Molecular Evidence for Gondwanan Origins of Multiple Lineages Within A
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Journal of Biogeography (J. Biogeogr.) (2009) 36, 2044–2055 ORIGINAL Molecular evidence for Gondwanan ARTICLE origins of multiple lineages within a diverse Australasian gecko radiation Paul M. Oliver1,2* and Kate L. Sanders1 1Centre for Evolutionary Biology and ABSTRACT Biodiversity, University of Adelaide and Aim Gondwanan lineages are a prominent component of the Australian 2Terrestrial Vertebrates, South Australian Museum, North Terrace, Adelaide, SA, terrestrial biota. However, most squamate (lizard and snake) lineages in Australia Australia appear to be derived from relatively recent dispersal from Asia (< 30 Ma) and in situ diversification, subsequent to the isolation of Australia from other Gondwanan landmasses. We test the hypothesis that the Australian radiation of diplodactyloid geckos (families Carphodactylidae, Diplodactylidae and Pygopodidae), in contrast to other endemic squamate groups, has a Gondwanan origin and comprises multiple lineages that originated before the separation of Australia from Antarctica. Location Australasia. Methods Bayesian (beast) and penalized likelihood rate smoothing (PLRS) (r8s) molecular dating methods and two long nuclear DNA sequences (RAG-1 and c-mos) were used to estimate a timeframe for divergence events among 18 genera and 30 species of Australian diplodactyloids. Results At least five lineages of Australian diplodactyloid geckos are estimated to have originated > 34 Ma (pre-Oligocene) and basal splits among the Australian diplodactyloids occurred c. 70 Ma. However, most extant generic and intergeneric diversity within diplodactyloid lineages appears to post-date the late Oligocene (< 30 Ma). Main conclusions Basal divergences within the diplodactyloids significantly pre-date the final break-up of East Gondwana, indicating that the group is one of the most ancient extant endemic vertebrate radiations east of Wallace’s Line. At least five Australian lineages of diplodactyloid gecko are each as old or older than other well-dated Australian squamate radiations (e.g. elapid snakes and agamids). The limbless Pygopodidae (morphologically the most aberrant living geckos) appears to have radiated before Australia was occupied by potential ecological analogues. However, in spite of the great age of the diplodactyloid radiation, most extant diversity appears to be of relatively recent origin, a pattern that is shared with other Australian squamate lineages. *Correspondence: Paul Oliver, Centre for Keywords Evolutionary Biology and Biodiversity, Australasia, Bayesian analysis, Carphodactylidae, Diplodactylidae, divergence University of Adelaide, Adelaide, 5005 SA, Australia. times, geckos, Gondwana, historical biogeography, Pygopodidae, relaxed-clock E-mail: [email protected] dating. evolved in relative geographical isolation, originating from INTRODUCTION either of two sources: (1) an ancient Gondwanan biota that The Australian biota is dominated by diverse and largely became isolated in Australasia as the northward-drifting Indo- endemic radiations (Keast, 1981; Heatwole, 1987; Crisp et al., Australian tectonic plate detached from Antarctica c. 55– 2004). It has become widely accepted that these lineages 32 Ma; and (2) a modern fauna derived from over-water 2044 www.blackwellpublishing.com/jbi ª 2009 Blackwell Publishing Ltd doi:10.1111/j.1365-2699.2009.02149.x Gondwanan origins of Australasian geckos dispersals, usually from the north after the Australian and Asian not yet been assessed comprehensively and directly using plates came in close enough proximity to allow island-hopping, molecular data. In this paper we use two nuclear sequences to beginning c. 30 Ma and continuing through to the Pliocene examine generic relationships for the Australian diplo- (Heatwole, 1987; Hall, 2001; Metcalfe, 2001). For any endemic dactyloids and estimate a time-scale for major divergence Australasian radiation, these biogeographical scenarios have events within the group. distinct, testable predictions for divergence times and phylog- eny. A vicariant Gondwanan hypothesis is supported if diver- MATERIALS AND METHODS gence from sister lineages found outside the Australasian region on Gondwanan landmasses occurred at least 55 (vs. < 35) Ma. Sampling and gene sequencing Gondwanan elements are a prominent feature of most major groups of Australian terrestrial vertebrates, including mam- We sampled 64 taxa, comprising 32 diplodactyloids (c. 25% of mals (marsupials and monotremes; Archer et al., 1999; Beck, the Australian species), eight gekkotan outgroups (Table 1) 2008), birds (e.g. ratites, passerines, parrots; Cooper et al., and 24 other (lepidosaur and archosaur) taxa spanning robust 2001; Barker et al., 2004; Schodde, 2006), frogs (pelodryadid calibration nodes (see Table S1 in Supporting Information). treefrogs, myobatrachids and possibly microhylids; Roelants Sequence data were obtained from all recognized genera of et al., 2007) and chelid turtles (Georges & Thompson, 2006). Australian diplodactyloid geckos with the exception of Orraya, Whereas lizards and snakes (squamates) are highly diverse in and multiple exemplars were obtained for species-rich and Australia (> 800 species: Wilson & Swan, 2008) and are among divergent genera in order to test their monophyly and the most species-rich endemic Australasian radiations (Rabo- minimize long-branch artefacts. While we did not include sky et al., 2007), phylogenetic and recent dating studies of any New Zealand genera, sequence data were obtained from most major extant Australian squamate lineages suggest these two genera of New Caledonian diplodactylids. Gekkonid are all Miocene immigrants that diverged from their nearest outgroups spanned three other gekkonoid families (Gekkon- extralimital (Old World) relatives within the last 10–35 Myr. idae, Sphaerodactylidae and Eublepharidae). This includes the venomous elapid snakes (Sanders & Lee, Whole genomic DNA was isolated from liver samples using 2008), pythons (Rawlings et al., 2008), agamid lizards (Hugall standard proteinase K protocols (Sambrook et al., 1989). et al., 2007), Sphenomorphus group skinks (Rabosky et al., Standard polymerase chain reaction (PCR) protocols were 2007; Skinner et al., 2008) and varanid lizards (Ast, 2001). followed using 25 or 50 ml reactions and TAQgold (Applied Geckos are a conspicuous component of the Australian Biosystems, Carlsbad, CA, USA) and buffer at concentrations squamate fauna. Molecular phylogenetic studies of the global recommended by the manufacturer for 34 cycles. Concentra- gecko radiation have uncovered deep divergences that are tions of buffer were varied depending on the initial reaction consistent with ancient Gondwanan vicariance (Gamble et al., success. Optimal thermal cycling temperatures for different 2008). The Australian gecko fauna is dominated by diplo- primer combinations and taxa ranged from 48 to 62°C. The PCR dactyloid geckos. This moderately diverse radiation consists of products were sequenced using the ABI PRISM BigDye Termi- three families: the Diplodactylidae, Carphodactylidae and nator Cycle Sequencing Ready Reaction Kit and an ABI 3700 Pygopodidae (Han et al., 2004), hereafter referred to jointly automated sequencer (Applied Biosystems). Two nuclear frag- as the Diplodactyloidea. The Carphodactylidae are entirely ments were selected: 1800 bp of RAG-1 (recombination reac- endemic to Australia, nearly all of the Pygopodidae are tivating gene 1) and 750 bp of c-mos (oocyte maturation factor). Australian endemics (two species occur in New Guinea) and These loci have been widely used in squamate studies; they are the Diplodactylidae comprise three relatively diverse radiations single copy, uninterrupted by introns and have a slow substi- in Australia, New Caledonia and New Zealand (Bauer, 1990; tution rate suitable for the time-scales of interest (e.g. Saint Wilson & Swan, 2008). Diplodactyloids include c. 15% of the et al., 1998; Townsend et al., 2004; Gamble et al., 2008). Primers Australasian squamate fauna and are the most ecologically and used are given in Table 2. Sequencing was outsourced to a morphologically diverse clade of gekkotan lizards in the world commercial firm (Macrogen, Seoul, South Korea) or the Insti- (Greer, 1989; Wilson & Swan, 2008). Notably aberrant are the tute of Medical and Veterinary Science (IMVS) in Adelaide. Pygopodidae, which are the world’s only limb-reduced geckos Sequence data were aligned by eye and then translated using and show a remarkable array of morphological and ecological MacClade (Maddison & Maddison, 2005) to check for adaptations to a limbless lifestyle within an only moderately mutations indicating the amplification of pseudogenes. species-rich clade (Greer, 1989; Webb & Shine, 1994). Previous phylogenetic studies that focused on the relation- Phylogenetic analyses ships within diplodactyloid clades (e.g. Kluge, 1987; Bauer, 1990; King, 1990; Couper et al., 2000; Jennings et al., 2003; Phylogenetic analysis using parsimony and likelihood methods Melville et al., 2004; Oliver et al., 2007a) or the higher-level was implemented in paup* version 4.0b10 (Swofford, 2002). phylogeny of gekkotans (King, 1987; Han et al., 2004; Gamble Bayesian inference was implemented in MrBayes version 3.1 et al., 2008) have all suggested that diplodactyloids have (Huelsenbeck & Ronquist, 2001). A maximum parsimony tree Gondwanan origins. However, divergence times and phylo- was estimated using unweighted heuristic searches with 50 genetic relationships across the diplodactyloid radiation have random step-wise