Author's personal copy

Molecular Phylogenetics and Evolution 65 (2012) 351–361

Contents lists available at SciVerse ScienceDirect

Molecular Phylogenetics and Evolution

journal homepage: www.elsevier.com/locate/ympev

Phylogeny and cryptic diversification in Southeast Asian flying geckos

Rafe M. Brown a,b,⇑, Cameron D. Siler a,b, L. Lee Grismer c, Indraneil Das d, Jimmy A. McGuire e,f
a
Biodiversity Institute, University of Kansas, Lawrence, KS 66045-7561, USA
b
Department of Ecology and Evolutionary Biology, University of Kansas, Lawrence, KS 66045-7561, USA
c
Department of Biology, La Sierra University, Riverside, CA 92515, USA
d
Institute of Biodiversity and Environmental Conservation, Universiti Malaysia Sarawak, 94300 Kota Samarahan, Sarawak, Malaysia
e
Museum of Vertebrate Zoology, University of California, Berkeley, CA 94720-3160, USA
f
Department of Integrative Biology, University of California, Berkeley, CA 94720-3160, USA

a r t i c l e i n f o a b s t r a c t

Article history: The closed-canopy forests of Southeast Asia are home to an impressive number of vertebrates that have
Received 8 March 2012 independently evolved morphologies that enhance directed aerial descent (gliding, parachuting). These
Revised 31 May 2012 assemblages include numerous mammal, frog, snake, and lizard clades. Several genera of gekkonid liz-
Accepted 13 June 2012
ards, in particular, have evolved specialized structures such as cutaneous expansions, flaps, and midbody
Available online 26 June 2012
patagia, that enhance lift generation in the context of unique gliding and parachuting locomotion. The
genus Ptychozoon represents arguably the most morphologically extreme, highly specialized clade of glid-
Keywords:
ing geckos. Despite their notoriety and celebrated locomotor ability, members of the genus Ptychozoon
Canopy specialists
Flying geckos
have never been the subject of a species-level molecular phylogenetic analysis. In this paper, we utilize
Parachute geckos molecular sequence data from mitochondrial and nuclear gene fragments to estimate the evolutionary
Southeast Asia relationships of this unique group of flying geckos. Capitalizing on the recent availability of genetic sam-
Vertebrate gliders ples for even the rarest of known species, we include the majority of known taxa and use model-based
phylogenetic methods to reconstruct their evolutionary history. Because one species, P. kuhli, exhibits
an unusually wide distribution coupled with an impressive range of morphological variation, we addi-
tionally use intensive phylogeographic/population genetic sampling, phylogenetic network analyses,
and Bayesian species delimitation procedures to evaluate this taxon for the possible presence of cryptic
evolutionary lineages. Our results suggest that P. kuhli may consist of between five and nine unrecog-
nized, distinct species. Although we do not elevate these lineages to species status here, our findings sug-
gest that lineage diversity in Ptychozoon is likely dramatically underestimated.
Ó 2012 Elsevier Inc. All rights reserved.

