original molecular evidence for gondwanan article origins of

ORIGINALARTICLE

Molecular evidence for Gondwananorigins of multiple lineages within adiverse Australasian gecko radiation

Paul M. Oliver1,2* and Kate L. Sanders1

INTRODUCTION

The Australian biota is dominated by diverse and largely

endemic radiations (Keast, 1981; Heatwole, 1987; Crisp et al.,

2004). It has become widely accepted that these lineages

evolved in relative geographical isolation, originating from

either of two sources: (1) an ancient Gondwanan biota that

became isolated in Australasia as the northward-drifting Indo-

Australian tectonic plate detached from Antarctica c. 55–

32 Ma; and (2) a modern fauna derived from over-water

1Centre for Evolutionary Biology and

Biodiversity, University of Adelaide and2Terrestrial Vertebrates, South Australian

Museum, North Terrace, Adelaide, SA,

Australia

*Correspondence: Paul Oliver, Centre for

Evolutionary Biology and Biodiversity,

University of Adelaide, Adelaide, 5005 SA,

Australia.

E-mail: [email protected]

ABSTRACT

Aim Gondwanan lineages are a prominent component of the Australian

terrestrial biota. However, most squamate (lizard and snake) lineages in

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.

Keywords

Australasia, Bayesian analysis, Carphodactylidae, Diplodactylidae, divergence

times, geckos, Gondwana, historical biogeography, Pygopodidae, relaxed-clock

dating.

Journal of Biogeography (J. Biogeogr.) (2009) 36, 2044–2055

2044 www.blackwellpublishing.com/jbi ª 2009 Blackwell Publishing Ltddoi:10.1111/j.1365-2699.2009.02149.x

dispersals, usually from the north after the Australian and Asian

plates came in close enough proximity to allow island-hopping,

beginning c. 30 Ma and continuing through to the Pliocene

(Heatwole, 1987; Hall, 2001; Metcalfe, 2001). For any endemic

Australasian radiation, these biogeographical scenarios have

distinct, testable predictions for divergence times and phylog-

eny. A vicariant Gondwanan hypothesis is supported if diver-

gence from sister lineages found outside the Australasian region

on Gondwanan landmasses occurred at least 55 (vs. < 35) Ma.

Gondwanan elements are a prominent feature of most major

groups of Australian terrestrial vertebrates, including mam-

mals (marsupials and monotremes; Archer et al., 1999; Beck,

2008), birds (e.g. ratites, passerines, parrots; Cooper et al.,

2001; Barker et al., 2004; Schodde, 2006), frogs (pelodryadid

treefrogs, myobatrachids and possibly microhylids; Roelants

et al., 2007) and chelid turtles (Georges & Thompson, 2006).

Whereas lizards and snakes (squamates) are highly diverse in

Australia (> 800 species: Wilson & Swan, 2008) and are among

the most species-rich endemic Australasian radiations (Rabo-

sky et al., 2007), phylogenetic and recent dating studies of

most major extant Australian squamate lineages suggest these

are all Miocene immigrants that diverged from their nearest

extralimital (Old World) relatives within the last 10–35 Myr.

This includes the venomous elapid snakes (Sanders & Lee,

2008), pythons (Rawlings et al., 2008), agamid lizards (Hugall

et al., 2007), Sphenomorphus group skinks (Rabosky et al.,

2007; Skinner et al., 2008) and varanid lizards (Ast, 2001).

Geckos are a conspicuous component of the Australian

squamate fauna. Molecular phylogenetic studies of the global

gecko radiation have uncovered deep divergences that are

consistent with ancient Gondwanan vicariance (Gamble et al.,

2008). The Australian gecko fauna is dominated by diplo-

dactyloid geckos. This moderately diverse radiation consists of

three families: the Diplodactylidae, Carphodactylidae and

Pygopodidae (Han et al., 2004), hereafter referred to jointly

as the Diplodactyloidea. The Carphodactylidae are entirely

endemic to Australia, nearly all of the Pygopodidae are

Australian endemics (two species occur in New Guinea) and

the Diplodactylidae comprise three relatively diverse radiations

in Australia, New Caledonia and New Zealand (Bauer, 1990;

Wilson & Swan, 2008). Diplodactyloids include c. 15% of the

Australasian squamate fauna and are the most ecologically and

morphologically diverse clade of gekkotan lizards in the world

(Greer, 1989; Wilson & Swan, 2008). Notably aberrant are the

Pygopodidae, which are the world’s only limb-reduced geckos

and show a remarkable array of morphological and ecological

adaptations to a limbless lifestyle within an only moderately

species-rich clade (Greer, 1989; Webb & Shine, 1994).

Previous phylogenetic studies that focused on the relation-

ships within diplodactyloid clades (e.g. Kluge, 1987; Bauer,

1990; King, 1990; Couper et al., 2000; Jennings et al., 2003;

Melville et al., 2004; Oliver et al., 2007a) or the higher-level

phylogeny of gekkotans (King, 1987; Han et al., 2004; Gamble

et al., 2008) have all suggested that diplodactyloids have

Gondwanan origins. However, divergence times and phylo-

genetic relationships across the diplodactyloid radiation have

not yet been assessed comprehensively and directly using

molecular data. In this paper we use two nuclear sequences to

examine generic relationships for the Australian diplo-

dactyloids and estimate a time-scale for major divergence

events within the group.

