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Molecular Phylogenetics and Evolution 51 (2009) 190–200 Molecular Phylogenetics and Evolution 51 (2009) 190–200 Contents lists available at ScienceDirect Molecular Phylogenetics and Evolution journal homepage: www.elsevier.com/locate/ympev Molecular phylogenetics reveals extreme morphological homoplasy in Brazilian worm lizards challenging current taxonomy Tamí Mott a,*, David R. Vieites b a Departamento de Zoologia, Instituto de Biociências, Universidade de São Paulo, Rua do Matão, travessa 14, No. 321, Cidade Universitária, CEP 05508-900 São Paulo, SP, Brazil b Museo Nacional de Ciencias Naturales, CSIC, C/ José Gutierrez Abascal 2, Madrid 28006, Spain article info abstract Article history: Amphisbaenians are fossorial squamate reptiles distributed mainly in South America and Africa. Brazilian Received 13 April 2008 worm lizards belong to the family Amphisbaenidae, which has far more recognized species than any of Revised 20 January 2009 the other five amphisbaenian families. Morphological datasets recovered Amphisbaenidae as paraphylet- Accepted 25 January 2009 ic, while previous molecular phylogenetic studies did not include enough taxa to solve the generic-level Available online 1 February 2009 relationships within this family. We present a molecular phylogenetic hypothesis based on a sample of 58 amphisbaenians, including representatives of six of the seven South American genera. Our molecular data Keywords: include sequences from two mitochondrial genes (16S, ND2; 1,184 characters) and three nuclear genes Molecular phylogeny (RAG-1, C-MOS, BDNF; 1,898 characters). Our phylogenetic hypothesis is not fully resolved, although it Systematics Amphisbaenidae does not support the monophyly of most genera except Leposternon. Morphological characters currently South America used to diagnose genera of South American amphisbaenians are homoplastic, and the taxonomy based on Morphological homoplasy them is not appropriate. We revise the taxonomy of this group and sink several South American genera of Amphisbaenidae (Cercolophia, Bronia, Aulura, Anops and Leposternon) into Amphisbaena. Ó 2009 Elsevier Inc. All rights reserved. 1. Introduction morphotypes evolved independently on different continents (Kearney and Stuart, 2004). Most authors claim that these charac- Morphological data are the base for the taxonomy of many ters may have evolved by convergence, although parallelism, groups, and have been widely used for phylogenetic reconstruc- understood as the independent evolution of similar traits starting tion. However, those data are prone to homoplasy (Hedges and from a similar ancestral condition, is another possibility (Kearney, Maxson, 1996; Wiens et al., 2003) and may mislead phylogenetic 2003; Kearney and Stuart, 2004). The morphological characters and taxonomic interpretations. Disagreement between molecular used to diagnose genera and species of amphisbaenians may be and morphological data as a result of homoplastic evolution of subject to homoplastic evolution, which may confound the infer- morphological characters is common in many groups. Inference ence of phylogeny in this group. of the phylogenetic relationships in squamates using morphologi- The Suborder Amphisbaenia contains the family Amphisbaeni- cal characters related to limb reduction or loss has been problem- dae. Among the six currently recognized families of amphisbae- atic. This is because those characters show homoplastic evolution nians, Amphisbaenidae has far more recognized species than any in squamate evolutionary history (Brandley et al., 2005; Estes other amphisbaenian family. It includes 15 recognized genera et al., 1988; Kearney, 2003; Kearney and Stuart, 2004; Whiting and at least 175 species. This family is geographically widespread, et al., 2003; Wiens et al., 2006). Limb loss and some degree of fos- with species occurring in Africa, South and Central America (Gans, soriality occurred at least 25 times during squamate evolution 1978, 1990, 2005; Kearney and Stuart, 2004; Macey et al., 2004; Vi- (Wiens et al., 2006). In amphisbaenians, a group of fossorial squa- dal et al., 2008), but no shared genera between continents. Inter- mates of which nearly all species are limbless, limb loss occurred at estingly, this family shows a high diversity of cranial least three times (Kearney and Stuart, 2004). Furthermore, cranial morphotypes. There are three main types of head shapes (round, features, which are also of taxonomic utility in amphisbaenians, shovel and keel-headed) in this family, with representatives of show homoplasy in this group (Kearney and Stuart, 2004). Differ- each morphotype on both continents (Kearney, 2003). Three gen- ent cranial morphotypes are associated with particular stereotyped era, Amphisbaena, Cercolophia and Bronia, show the