Anniellidae; Shinisauridae; Xenosauridae; Asia; Eu- statistical support. The Diploglossinae*, which ap- rope; Morocco; North America; historical biogeogra- pears monophyletic, is retained as a metataxon (de- phy; mitochondrial DNA; cysteine tRNA; phylogenetics noted with an asterisk) because its monophyly is statis- tically neither supported nor rejected. The family Anguidae appears monophyletic in analyses of the Anguid lizards, found predominately in the northern DNA sequence data, and statistical support for its hemisphere, are an exciting group for a molecular monophyly is provided by reanalysis of previously phylogenetic study of biogeographic fragmentation be- published allozymic data. Anguid lizards appear to tween North America and Eurasia. The anguimorph have had a northern origin in Laurasia. Taxa currently located on Gondwanan plates arrived there by dis- family Anguidae contains three subfamilies. The sub- persal from the north in two separate events, one from family Gerrhonotinae occurs strictly in North America the West Indies to South America and another from a and Central America. The subfamily Diploglossinae Laurasian plate to Morocco. Because basal anguine ranges from Mexico and the West Indies to South lineages are located in western Eurasia and Morocco, America. The subfamily Anguinae, comprising the gen- formation of the Atlantic Ocean (late Eocene) is impli- era Anguis and Ophisaurus, is the only anguid subfam- cated in the separation of the Anguinae from its North ily that occurs in the Old World. Anguis is restricted to American sister taxon, the Gerrhonotinae. Subsequent Europe, whereas Ophisaurus is found in eastern North dispersal of anguine lizards to East Asia and North America, eastern Asia, western Asia, and adjacent America appears to have followed the Oligocene dry- Europe and Morocco. ing of the Turgai Sea. The alternative hypothesis, that Some taxonomists consider the anguimorph lizard anguine lizards originated in North America and dis- family Anniellidae a fourth subfamily of the Anguidae (see Gauthier, 1982). It comprises two species, Anniella 1 To whom correspondence should be addressed. Fax: (314) 935- geronimensis and A. pulchra, from the west coast of 4432. E-mail: [email protected]. North America. Three major hypotheses have been 250 1055-7903/99 $30.00 Copyright ௠ 1999 by Academic Press All rights of reproduction in any form reserved. PHYLOGENETICS AND BIOGEOGRAPHY OF ANGUID LIZARDS 251 considered for the phylogenetic position of the Annielli- phylogenetic analyses of morphological data (Estes et dae relative to the Anguidae: (1) Anniella is the sister al., 1988; Macey et al., 1997a; Schwenk, 1988) cannot group to all anguid taxa (Good, 1987), (2) Anniella is determine whether the Varanoidea or the Xenosauri- the sister group to the Anguinae (Gauthier, 1982), and dae is closer to the anguid and anniellid clade; this (3) Anniella is the sister taxon to Anguis of Europe question, however, is not a focus of this study. (Keqin and Norell, 1998). Phylogenetic relationships are examined using 2001 The anguimorph family Xenosauridae occurs in the aligned positions (1013 informative) of mitochondrial New World (Xenosaurus) and Old World (Shinisaurus). DNA sequence. The region sequenced extends from Some authors consider Shinisaurus a separate mono- the protein-coding gene, ND1 (subunit one of NADH typic family, the Shinisauridae (see Zhao and Adler, dehydrogenase), through the genes encoding tRNAIle, 1993). No previous molecular study has examined the tRNAGln, tRNAMet, ND2, tRNATrp, tRNAAla, tRNAAsn, relationships of these taxa. tRNACys, and tRNATyr to the protein-coding gene COI A major question within the Anguinae is the phyloge- (subunit I of cytochrome c oxidase), and includes the netic position of Anguis fragilis, Ophisaurus apodus, replication origin for the light strand (O ) between the and O. koellikeri, which occur between two extremely L tRNAAsn and the tRNACys genes. large barriers to faunal distributions, the Atlantic Previously published allozymic data (Good, 1987, Ocean and the Tibetan Plateau. Current taxonomy 1988) are reanalyzed and compared with the results implies at least two separate origins of anguid lizards in this region. Mitochondrial DNA sequences are re- obtained from the new DNA sequence data to provide a ported for these taxa as well as for the East Asian comprehensive assessment of relationships among the O. harti and the North American O. attenuatus and taxa investigated. O. ventralis. The mitochondrial genomic region sequenced demon- We examine all genera within the Gerrhonotinae strates several unusual characteristics among squa- except Coloptychon, which is known from only three mate reptiles (Kumazawa and Nishida, 1995; specimens. Abronia oaxacae, Barisia imbricata, Gerrho- Kumazawa et al., 1996; Macey et al., 1997a,b,c; Seutin notus liocephalus, and Mesaspis moreleti represent the et al., 1994). Within anguimorph lizards, gene se- four tropical genera in our analysis. Five species of quences encoding tRNACys lack a dihydrouridine (D) Elgaria, primarily from the temperate part of North stem and instead contain a D-arm replacement loop in America, are examined. Elgaria coerulea, sampled from Varanus (Macey et al., 1997b). A model involving repli- coastal California, is the most northern species of the cation slippage has been proposed for the formation of Gerrhonotinae. Two other species are included from D-arm replacement loops in mitochondrial tRNAs California, E. multicarinata collected from the east side (Macey et al., 1997b). Under this model direct repeats of the Sierra Nevada and E. panamintina from an are expected and the size of the D-arm replacement adjacent population in the Inyo Mountains. Elgaria loop should be less than 12 bases, the minimum num- kingii, sampled from Arizona, occurs along the west ber of bases normally found between the amino acid- coast of Mexico in the Sierra Madre Occidental opposite acceptor (AA) and the anticodon (AC) stems when a Baja California where E. paucicarinata was obtained D-stem is present (Macey et al., 1997b). Alternatively, from the Sierra de La Laguna. This choice of species gradual relaxation of pairing among bases in the allows an examination of taxa from both sides of the D-stem would not produce either repeats or deletion of Gulf of California, a region of rifting between tectonic bases. Gradual deletion of bases or base pairs would not plates. produce repeats but could produce length variation All genera of the neotropical subfamily Diploglossi- that may result in less than 12 bases between the AA- nae are examined. Ophiodes striatus represents the and AC stems. Under a model of gradual deletion of only endemic anguid genus in South America. Celestus enneagrammus from Mexico and Diploglossus biloba- bases or base pairs, a tRNA that has a single base tus from Costa Rica represent mainland North Ameri- pairing in the D-stem could result, as has been ob- Asn can diploglossines, and Diploglossus pleei, Sauresia served in the tRNA gene of the