MATERIALS AND METHODS

Sampling and gene sequencing

We sampled 64 taxa, comprising 32 diplodactyloids (c. 25% of

the Australian species), eight gekkotan outgroups (Table 1)

and 24 other (lepidosaur and archosaur) taxa spanning robust

calibration nodes (see Table S1 in Supporting Information).

Sequence data were obtained from all recognized genera of

Australian diplodactyloid geckos with the exception of Orraya,

and multiple exemplars were obtained for species-rich and

divergent genera in order to test their monophyly and

minimize long-branch artefacts. While we did not include

any New Zealand genera, sequence data were obtained from

two genera of New Caledonian diplodactylids. Gekkonid

outgroups spanned three other gekkonoid families (Gekkon-

idae, Sphaerodactylidae and Eublepharidae).

Whole genomic DNA was isolated from liver samples using

standard proteinase K protocols (Sambrook et al., 1989).

Standard polymerase chain reaction (PCR) protocols were

followed using 25 or 50 ml reactions and TAQgold (Applied

Biosystems, Carlsbad, CA, USA) and buffer at concentrations

recommended by the manufacturer for 34 cycles. Concentra-

tions of buffer were varied depending on the initial reaction

success. Optimal thermal cycling temperatures for different

primer combinations and taxa ranged from 48 to 62�C. The PCR

products were sequenced using the ABI PRISM BigDye Termi-

nator Cycle Sequencing Ready Reaction Kit and an ABI 3700

automated sequencer (Applied Biosystems). Two nuclear frag-

ments were selected: 1800 bp of RAG-1 (recombination reac-

tivating gene 1) and 750 bp of c-mos (oocyte maturation factor).

These loci have been widely used in squamate studies; they are

single copy, uninterrupted by introns and have a slow substi-

tution rate suitable for the time-scales of interest (e.g. Saint

et al., 1998; Townsend et al., 2004; Gamble et al., 2008). Primers

used are given in Table 2. Sequencing was outsourced to a

commercial firm (Macrogen, Seoul, South Korea) or the Insti-

tute of Medical and Veterinary Science (IMVS) in Adelaide.

Sequence data were aligned by eye and then translated using

MacClade (Maddison & Maddison, 2005) to check for

mutations indicating the amplification of pseudogenes.

Phylogenetic analyses

Phylogenetic analysis using parsimony and likelihood methods

was implemented in paup* version 4.0b10 (Swofford, 2002).

Bayesian inference was implemented in MrBayes version 3.1

(Huelsenbeck & Ronquist, 2001). A maximum parsimony tree

was estimated using unweighted heuristic searches with 50

random step-wise sequence addition replicates and tree

Gondwanan origins of Australasian geckos

Journal of Biogeography 36, 2044–2055 2045ª 2009 Blackwell Publishing Ltd

bisection–reconnection (TBR) branch swapping. Bootstrap

support was calculated using 1000 bootstrap replicates. Bayes

factors (see Kelchner & Thomas, 2007) and average likelihoods

were used to assess the effect of partitioning data by gene

and codon. Alternative partitioning strategies were run with

four incrementally heated chains for 3,000,000 generations

(sampled every 1000th generation) using best-fit models of

nucleotide substitution for each partition identified using the

Table 1 Specimen numbers, GenBank accession numbers and localities for gekkonid lizards included in analyses, pre-existing GenBank

accession numbers are indicated in bold.

Taxon Specimen Locality RAG-1 c-mos

Carphodactylids

Carphodactylus laevis QMJ 8944 Lake Barrine, Qld, Australia FJ855442 AF039467

Nephurus milii SAMA R38006 17 km SE Burra, South Australia FJ571622 FJ571637

Nephurus stellatus SAMA R36563 19.3 km NE Courtabie, South Australia FJ855446 FJ855466

Nephrurus asper SAMA R55649 10 km W Isaac River, Qld, Australia FJ855445 FJ855465

Phyllurus platurus ABTC 51012 Bents Basin, Sydney, Australia FJ855443 _

Phyllurus platurus NA NA __ AY172942

Saltuarius swaini SAMA R29204 Wiangaree, NSW, Australia FJ855444 FJ855464

Diplodactylids

Bavayia sauvagei AMS R125814 Mare Island, New Caledonia FJ855448 FJ855468

Crenadactylus ocellatus horni SAMA R22245 10 km S Barrow Creek, NT, Australia AY662627 FJ571641

Crenadactylus ocellatus naso AMS R126186 Mitchell Plateau, Western Australia FJ855458 FJ855479

Crenadactylus ocellatus ocellatus WAM R135495 False Entrance Well, Western Australia FJ855457 FJ855478

Diplodactylus granariensis WAM R127572 Goongarrie, Western Australia FJ855452 FJ855473

