original molecular evidence for gondwanan article origins of
TRANSCRIPT
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)
Gondwanan origins of Australasian geckos
Journal of Biogeography 36, 2044–2055 2047ª 2009 Blackwell Publishing Ltd
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.
P. M. Oliver and K. L. Sanders
2048 Journal of Biogeography 36, 2044–2055ª 2009 Blackwell Publishing Ltd
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.
Gondwanan origins of Australasian geckos
Journal of Biogeography 36, 2044–2055 2049ª 2009 Blackwell Publishing Ltd
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).
P. M. Oliver and K. L. Sanders
2050 Journal of Biogeography 36, 2044–2055ª 2009 Blackwell Publishing Ltd
(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
Gondwanan origins of Australasian geckos
Journal of Biogeography 36, 2044–2055 2051ª 2009 Blackwell Publishing Ltd
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.
Please note: Wiley-Blackwell is not responsible for the
content or functionality of any supporting materials supplied
by the authors. Any queries (other than missing material)
should be directed to the corresponding author for the
article.
P. M. Oliver and K. L. Sanders
2054 Journal of Biogeography 36, 2044–2055ª 2009 Blackwell Publishing Ltd
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.
Gondwanan origins of Australasian geckos
Journal of Biogeography 36, 2044–2055 2055ª 2009 Blackwell Publishing Ltd