1. Introduction by biologists and, as a consequence, are poorly represented in natu-

ral history collections (Brown et al., 1997). The majority of species
Among Southeast Asia’s myriad of highly specialized gliding are known from the Southeast Asian mainland and adjacent Sunda-
vertebrates (Colbert, 1967; Russell, 1979a; Emerson and Koehl, land island archipelagos (Taylor, 1915, 1922a,b; Brown et al., 1997;
1990; Goldingay and Scheibe, 2000; Dudley et al., 2007), flying Das and Vijayakumar, 2009), but three taxa, P. kuhli, P. lionotum, and
geckos of the genus Ptychozoon have inspired more awe1 and spec- P. trinotaterra, possess geographic ranges extending northward well
ulation than perhaps any other group of Asian geckos (Annandale, into Indochina (Taylor, 1963; Inger and Colwell, 1977; Biswas and
1904, 1905; Barbour, 1912; de Rooij, 1915; Tweedie, 1954; Tiwari, Sanyal, 1980; Das, 1994a,b; Brown, 1999; Pauwels et al., 2000; Stu-
1961; Pong, 1974; Tho, 1974; Taylor, 1975; Russell, 1979a,b). Most art and Emmett, 2006; Grismer et al., 2008; Grismer, 2011), and a
species in this small genus (seven species) are rarely encountered single species (P. lionotum) has been recorded from the Indian sub-
continent (Pawar and Biswas, 2001; Venugopal, 2010).
Until recent taxonomic work clarified species boundaries
⇑ Corresponding author at: Biodiversity Institute, 1345 Jayhawk Blvd., Lawrence,
KS 66045, USA. Fax: +1 785 864 5335.
(Brown et al., 1997; Brown, 1999; Das and Vijayakumar, 2009),
E-mail addresses: [email protected] (R.M. Brown), [email protected] (C.D. Siler), specimen identification in museum collections was often tenuous
[email protected] (L. Lee Grismer), [email protected] (I. Das), mcguirej@ (RMB, pers. obs), with numerous conflicting literature accounts
berkeley.edu (J.A. McGuire). resulting in general confusion regarding species identifications
1
‘‘Tiny wing like flaps were on either side of the face, other broad winglike and geographic distributions (Cantor, 1847; Günther, 1864, 1885;
expansions were along the side of its body, a similar flap bordered the back of the
thigh. The long tail had a series of frills on each side, as if scalloped lace had been
Boulenger, 1885; Flower, 1896; Stejneger, 1907; Smith, 1930,
sewn on the sides for decoration’’ (Taylor, 1975, p. 47). 1935; Taylor, 1928, 1963; Wermuth, 1965; Dring, 1979; Bobrov,

1055-7903/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.ympev.2012.06.009
 Author's personal copy