Diplodactylus tessellatus SAMA R41130 Nr Stuart Hwy, South Australia FJ571624 FJ571639

Lucasium byrnei SAMA R52296 Camel Yard Spring, South Australia FJ855453 FJ855474

Luscasium stenodactylum NTM R26116 Mann River, NT, Australia FJ855454 FJ855475

Oedura marmorata SAMA R34209 Lawn Hill NP, Qld, Australia FJ571623 FJ571638

Oedura reticulata SAMA R23035 73 km E. Norseman, Western Australia FJ855450 FJ855471

Oedura rhombifer SAMA R34513 Townsville area, Qld, Australia FJ855451 FJ855472

Pseudothecadactylus australis QMJ 57120 Heathlands, Qld, Australia FJ855449 FJ855470

Pseudothecadactylus lindneri AMS 90915 Liverpool River, NT, Australia AY662626 FJ855469

Rhacodactylus leachianus AMS R118009 Mt Gouemba, New Caledonia. FJ855447 FJ855467

Rhychoedura ornata SAMA R36873 Mern Merna Station, South Australia FJ855455 FJ855476

Strophurus intermedius SAMA R28963 Gawler Ranges, South Australia FJ571625 FJ571640

Strophurus jeanae SAMA R53984 11 km S. of Wycliffe Well, Qld FJ855456 FJ855477

Pygopodids

Aprasia inaurita SAMA R40729 2 km E of Burra, South Australia FJ571632 FJ571646

Delma australis SAMA R22784 Mt Remarkable NP, South Australia FJ571633 FJ571647

Delma molleri SAMA R23137 Mt Remarkable NP, South Australia FJ571635 FJ571649

Lialis jicari TNHC 59426 NA AY662628 _

Lialis jicari NA Irian Jaya _ AY134564

Ophidiocephalus taeniatus SAMA R44653 Todmorden Stn, South Australia FJ571630 FJ571645

Pletholax gracilis WAM R104374 Victoria Park, Western Australia FJ571631 _

Pletholax gracilis WBJ-2483 Lesueur NP, Western Australia _ AY134566

Paradelma orientalis QMJ 56089 20 km N Capella, Qld, Australia FJ571626 FJ571642

Pygopus lepidopodus WAM R90378 Walpole-Nornalup NP, Western Australia FJ571627 FJ571643

Pygopus nigriceps SAM R23908 134 km ENE Laverton, Western Australia FJ571628 FJ571644

Other gekkonids

Christinus marmoratus SAMA R42098 Wedge Is, South Australia FJ855440 FJ855461

Cyrtodactylus marmoratus AMS R126129 Cibodas forest, Java, Indonesia FJ855438 FJ855459

Gehyra variegata SAMA R54022 Brunette Downs, NT, Australia FJ855439 FJ855460

Gekko gekko MVZ 215314 NA AY662625 _

Gekko gekko FMNH 258696 NA _ AY444028

Hemidactylus frenatus SAMA R34178 Daly Waters, NT, Australia FJ855441 FJ855462

Teratoscincus przewalski CAS 171010 South Gobi Desert, Mongolia AY662624 AY662569

Eublepharis turkmenicus CAS 184771 NA AY662622 _

Eublepharis macularius ABTC 32296 Pet trade _ FJ855463

Sphaerodactylus shreveri SBH 194572 Haiti AY662623 AY662547

Qld, Queensland; NSW, New South Wales; NT, Northern Territory; NP, national park; NA, not applicable.

P. M. Oliver and K. L. Sanders

2046 Journal of Biogeography 36, 2044–2055ª 2009 Blackwell Publishing Ltd

Akaike information criterion implemented in MrModeltest

(Nylander, 2004) and paup* (Swofford, 2002). A three-

partition model with both genes combined and partitioned

by codon position (1st + 2nd + 3rd) was selected as optimum

based on a Bayesian information criterion approximation

(Schwarz, 1978). The best-fit substitution model for each of

these partitions was GTRig. This model was then run with four

chains for 5,000,000 generations, sampling every 1000 gener-

ations. Values for all model parameters were unlinked, i.e.

allowed to vary independently across partitions. The first

1,000,000 generations were discarded as burn-in. MrBayes

analyses were run in parallel across four nodes on a SGI Altix

XE1300 supercomputer (SGI Sunnyvale, CA, USA).

Molecular dating

Divergence times were estimated using Bayesian inference as

implemented in beast version 1.4 (Drummond & Rambaut,

2006) and penalized likelihood rate smoothing (PLRS) in r8s

version 1.7 (Sanderson, 2002, 2003) and paup* version 4.0b10

(Swofford, 2002). Preliminary beast runs failed to resolve the

split between squamates (lizards and snakes) and rhyncho-

cephalians (tuataras). However, because this relationship is

well supported by previous molecular studies (e.g. Hugall

et al., 2007), it was constrained in all further beast analyses.