round-headed burrowing behaviors (Gans, 1974), and these different cranial morphotype in Central and South America, as Chirindia, Cynisca, Loveridgea and Zygapsis do in Africa. Among shovel-headed amphisbaenids, Aulura and Leposternon occur in South America, * Corresponding author. E-mail addresses: [email protected] (T. Mott), [email protected] (D.R. and Dalophia and Monopeltis in Africa. Anops and Mesobaena are Vieites). representatives of keel-headed amphisbaenids that occur in South 1055-7903/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.ympev.2009.01.014 T. Mott, D.R. Vieites / Molecular Phylogenetics and Evolution 51 (2009) 190–200 191 America, and Ancylocranium and Geocalamus show this condition in keel-headed amphisbaenid restricted to Venezuela and Colombia Africa. was the only South American genus not included in this study. Cladistic analysis of morphological data suggests paraphyly of We also included in the analyses an African genus of amphisbaenid the family Amphisbaenidae and of the South American amphisbae- (Geocalamus) and at least one representative of all other families, nids (Kearney, 2003). However, the classification according to cra- except Cadeidae, summarizing 34 species of amphisbaenians. Table nial morphotypes was tentative because of potential homoplasy 1 provides a list of the taxa included, including voucher, museum (Kearney, 2003). Molecular phylogenetic hypotheses confirmed and locality information. homoplasy of head shapes (Kearney and Stuart, 2004; Macey We assembled a comprehensive multi-locus dataset. We et al., 2004; Vidal et al., 2008), and disputed the classification based sequenced two mitochondrial and three nuclear markers. Mito- on head morphology (Kearney and Stuart, 2004). Those molecular chondrial genes included the 16S ribosomal RNA (16S) and the studies had a limited representation of Amphisbaenidae (two and NADH dehydrogenase subunit 2 (ND2). Nuclear markers included ten species included in Macey et al., 2004 and Kearney and Stuart, the recombination-activating gene 1 (RAG-1), the nuclear proto- 2004, respectively), and the statistical support for many amphis- oncogene c-mos (C-MOS) and the brain-derived neurotrophic fac- baenid phylogenetic relationships was low. A recent biogeograph- tor (BDNF). Among the genes present in the mitochondrion the ical study on amphisbaenians using genetic data (Vidal et al., 2008) 16S is the most slowly evolving one (Mueller et al., 2004). The included 13 species of West Indian amphisbaenids. Their results ND2 gene has performed well in previous phylogenetic estimations supported monophyly of the family Amphisbaenidae and of the (Zardoya and Meyer, 1996), and it has been used in squamate phy- New World amphisbaenids, but lack most of the genera. The study logenetics (Macey et al., 1997; Townsend et al., 2004). The three included only one genus of New World worm lizards from Central nuclear markers used for phylogenetic inference are functionally America. The phylogenetic relationships of the South American independent, unlinked, and present unique evolutionary patterns. amphisbaenids are still not resolved, both at the species and gen- Previous phylogenetic studies used RAG-1 and C-MOS in squa- eric levels, and a comprehensive study investigating this question mates, including amphisbaenians (Townsend et al., 2004; Kearney is lacking. and Stuart, 2004). BDNF performed well for phylogenetic inference The foundations of the current taxonomy in this clade are mor- in several tetrapod groups (Noonan and Chippindale, 2006; Vieites phological characters used to diagnose genera and species. We et al., 2007). Many studies applied mitochondrial markers to infer analyze the phylogenetic relationships of South American genera species-level phylogenies in squamates, while the use of nuclear of the family Amphisbaenidae by assembling a comprehensive markers is more recent. We analyzed both nuclear and mitochon- multi-locus molecular dataset. The taxa included represent all dif- drial datasets to test for differences in the phylogenetic signal, and ferent cranial morphotypes and include the type species of each performed combined phylogenetic analyses. genus. Most amphisbaenian species are known only from old type We included published information from Genbank for three series, making tissues for molecular work unavailable. In addition, species, Rhineura floridana, Bipes canaliculatus, and Blanus strauchi. the extent and sampling difficulties of the South American conti- (Accession numbers AY605473, AY605484 and AY444024, respec- nent make a continental analysis not yet possible. Therefore, we tively). These species were the only ones for which mitochondrial have focused our efforts mainly in Brazil for several reasons. Nearly and nuclear sequences were not
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