352 R.M. Brown et al. / Molecular Phylogenetics and Evolution 65 (2012) 351–361

1995; Nabhitabhata et al., 2000; Cox et al., 1998; Chan-ard et al., We undertook the present study to estimate phylogenetic rela-
1999). Only recently have naturalists developed a reliable under- tionships of the members of the genus Ptychozoon using a multilo-
standing of the taxonomy, ranges of morphological variation with- cus dataset of mitochondrial and nuclear DNA sequences. Although
in, and geographic distributions of each species (Manthey and some studies have hinted at the presence of geographic variation in
Grossmann, 1997; Das, 2004; Malkmus et al., 2002; Nguyen various morphological characters that might eventually prove use-
et al., 2009; Grismer, 2011). ful for diagnosing additional evolutionary lineages (Taylor, 1963;
Flying geckos possess highly derived morphological specializa- Brown et al., 1997, 1999; Das and Vijayakumar, 2009), no compre-
tions, which are present in only a few other groups of gekkonid liz- hensive analysis of morphology or molecular variation has ever
ards (Southeast Asian genus Hemidactylus and possibly the been performed across the geographical range of the most wide-
Malagasy genus Uroplatus). They are characterized by highly dis- spread species, P. kuhli (Fig. 1). At present, an analysis of potential
tinctive and elaborate cutaneous expansions bordering the nuchal diagnostic morphological characters is beyond the scope of this
region and anterior and posterior margins of the limbs, a broadly paper and may be prevented by a lack of adequate numbers of
expanded cutaneous flap bordering both sides of the body (the specimens in museum collections. However the aim of this study
midbody patagium), and a series of serrated, denticulate lobes was to use genetic data, phylogenetic network analysis, and a
extending the length of the tail (terminating in an ornate distal tail recently developed Bayesian lineage delimitation method (Yang
flap in some species; Manthey, 1985; Brown et al., 1997; Brown, and Rannala, 2010) to screen the widespread species P. kuhli for
1999). Lizards of the genus Ptychozoon are so morphologically dis- the presence of genetic partitions that might identify highly
tinctive, that it has come as a surprise to herpetologists that recent divergent lineages corresponding to putative taxonomic entities
molecular phylogenetic studies have found this genus to be nested warranting additional scrutiny by taxonomists.
within the morphologically generalized widespread Eurasian In this study we provide the first estimate of phylogenetic
genus Gekko (Brown et al., 2012). relationships for members of the genus Ptychozoon. Our results
The hypothesized flight structures (the midbody patagia and additionally suggest that the taxon currently recognized as P. kuhli
other extensive cutaneous flaps throughout lateral surfaces of consists of at least five (and possibly as many as nine) divergent
the body) of species in the genus actually have been the subject lineages (possible new species), and this taxon should be the
of historical debate. Although some authors have maintained subject of a future comprehensive taxonomic review aimed at
that they serve no purpose2 or contribute to camouflage ability exploring patterns of morphological variation in this group and
by breaking up the body’s outline against similarly-colored sub- its correlation with patterns of genetic variation identified here.
strates (Gadow, 1901; Barbour, 1912; Pong, 1974; Medway,
1975; Vetter and Brodie, 1977; Kiew, 1987; Russell, 1979a), the
ability of Ptychozoon species to glide or parachute has been well 2. Materials and methods
documented (Tweedie, 1954; Heyer and Pongsapipatana, 1970;
Marcellini and Keefer, 1976; Young et al., 2002). More recent stud- 2.1. Taxon sampling and data collection
ies, however, have acknowledged that lift-generating surfaces
other than just the patagia may contribute to directed aerial des- Ingroup sampling included 32 individuals collected from
cent (Brown et al., 1997; Russell et al., 2001; Young et al., 2002; numerous localities throughout Sundaland (the land-bridge islands
Dudley et al., 2007); these may include the ventrally flattened sur- adjacent to the Malay Peninsula and the Asian mainland) and the
faces of the body, limbs, and tail, as well as extensive interdigital island archipelagos of Indonesia and the Philippines. We collected
webbing of the hands and feet. In the absence of underlying skel- or gained access to genetic samples of six of the seven currently
etal elements or striated muscle, the patagia are not under volun- recognized species of Ptychozoon (Fig. 1; Table 1); to the best of
tary muscle control (Russell, 1972, 1979a; Russell et al., 2001). our knowledge, no tissues have ever been collected for Ptychozoon
Instead they curl under the body at rest, and only extend pas- nicobarensis (Das and Vijayakumar, 2009). To assess the mono-
sively in response to air resistance during gliding or parachuting phyly of the genus and investigate appropriate outgroup taxa, a
(Marcellini and Keefer, 1976; Brown et al., 1997; Young et al., broad sampling from the family Gekkonidae was included, as well
2002). Additionally, Brown et al. (1997) argued that the enlarged, as a single outgroup sample from the Gekkotan family Phyllodac-
imbricate scales on the dorsal surfaces of the midbody cutaneous tylidae (Table 1).
expansion may assist in supporting the expanded patagia during We extracted genomic DNA from liver tissues stored in 95–
flight by preventing inversion (see also Russell, 1979a; Russell 100% ethanol using the guanidine thiocyanate method of Esselstyn
et al., 2001). et al. (2008). We sequenced the mitochondrial gene NADH Dehy-
To date, exemplars of only a few species have ever been in- drogenase Subunit 2 (ND2) and components of three flanking
cluded as outgroups in phylogenetic studies of presumably related transfer RNA genes (tRNAtrp, tRNAala, tRNAasn) using the primers
genera (e.g., Luperosaurus, Brown et al., 2000a,b) or in higher-level and protocols of Brown et al. (2009a, 2012) for 31 vouchered
gekkonid phylogenies (Brown et al., 2012). No species-level specimens (Table 1). For the same specimens, plus one additional
hypothesis of relationships within Ptychozoon has ever been at- vouchered species, we also sequenced the nuclear Phosducin
tempted, although the relevant taxonomic literature contains a (PDC) gene using the primers and protocols of Gamble et al.
variety of predictions with regard to species phenotypic similarity (2008, 2011). Thermal profiles and PCR and sequencing protocols
and implied systematic affinities (Taylor, 1963; Russell, 1972, followed Siler et al. (2012). Amplified products were visualized
1979a,b; Brown and Alcala, 1978; Dring, 1979; Manthey, 1985; on 1.5% agarose gels. PCR products were purified with 1 lL of a
Brown et al., 1997; Brown, 1999; Manthey and Grossmann, 1997). 20% dilution of ExoSAP-IT (US78201, Amersham Biosciences, Pis-
cataway, NJ). Cycle sequencing reactions were run using ABI Prism
BigDye Terminator chemistry (Ver. 3.1; Applied Biosystems, Foster
City, CA), and purified with Sephadex (NC9406038, Amersham Bio-
2
Barbour (1912), on the topic of Ptychozoon cutaneous expansions, stated ‘‘They sciences, Piscataway, NJ) in Centri-Sep 96 spin plates (CS-961,
may possibly assist in rendering the creature less conspicuous at certain rare Princeton Separations, Princeton, NJ). Purified products were
momentary crises. I believe it is far more probable that these developments serve at
present no purpose whatever. They may be taken to represent, perhaps, the result of
analyzed with an ABI Prism 3130xl Genetic Analyzer (Applied Bio-
an inherent tendency to vary in a definite direction, coupled with what Cope has systems). Continuous gene sequences were assembled and edited
called superabundant growth force.’’ using Sequencher 4.8 (Gene Codes Corp., Ann Arbor, MI). To these
 Author's personal copy