All other relationships were left free to vary so that topological

uncertainty was incorporated into posterior estimates of

divergence dates. A Yule branching process (appropriate for

divergent, interspecific relationships) with a uniform prior was

adopted. A relaxed clock was used with branch rate variation

modelled using a lognormal distribution and initially assumed

to be uncorrelated (Drummond et al., 2006; see below). These

settings allow the pattern of rate variation to be quantified to

ascertain whether more restricted models of rate variation (e.g.

strict clock, correlated lognormal) would be more appropriate.

The combined loci were partitioned by codon position

(1st + 2nd vs. 3rd) with unlinked parameter values. The final

analysis consisted of two independent Markov chain Monte

Carlo (MCMC) analyses; each chain was run for 15,000,000

generations with parameters sampled every 1000 steps. Inde-

pendent runs converged on very similar posterior estimates

and were combined using LogCombiner version 1.4 (Drum-

mond & Rambaut, 2006). Tracer 1.2 (Drummond &

Rambaut, 2006) was used to confirm adequate mixing of the

MCMC chain, appropriate burn-in (25%) and acceptable

effective sample sizes (> 200).

Most available gecko fossils calibrate shallow divergences

(< 20 Myr) between taxa that are not included in the present

study (see Gamble et al., 2008). The fossil Pygopus hortulanus

is thought to be close to the origin of extant pygopodid genera

Pygopus and Paradelma (available in this study) and is from a

site dated as early–middle Miocene (c. 20 Ma; Hutchinson,

1998). Our phylogenetic analyses (see below) recovered

Pygopus as paraphyletic with respect to Paradelma; the

P. hortulanus calibration was therefore used conservatively to

constrain the node containing all sampled Pygopus and

Paradelma. Relaxed-clock dating benefits from multiple cali-

brations spanning the divergences of interest (Drummond

et al., 2006). We therefore used the P. hortulanus calibration in

combination with two well-corroborated external calibrations:

Ornithodira (birds and relatives) vs. Crurotarsi (crocodiles and

relatives), and scincomorph lizards vs. lacertoid plus toxicof-

eran lizards (Table 3; see Hugall et al., 2007; Sanders & Lee,

2007, 2008). All calibration priors were given a translated

lognormal distribution since this best reflects the asymmetrical

Table 2 Primer combinations used in this study (IUB redundancy codes are Y ¼ C, T: R ¼ A, G: M ¼ A, C: N ¼ A, T, G, C). [Correction

added after online publication on 6 July 2009: values for N were corrected.]

Gene Primer References

RAG-1 G755 5¢-AAGTTTTCAGAATGGAAGTTYAAGCTNTT-3¢ Hugall et al. (2007)

G756 5¢-TCTCCACCTTCTTCYTTNTCAGCAAA-3¢ Hugall et al. (2007)

G1278 5¢-TGATGCAARAAYCCTTTCAGA-3¢ This study

G1279 5¢-TCTCCACCTTCTTCTTTCTCAG-3¢ This study

G889 5¢-AAAGGTGGACGCCCTAGGCARCA-3¢ Hugall et al. (2007)

G883 5¢-TCATGGTCAGATTCATCAGCNARCAT-3¢ Hugall et al. (2007)

c-mos G303 5¢-ATTATGCCATCMCCTMTTCC-3¢ Saint et al. (1998)

G74 5¢-TGAGCATCCAAAGTCTCCAATC-3¢ Saint et al. (1998)

G708 5¢-GCTACATCAGCTCTCCARCA-3¢ Hugall et al. (2008)

G1092 5¢-CTTTTGTCCGATGGCTGAGTC-3¢ This study

G1163 5¢-CTGCCTGCCAAAGTGGAAAG-3¢ This study

Table 3 beast prior probability distributions (Ma) for calibra-

tion nodes used in this study. The r8s analysis used the zero offset

and upper 95% confidence intervals (CIs) of the lognormal priors

as minimum and maximum constraints.

Calibration node

Prior distribution

Lognormal:

mode [zero offset,

upper 95% CI]

Normal:

mode [95% CIs]

Pygopus–Paradelma 19 [16, 25] 20 [15, 25]

Scincomorphs vs.

lacertoids + toxicoferans

168 [155, 200] 168 [135, 200]

Bird–crocodile 240 [228, 271] 240 [207, 273]

Root (mammal–bird) 255–310 (uniform) 255–310 (uniform)



bias in the fossil record (the true divergence date is more likely

to be older than younger due to non-preservation). Additional

analyses were performed assuming normally distributed

(symmetrical) calibration priors; these returned very similar

results (Table 4). In all beast analyses, a wide uniform

constraint of 255–310 Ma (e.g. Benton & Donoghue, 2006)

was placed on the root of the tree (mammal–bird split) to

prevent the chain from becoming fixed on unrealistic inflated

values (Drummond et al., 2006).