R.M. Brown et al. / Molecular Phylogenetics and Evolution 65 (2012) 351–361 353

Fig. 1. Sampling for this study, with various symbols representing the species included (key).

data, we added 29 published sequences of other gekkonid taxa as with parameters and topologies sampled every 5000 generations.
outgroup samples (Bauer et al., 2008; Albert et al., 2009; Siler We assessed stationarity with Tracer v1.4 (Rambaut and Drum-
et al., 2012). All sequences were deposited in GenBank (Table 1). mond, 2007) and confirmed convergence with AWTY (Wil-
genbusch et al., 2004). Stationarity was achieved after 3 million
2.2. Sequence alignment and phylogenetic analyses generations (i.e., the first 15%), and we conservatively discarded
the first 20% of samples as burn-in.
Initial alignments were produced in Muscle (Edgar, 2004) with Partitioned maximum likelihood (ML) analyses were conducted
minimal manual adjustments. To assess phylogenetic congruence in RAxMLHPC v7.0 (Stamatakis, 2006) on the concatenated dataset
between the mitochondrial and nuclear data, we inferred the phy- with the same partitioning strategy as for the Bayesian analysis.
logeny for each gene independently using maximum likelihood The more complex model (GTR + I + C) was used for all subsets
and Bayesian analyses and assessed all strongly supported nodes (Table 2), and 100 replicate ML inferences were performed for each
for differences in relationships between mitochondrial and nuclear analysis. Each inference was initiated with a random starting tree
gene partitions. After observing no statistically significant incon- and nodal support was assessed with 100 bootstrap pseudorepli-
gruence between datasets, we concatenated our data for subse- cates (Stamatakis et al., 2008).
quent analyses. Exploratory analyses of the combined dataset of
61 individuals (including outgroup taxa with missing data for 2.3. Population structure
PDC) and a reduced dataset of individuals with no missing data
exhibited identical relationships; we therefore chose to include In order to visualize population genetic structure and possible
all available data for subsequent analyses of the concatenated reticulating relationships within the widespread (Figs. 1 and 2)
dataset. species, P. kuhli, the NeighborNet algorithm (Bryant and Moulton,
Partitioned Bayesian analyses were conducted in MrBayes 2004) was implemented in the program SplitsTree version 4.10
v3.1.2 (Ronquist and Huelsenbeck, 2003). Both datasets (ND2, (Huson, 1998; Huson and Bryant, 2006) to generate phylogenetic
PDC) were partitioned by codon position and the three flanking networks for the ND2 and PDC datasets, independently. To assess
tRNAs were analyzed as a single subset. The Akaike Information the support for the observed structure, a bootstrap analysis was
Criterion (AIC), as implemented in jModeltest v0.1.1 (Posada, conducted with 1000 replicates. Finally, the pairwise homoplasy
2008), was used to select the best model of nucleotide substitution index (PHI) statistic (Bruen et al., 2006) was calculated in Splits-
for each partition (Table 2). A rate multiplier model was used to al- Tree 4.10 to test for recombination within the mitochondrial and
low substitution rates to vary among subsets, and default priors nuclear loci.
were used for all model parameters. We ran eight independent
MCMC analyses, each with four Metropolis-coupled chains, an 2.4. Bayesian delimitation of putative species
incremental heating temperature of 0.02, and an exponential dis-
tribution with a rate parameter of 25 as the prior on branch lengths With no a priori hypotheses concerning species diversity within
(Marshall, 2010). All analyses were run for 20 million generations, the Ptychozoon kuhli Complex, and low numbers of individuals
 Author's personal copy