An additional dating analysis was performed with the

program r8s version 1.7 (Sanderson, 2002), using PLRS with

the Truncated Newton (TN) algorithm and an additive penalty

function (Sanderson, 2002). Non-squamate taxa (archosaurs

and turtles) were removed from the final r8s analysis because

the inclusion of these phylogenetically very distant taxa

appeared to hinder optimization of rate smoothing levels in

preliminary r8s runs. The smoothing parameter was chosen

using cross-validation. The initial maximum-likelihood tree

was generated in paup* version 4.0b10 (Swofford, 2002) using

a GTRig substitution model (identified by MrModeltest);

parameters were optimized using an iterative process of

estimating parameter values, then performing new searches

using estimated values until the tree likelihood stabilized and

topology did not change significantly. To maximize similarity

with the beast analyses, we used zero offset and upper 95%

confidence interval (CI) values from the beast lognormal

calibration priors (Table 3) as the upper and lower constraints

in the r8s analysis.

RESULTS

Phylogenetic relationships

The final data matrix consisted of 2367 sites (1740 RAG-1 and

627 c-mos) of which 934 (689 RAG-1 and 245 c-mos) were

variable within geckos and 553 (402 RAG-1 and 151 c-mos)

were parsimony informative within geckos. All sequences

could be translated into amino acids with no evidence of

pseudogenes. Our c-mos sequence for the diplodactylid Oedura

marmorata contained a 12 bp indel. The model-based and

parsimony results were highly concordant, showing no con-

flicting node support; nodes that were strongly supported in

the model-based methods were also strongly supported by

parsimony, and nodes with low support were collapsed in the

parsimony consensus tree (Fig. 1). Relationships amongst

squamate and gekkonid outgroup taxa were consistent with

previously published studies (Townsend et al., 2004; Hugall

et al., 2007; Gamble et al., 2008). The diplodactyloid geckos

formed a strongly supported sister clade (node A) to all other

sampled geckos (gekkonids, sphaerodactylids and Eublepharis)

(node I). The monophyly of each of the three diplodactyloid

families [Carphodactylidae (node B), Pygopodidae (node C)

and Diplodactylidae (node D)] was strongly supported,

although their interrelationships were unresolved.

Within the diplodactyloids, multiple exemplars from single

genera formed strongly supported monophyletic groups with

two exceptions: Oedura may be paraphyletic with respect to

Table 4 Mean and range of divergence time estimates for selected gekkotan and calibration nodes obtained using outgroup (squamate and

archosaur) calibrations alone, and outgroup calibrations combined with the Pygopus–Paradelma calibration. Values obtained for normal and

lognormal priors are shown. All estimates are given in millions of years ago (Ma). Letters alongside major gecko splits correspond to node

labels in Fig. 1.

Node

beast posterior distributions: mean [95% highest posterior density] r8s

Lognormal calibration priors Normal priorsOutgroup

onlyOutgroup only Outgroup + Pygopus Outgroup only Outgroup + Pygopus

Gekkotans

(A) Diplodactyloids 71.5 [53.2, 91.2] 79.1 [58.1, 101.7] 77.4 [56.1, 101.8] 83.5 [59.8, 110.1] 55.1

(B) Pygopodidae 31.3 [20.4, 44.9] 39.2 [27.0, 52.4] 33.7 [20.7, 48.6] 38.2 [25.4, 53.5] 23.7

(C) Carphodactylidae 33.4 [20.8, 46.1] 37.6 [22.3, 53.5] 36.4 [21.9, 52.8] 38.8 [23.4, 55.9] 25.7

(D) Diplodactylidae 56.9 [41.0, 73.2] 62.4 [44.6, 80.8] 61.4 [42.4, 81.4] 66.2 [46.6, 87.0] 45.8

(E) Most Australian Diplodactylidae 34.5 [25.1, 44.9] 37.7 [26.8, 49.5] 37.2 [25.7, 49.8] 39.7 [27.5, 53.2] 26.8

(F) Australian Diplodactylidae vs. New

Caledonia + Pseudothecadactylus

51.2 [37.4, 66.2] 56.6 [40.1, 73.9] 55.9 [37.9, 74.3] 60.3 [41.5, 79.2] 42.9

(G) Australia vs. New Caledonia 42.8 [27.9, 58.7] 47.4 [29.8, 63.8] 47.5 [30.1, 66.6] 51.0 [32.2, 69.8] 38.5

(H) Diplodactyloids vs. other gekkonids 118.1 [88.9, 147.3] 125.4 [97.4, 155.8] 125.4 [91.3, 162.9] 134.7 [98.7, 172.2] 101.4

(I) Gekkonindae (sensu Han et al., 2004)

vs. Eublepharis

101.8 [74.6, 131.7] 108.6 [80.3, 140.4] 109.6 [75.4, 142.1] 116.9 [82.6, 152.7] 91.6

Calibrations

Pygopus–Paradelma 10.8 [5.5, 17.3] 20.0 [17.8, 22.7] 11.9 [5.9, 19.2] 17.0 [11.9, 22.3] 9.6

Scincomorphs vs. lacertoids + toxicoferans 172.5 [159.4, 188.2] 174.7 [160.2, 192.8] 183.8 [149.9, 217.5] 189.5 [156.7, 221.7] 170.5

Crown squamates 189.2 [169.3, 212.6] 192.5 [170.4, 216.5] 206.3 [163.1, 250.8] 215.4 [172.4, 260.6] 181.8

Bird–crocodile 239.6 [230.3, 252.8] 239.4 [230.1, 252.3] 221.9 [184.8, 259.4] 226.0 [189.9, 262.7] 271.2

Root 298.9 [281.7, 309.9] 300.8 [285.5, 310.0] 341.5 [268.8, 423.1] 359.3 [280.9, 444.3] NA

NA, not applicable.