354 R.M. Brown et al. / Molecular Phylogenetics and Evolution 65 (2012) 351–361

Table 1
Summary of gekkonid specimens corresponding and genetic samples included in the study. ACD = Arvin C. Diesmos field number, uncataloged specimen deposited at the National
Museum of the Philippines; AMB = Aaron M. Bauer field series; AMS = Australian Museum, Sydney, Australia; CAS = California Academy of Sciences Herpetological Collections;
CMNH = Cincinnati Museum of Natural History (Cincinnati Museum Center); DSM = David McLeod field series, specimen deposited Chulalongkorn University (Thailand) reference
collection; HOFH = Hidetoshi Ota genetic samples deposited in the Museum of Nature and Human Activities, University of Hyogo, Japan; JAM = Jim McGuire field series, specimen
deposited in the Forest Research Institute of Malaysia reference collection); JB = Jon Boone captive collection; JFBM = James Ford Bell Museum of Natural History; KU = University
of Kansas Natural History Museum; LSUHC = La Sierra University Herpetological Collections; UNIMAS P = Pui Yong Min field series, deposited at Universiti Malaysia Sarawak, Kota
Samarahan (UNIMAS); PNM = National Museum of the Philippines (Herpetology collection); RMB = Rafe Brown field number, uncataloged specimen deposited at the National
Museum of the Philippines; TNHC = Texas Natural History Collections of the Texas Memorial Museum, University of Texas at Austin; USNM = United States National Museum;
ZRC = Zoological Reference Collection of the Raffles Museum Collections at the National University of Singapore;  = no voucher/locality information provided by source
publication.

Figure Genus Species Voucher Country Island/ General locality Specific locality ND2 PDC
code number landmass
N/A Ptychozoon horsfieldii ZRC 2.5339 Malaysia Borneo Sarawak Lambir Hills JQ437907 JQ437949
N/A Ptychozoon intermedium PNM 2501 Philippines Mindanao Davao City Municipality of Calinan, JQ437908 JQ437950
Island Province Barangay Malagos
N/A Ptychozoon intermedium CMNH 4747 Philippines Mindanao Davao City Municipality of Calinan, JQ437909 JQ437951
Island Province Barangay Malagos
N/A Ptychozoon intermedium TNHC 56144 Philippines Mindanao Davao City Municipality of Calinan, JQ437911 JQ437953
Island Province Barangay Malagos
16 Ptychozoon kuhli LSUHC 3835 Malaysia Malay Pahang Pulau Tioman, Tekek-Juara JQ437935 JQ437977
Peninsula Trail
14 Ptychozoon kuhli LSUHC 4679 Malaysia Malay Pahang Pulau Tioman, Tekek-Juara JQ437918 JQ437960
Peninsula Trail
15 Ptychozoon kuhli LSUHC 6433 Malaysia Malay Pahang Pulau Tioman, Tekek-Juara JQ437931 JQ437973
Peninsula Trail
18 Ptychozoon kuhli LSUHC 5042 Malaysia Malay Pahang Pulau Tioman, Sungai JQ437930 JQ437972
Peninsula Mentawak
8 Ptychozoon kuhli LSUHC 7141 Malaysia Malay Kedah Pulau Langkawi, Air Terjun JQ437924 JQ437966
Peninsula below Telaga Tuju
9 Ptychozoon kuhli LSUHC 4819 Malaysia Malay Selangor Kepong JQ437920 JQ437962
Peninsula
10 Ptychozoon kuhli LSUHC 3518 Malaysia Malay Pahang Pulau Tioman JQ437936 JQ437978
Peninsula
22 Ptychozoon kuhli LSUHC 5587 Malaysia Malay Johor Pulau Babi, Besar JQ437939 JQ43798
Peninsula
11 Ptychozoon kuhli LSUHC 5055 Malaysia Malay Pahang Pulau Tulai JQ437929 JQ437971
Peninsula
12 Ptychozoon kuhli LSUHC 6273 Malaysia Malay Johor Pulau Tulai JQ437938 JQ437980
Peninsula
17 Ptychozoon kuhli LSUHC 5199 Malaysia Malay Johor Pulau Sembilan JQ437925 JQ437967
Peninsula
21 Ptychozoon kuhli LSUHC 6321 Malaysia Malay Johor Pulau Tinggi, Pasir Panjang JQ437934 JQ437976
Peninsula
5 Ptychozoon kuhli ZRC 2.5332 Malaysia Borneo Sarawak Kapit. Kelep, Asap JQ437928 JQ437970
Island
6 Ptychozoon kuhli RMB T-1134 ‘‘Malaysia’’ ‘‘Malay ‘‘Pahang’’ Pet trade sample: JQ437919 JQ437961
Peninsula’’ reportedly ‘‘Gua Musang’’
7 Ptychozoon kuhli RMB T-1139 ‘‘Malaysia’’ ‘‘Malay ‘‘Pahang’’ Pet trade sample: JQ437937 JQ437979
Peninsula’’ reportedly ‘‘Gua Musang’’
1 Ptychozoon kuhli RMB T-0001 ‘‘Indonesia’’ ‘‘Java Unknown Pet trade sample: JQ437927 JQ437969
Island’’ reportedly ‘‘Java’’
20 Ptychozoon kuhli LSUHC 5708 Malaysia Malay Pahang Pulau Aceh JQ437932 JQ437974
Peninsula
13 Ptychozoon kuhli LSUHC 8024 Malaysia Malay Johor Pulau Pemanggil JQ437933 JQ437975
Peninsula
19 Ptychozoon kuhli LSUHC 7640 Malaysia Malay Johor Endau-Rompin JQ437926 JQ437968
Peninsula
3 Ptychozoon kuhli MVZ 239588 Indonesia Sumatra Kabupaten Kecematan Kepahiang, JQ437922 JQ437964
Island Bengkulu 46 km E of Bengkulu, Cagar
Alam Tabapenangjung
2 Ptychozoon kuhli MVZ 239358 Indonesia Enggano Kecematan vicinity Malakoni village JQ437921 JQ437963
Island Enggano
4 Ptychozoon kuhli JAM 6445 Indonesia Sulawesi Sulawesi Barat Kecamatan Tapalang, Desa JQ437923 JQ437965
Island Province, Takandeang,
Kabupaten
Mumuju
N/A Ptychozoon lionotum JAM 1426 Malaysia Malay Selangor Ulu Gombak Field Studies JQ437916 JQ437958
Peninsula Centre, km 30N of Kuala
Lumpur via Rt. 68
N/A Ptychozoon lionotum LSUHC 6437 Malaysia Malay Pahang JQ437917 JQ437959
Peninsula
N/A Ptychozoon lionotum DSM 798 Thailand Southern Nakhon Si Khao Luang National Park JQ437914 JQ437956
Malay Thammarat
Peninsula Province
N/A Ptychozoon lionotum FMNH Cambodia Asian Kampong Speu Phnom Sruoch District JQ437915 JQ437957
261852 mainland Province
 Author's personal copy

R.M. Brown et al. / Molecular Phylogenetics and Evolution 65 (2012) 351–361 355

Table 1 (continued)