Strophurus, and Pygopus is paraphyletic with respect to

Paradelma. Within the Diplodactylidae, three highly divergent

Australian lineages were recovered: (1) a monotypic Crena-

dactylus sister to all remaining diplodactylids (node D); (2) the

northern Australian genus Pseudothecadactylus plus the New

Caledonian radiation (Bavayia + Rhacodactylus) (node G);

and (3) all other sampled diplodactylids separated by short

internodes (node E). Within this third lineage the small

bodied, predominantly terrestrial forms Diplodactylus, Luca-

sium and Rhynchoedura formed a strongly supported clade and

the hypothesis that Rhynchoedura is sister to Lucasium

(Melville et al., 2004; Oliver et al., 2007b) was also supported.

Among the remaining arboreal species in this third lineage,

the monophyly of Strophurus was supported, but other inter-

and intra-generic relationships were unresolved (mainly

involving the genus Oedura). Relationships between most

genera within the Carphodactylidae and Pygopodidae were

also relatively poorly resolved; however, there was strong

support for a basal dichotomy between Delma and all other

pygopodid genera.

Divergence date estimates for the diplodactyloid

geckos

The two combined beast MCMC runs yielded high effective

sample sizes (> 500) for all relevant parameters (e.g. branch

lengths, topology and clade posteriors), indicating adequate

sampling of the posterior distribution. Levels of rate hetero-

geneity were moderate (coefficients of rate variation 0.53) and

rates were weakly correlated between adjacent branches

(branch rate covariance c. 0.06). The maximum credibility

tree (Figs 2 & S1) retrieved from the combined analyses

(TreeAnnotator version 1.4; Drummond & Rambaut, 2006)

is nearly identical to the MrBayes consensus tree (Fig. 1) in

topology and posterior support values. Bayesian and PLRS

date estimates are presented in Table 4. Both methods yielded

broadly similar date estimates, with PLRS giving consistently

shallower dates for all nodes of interest.

DISCUSSION

Phylogeny

The major gekkonid relationships recovered here are consis-

tent with previously published molecular studies (Donnellan

et al., 1999; Han et al., 2004; Townsend et al., 2004; Gamble

et al., 2008). In contrast to previous morphologically derived

hypotheses (in which the Eublepharidae were regarded as the

basal lineage of gekkotans), these studies all indicate that the

diplodactyloids are sister to all other extant gekkotans. Our

relatively complete sampling of genera provides strong support

for the monophyly of each of the three diplodactyloid families,

Figure 1 Bayesian all compatible consensus of 40,000 trees sampled post-burn-in for the three families of Australian diplodactyloid geckos

(shown in bold) and gekkotan outgroups. Support values > 0.98 for Bayesian and > 75% for parsimony analyses are shown. Letters at key

nodes correspond to those in Table. 4.



but like previous molecular studies (Han et al., 2004; Gamble

et al., 2008) failed to resolve their interrelationships.

This study has revealed the existence of the three highly

divergent Australian lineages within the family Diplodactyli-

dae. Previous studies that sampled these taxa were based on

mitochondrial DNA (mtDNA) (e.g. Melville et al., 2004;

Oliver et al., 2007b); substitutional saturation at this rapidly

evolving locus may have impeded resolution of these deep

divergences. Our data indicate that the monotypic genus

Crenadactylus is the most basal lineage of the family Diplo-

dactylidae. Additional sampling of extralimital taxa and

additional genes are required to test this hypothesis further.

Likewise Pseudothecadactylus was found to be highly divergent

from all other Australian lineages and sister to the New

Caledonian radiation (as demonstrated by Bauer, 1990, using

morphological data).

Jennings et al. (2003) were unable to robustly resolve

generic relationships among pygopods but suggested that the

comparatively conservative genus Delma is sister to all other

genera. Our data strongly support this hypothesis; a result that

underlines the high levels of ecological and morphological

variation shown by the clade containing the six remaining

genera (Greer, 1989). In most other instances intergeneric

relationships in all three families of diplodactyloid gecko are

relatively poorly resolved. This is particularly striking within

the Carphodactylidae, the Pygopodidae exclusive of Delma and

the major Australian radiation of arboreal Diplodactylidae

(Oedura and Strophurus). In the two latter clades this poor

resolution and short internode branches have also been found

in studies employing rapidly evolving mitochondrial markers

(Jennings et al., 2003; Oliver et al., 2007b). While additional

and larger datasets are required, these findings are suggestive of

relatively rapid cladogenesis.