Figure Genus Species Voucher Country Island/ General locality Specific locality ND2 PDC
code number landmass
N/A Ptychozoon rhacophorus UNIMAS P- Malaysia Borneo Sarawak Gunung Penrissen JQ437913 JQ437955
0501
N/A Ptychozoon trinotaterra ROM 31912 Vietnam Asian Yok Don Yok Don National Park JQ437912 JQ437954
mainland Province
N/A Gehyra australis AMS 139934 Australia Australia Western El Questro Station, JN019081 JN019113
Australia, Jackeroos Waterhole,
N/A Gehyra mutilata AMB 7515 Sri Lanka Sri Lanka Nimalawa JN019082 JN019114
N/A Hemidactylus aquilonius CAS 206649 Myanmar Asian Sagaing Division Alaungdaw Kathapa EU268373 –
mainland National Park
N/A Cyrtodactylus annulatus KU 314944 Philippines Mindanao Agusan del Sur Municipality of San GU366088 –
Island Province Francisco, Barangay
Kaimpugan
N/A Cyrtodactylus philippinicus KU 304784 Philippines Babuyan Cagayan Municipality of Calayan, GU550900 –
Claro Province Barangay Babuyan Claro
Island
N/A Luperosaurus joloensis KU 314947 Philippines Mindanao Zamboanga City Barangay Pasanonca JQ437900 See Dryad
Island Submission
N/A Luperosaurus cumingii TNHC 61910 Philippines Luzon Albay Province Municipality of Tiwi, Mt. JQ437902 –
Island Malinao
N/A Luperosaurus angliit KU 322189 Philippines Luzon Aurora Province Municipality of Baler, JQ437903 JQ439744
Island Barangay Zabali
N/A Pseudogekko smaragdinus KU 302819 Philippines Polillo Quezon Province Municipality of Polillo, JQ437898 JQ437941
Island Barangay Pinaglubayan
N/A Pseudogekko compressicorpus KU 324426 Philippines Bohol Bohol Province Municipality of Sierra JQ437897 JQ437940
Island Bullones, Barangay Danicop
N/A Lepidodactylus herrei RMB 4330 Philippines Leyte Leyte Province Municipality of Baybay JQ173539 –
Island
N/A Lepidodactylus moestus USNM Palau Ngerur JN019079 JN019111
521730 Island
N/A Gekko athymus KU 314944 Philippines Palawan Palawan Municipality of Brooke’s JQ173403 JQ173559
Island Province Point, Barangay Mainit
N/A Gekko crombota KU 304825 Philippines Babuyan Cagayan Municipality of Calayan, JQ173410 JQ173549
Claro Province Barangay Babuyan Claro
Island
N/A Gekko romblon KU 315348 Philippines Tablas Romblon Municipality of Calatrava, JN710497 JN710506
Island Province Barangay Balogo
N/A Gekko mindorensis KU 302668 Philippines Mindoro Oriental Municipality of Bongabong, JN710490 JN710501
Island Mindoro Barangay Formon
Province
N/A Gekko monarchus ACD 1278 Philippines Palawan Palawan Municipality of Brooke’s JQ173501 JQ173594
Island Province Point, Mt. Mantalingajan
N/A Gekko smithii LSUHC 6095 Malaysia Malay Pahang Pekan JQ173534 JQ173616
Peninsula
N/A Gekko gecko CAS 204952 Myanmar Asian Ayeyarwady Myaungmya District JQ173416 –
mainland Division
N/A Gekko chinensis LSUHC 4210 China Hainan Wuzhi Shan JQ173409 JQ173547
Island
N/A Gekko japonicus HOFH Japan Ryukyu JQ173424 JQ173558
10061402 Islands
N/A Gekko swinhonis NNU Z China Asian Szechuan Chengdu JN019061 JN019095
20051124.001 mainland
N/A Gekko subpalmatus AMB 6567 China Asian Szechuan Chengdu JN019063 JN019097
mainland
N/A Gekko hokouensis HOFH China Orchid Lanyu Township JQ173422 JQ173556
89053103 Island
N/A Gekko vittatus USNM Solomon Santa Cruz Temotu Luesalo JN019073 JN019106
533255 Islands Island
N/A Gekko petricolus JB 70 Unknown Pet trade JN019066 JN019100
N/A Gekko badenii JB 13 Unknown Pet trade JN019065 JN019099
N/A Gekko grossmanni JFBM 9 Unknown Pet trade JN019064 JN019098