Age and origin of the diplodactyloid geckos

We focus the following discussion on the results of our

Bayesian dating analyses because: (1) beast better incorporates

uncertainty in calibration priors and rate smoothing (Drum-

mond et al., 2006); and (2) PLRS performs best if rates are

strongly autocorrelated across the tree (r8s documentation in

Sanderson, 2003), whereas our data show weak autocorrelation

Figure 2 beast maximum credibility ultra-

metric tree for diplodactyloids and gekkotan

outgroups. Nodes with posterior support

below 0.98 are indicated with an asterisk (*).

Australian diplodactyloid lineages are shown

in bold. Letters at key nodes correspond to

those in Table. 4. Node bars indicate 95%

highest posterior age distributions for three

strongly supported ingroup divergences that

pre-date estimates of the last stages of the

break-up of Australia and Antarctica (c. 50–

40 Ma; indicated by the light grey bar): (A)

the crown diplodactyloid divergence; (D) the

basal split in the family Diplodactylidae;

(F) the split between the New-Caledonian/

Pseudothecadactylus lineage and the main

Australian radiation of diplodactyloids.

Divergence date estimates for other major

divergences are given in Table. 1. The

time-scale is in millions of years ago (Ma).



(see branch rate covariance above). However, adopting the

PLRS results would not change our overall interpretations of

the biogeographical history of diplodactyloid geckos.

Analyses that did not enforce the Pygopus–Paradelma

calibration dated this divergence at 10.8 Ma [95% highest

posterior density (HPD) 5.5, 17.3], almost half the minimum

fossil-based age (based on P. hortulanus; Hutchinson, 1998).

Recent morphological and phylogenetic reanalysis of this fossil

suggests that there is considerable error associated with its

phylogenetic placement (Lee et al., 2009). Enforcing the

Pygopus–Paradelma calibration only moderately inflated in-

group age estimates (Table 4) and we focus our discussion on

date estimates derived using only the better corroborated

external calibrations.

For both external calibration nodes, mean posterior age

estimates were close to the priors: the scincomorphs vs.

lacertoids + toxicoferans split is dated at 172.5 Ma (95% HPD

159.4–188.2), and the bird–crocodile split is dated at 239.6 Ma

(95% HPD 230.3–252.8). The diplodactyloid–gekkonid diver-

gence (node H) is dated at 125.4 Ma (95% HPD 97.4–155.8),

and is consistent with recent studies using relaxed-clock dating

and different combinations of data, taxa and fossil calibrations

to those applied in the present study (Hugall et al., 2007;

Gamble et al., 2008). Our date for the diplodactyloid crown

group (node A), 71.5 Ma (95% HPD 53.2–91.2), is also highly

congruent with the relaxed clock estimate of Gamble et al.

(2008) and a study that used independent immunological data

(King, 1990). The proximity of our dates to those of previous,

independently calibrated studies suggests that although our

dating analyses are based on two distant external calibrations,

the results have not been seriously affected by substantial rate

variation between the calibration and ingroup taxa.

The diplodactyloid lineage is estimated to have diverged

from all other extant geckos before the mid-Cretaceous

> 100 Ma (Table 4; see also Gamble et al., 2008). The

subsequent diversification of the crown diplodactyloid lineage

is estimated to have occurred in the late Cretaceous, c. 70 Ma,

with five lineages diverging by at least 45 Ma. These dates

strongly imply diversification in East Gondwana, with

subsequent persistence of multiple diplodactyloid lineages on

the newly isolated Australian continent after the final split

from Antarctica c. 32 Ma (Lawver & Gahagan, 2003; Wei,

2004). We date the divergence between the New Caledonian

diplodactyloids and their Australian sister lineage at c. 43 Ma

(95% HPD 27.9–58.7). Regardless of whether diplodactyloids

reached New Caledonia via dispersal or vicariance (see

Discussion below) this date provides further support for the

long-term presence of diplodactyloids in East Gondwana.

Thus, even in the absence of further biogeographical support

from the distribution of extralimital sister lineages, our date

estimates for the basal diplodactyloid divergences both within

Australia and between Australia and New Caledonia are highly

inconsistent with short-range dispersal from Asia in the last

30 Myr.

Recent advances in our understanding of the geological

history of New Caledonia and Australia strongly suggest that

these landmasses separated considerably later than 80 Ma, as

previously inferred from vicariance events and used to

calibrate dating studies of diplodactyloid geckos (e.g. Couper

et al., 2000). Our mean estimate of c. 43 Ma (95% HPD 27.9–

58.7) for the divergence between New Caledonian diplodacty-

loids and their Australian sister lineage is not inconsistent with

vicariance under newer models for the opening of the Tasman

Sea during the Palaeogene (see Gaina et al., 1998; Ladiges &

Canttril, 2007). Neither does this relatively young date conflict

with an alternative scenario of at least limited dispersal

between Gondwanan fragments during the Oligocene.

The Australasian diplodactyloid gecko radiation includes

multiple lineages with Gondwanan origins that almost cer-

tainly diversified before Australia became isolated from other

Gondwanan continents (specifically Antarctica and South

America). These ancient gecko lineages have persisted through

extreme changes in environment and climate (Byrne et al.,

2008) and are among the oldest radiations of vertebrates

restricted to the Australasian region – contemporaneous with

marsupials (Beck, 2008) and passeriform birds (Barker et al.,

2004), and significantly exceeded only by myobatrachid frogs

(Roelants et al., 2007). Many of these old Gondwanan clades

are absent or ecologically depauperate outside the Australasian

region. Most famously, the marsupials and monotremes are

widely regarded to have persisted and radiated in Australia

through a combination of isolation and the absence of more

widespread and competitive groups of placental mammals

(Lillegraven et al., 1987). In parallel with this, other families of

geckos (particularly Gekkonidae), whose ancestors diverged

from the diplodactyloids at least 100 Ma, dominate gecko

faunas elsewhere in the world but are depauperate in the three

Gondwanan fragments inhabited by diplodactyloids.

Comparison with other Australian squamate

radiations

The five main lineages of diplodactyloids are the only extant

squamate lineages that can convincingly be shown to have been

present at the time of Australia’s final rifting from Antarctica

(c. 40–30 Ma) (Fig. 2). There are currently more than 800

described species of Australian squamates and all available

evidence (phylogeny, fossils, diversity distributions and molec-

ular divergence dates) indicates that these largely stem from 10

to 15 over-water invasions of Australia (Table 5). Almost all of

these invasions have occurred since the Oligocene, in the last c.

30 Myr (including multiple post-Pliocene entries by colubroid

snakes and gekkonid geckos). However, further data are

required for three additional squamate groups. Current data

do not rule out a Gondwanan origin for Australian Egernia and

Eugongylus skinks, although low molecular distances (Austin &

Arnold, 2006; Smith et al., 2007) and the absence of fossils pre-

dating the late Oligocene provide no indication of an ancient

origin. The history and origin of the Australian Scolecophidian

(blindsnake) radiation is virtually unknown.

The age and isolated history of the diplodactyloids may

explain their high morphological and ecological diversity



relative to other gekkonids. Most notably, pygopods are the

world’s only limb-reduced geckos and probably evolved in the

absence of almost all other extant Australian squamate

lineages, including limb-reduced and ecologically equivalent

groups [Sphenomorphus group skinks (Skinner et al., 2008)

and elapid snakes (Sanders & Lee, 2008)].

In contrast to the deep splits between the five major clades

of Australian diplodactyloids, the majority of extant intergen-

eric and generic diversity appears to have accumulated

relatively recently. Mean crown group age estimates for the

three most diverse Australian lineages (pygopodids, carpho-

dactylids and the core Australian diplodactylids) are c. 30–

35 Ma. All three groups differ in ecology, and their relatively

contemporaneous diversification is suggestive of extrinsic

environmental change at this time. These divergence dates

are roughly coincident with age estimates for the formation of

the Antarctic Circumpolar Current (Barker et al., 2007) and

associated onset of aridification in Australia. This process is

thought to have been the predominant abiotic driver of

evolution and extinction in the Australian biota for the last

30 Myr (Heatwole, 1987; Jennings et al., 2003; Crisp et al.,

2004; Rabosky et al., 2007; Byrne et al., 2008). Our observa-

tions support a picture of an extant Australian biota (and

squamate fauna in particular) that is characterized by major

radiations of both Gondwanan and Asian groups no older than

the late Oligocene or early Miocene (e.g. Crisp et al., 2004;

Beck, 2008; Table 5).

ACKNOWLEDGEMENTS

This work was supported by an Australia Pacific Science

Foundation grant to Paul Doughty, Paul Oliver, Andrew

Hugall, Mark Adams and Mike Lee, and an Australian

Research Council grant to Mike Lee and Mark Hutchinson.

We thank Andrew Hugall, Mike Lee, Mark Hutchinson, Paul

Doughty, Aaron Bauer, Steve Cooper and Adam Skinner

for advice and comments; Andrew Hugall also provided

unpublished sequence data. Bayesian analyses were performed

using the supercomputer facilities at e-Research SA. We thank

Pauline Ladiges and two anonymous reviewers for their

constructive comments on the original manuscript.

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SUPPORTING INFORMATION

Additional Supporting Information may be found in the

online version of this article:

Figure S1 Maximum credibility beast chronogram of

relationships between diplodactyloids and both gekkotan and

non-gekkotan outgroups showing (a) support values, (b) mean

node ages in millions of years, and (c) 95% confidence

intervals for age estimates.

Table S1 GenBank accession details for non-gekkotan

outgroup sequences.

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BIOSKETCHES

Paul Oliver is a postgraduate student at the University of Adelaide and South Australian Museum. His research is focused on the

origin, evolution and systematics of the Australasian herpetofauna, especially gekkotan lizards and Melanesian frogs.

Kate Sanders is a postdoctoral researcher at the University of Adelaide. Her main research interests concern the evolutionary and

conservation biology of squamate reptiles in Southeast Asia and Australia.

Editor: Pauline Ladiges

Note added in press: The clade of geckos herein informally referred to as the diplodactyloids, has recently been formally named

Pygopoidea, see Vidal & Hedges, 2009.



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