Table 2
sampled per population, we approached the question of species-le- Models of evolution selected by AIC and applied for partitioned, model-based
vel diversity from the most liberal perspective; we treated each of phylogenetic analyses.
the nine sampled localities as separate populations. We then ex- Partition AIC model Number of characters
plored support for hypothesized species boundaries using the pro-
NADH 2, 1st codon position HKY + I + C 346
gram Bayesian Phylogenetics and Phylogeography (BPP v.2.0; Yang NADH 2, 2nd codon position HKY + C 346
and Rannala, 2010). In order to provide an objective starting topol- NADH 2, 3rd codon position GTR + C 346
ogy for BPP, we used the multi-species coalescent model imple- All tRNAs (Trp, Ala, Asn) HKY + C 223
mented in the program BEAST (v1.6.2; Drummond and Rambaut, Phosducin, 1st codon position HKY + I 137
Phosducin, 2nd codon position HKY + I 136
2007; Heled and Drummond, 2010) to estimate relationships
Phosducin, 3rd codon position GTR + I 136
among the nine divergent populations observed in phylogenetic
 Author's personal copy

356 R.M. Brown et al. / Molecular Phylogenetics and Evolution 65 (2012) 351–361

Fig. 2. Hypothesized species level relationships of the genus Ptychozoon, illustrated by the maximum clade credibility tree resulting from Bayesian analyses. Nodes supported
by P0.95 Bayesian posterior probabilities and P70% ML bootstrap support were considered significantly supported. Numbered and differently colored terminals within P.
kuhli correspond to numbered localities on the map and the same color scheme is utilized in Fig. 3. Letters refer to inferred genetic lineages (hypothesized species) from the
BPP analyses (Fig. 3).

analyses. Individual sequences were assigned to lineages on the simple while still capturing the important aspects of the system.
basis of sampling locality, which also corresponded to well-sup- To investigate support for species relationships under a different
ported lineages observed in phylogenetic analyses (Fig. 2). tree prior, we reanalyzed the data under a piecewise linear prior,
For BEAST analyses, we applied the following settings: (1) sep- or unconstrained Ne across all branches on the tree.
arate GTR + C nucleotide substitution models and lognormal-dis- We ran two independent analyses for 80 million generations,
tributed relaxed clock models to nuclear and mitochondrial sampling every 15,000 generations. Using the program Tracer
subsets, (2) a mean rate of the mitochondrial relaxed clock hy- (Rambaut and Drummond, 2005), we assessed stationarity and
per-parameter to 1.0, (3) estimates of the rate of the nuclear re- convergence of each run by plotting all parameters and likelihood,
laxed clock relative to the mitochondrial clock, (4) a uniform prior, and posterior scores over generations. Both of our analyses
prior (U(0, 2.0)) on the mean of the lognormal-distributed nuclear showed patterns consistent with convergence, and we observed
relaxed-clock hyper-parameter, (5) an exponentially distributed effective sample sizes >200 after conservatively removing the first
prior (Exp(20)) on the standard deviation of both lognormal-dis- 20 million generations as burn-in.
tributed relaxed-clock hyper-parameters, and (6) default priors Using the topology observed in the resulting chronogram from

for the parameters of both GTR models. For each of the two loci, BEAST analyses, we evaluated the statistical support for our liberal
we implemented appropriate ploidy levels and random starting hypothesis of putative species boundaries by employing the mod-
trees to infer gene trees conditional on the species tree. For the el-based program BPP. We phased the nuclear data, using the pro-
species tree we implemented a Yule process prior, and constrained, gram PHASE v. 2.1.1 (Stephens and Donnelly, 2003), and retained
constant effective population size (Ne) along each branch. We haplotypes with the highest probabilities for subsequent analyses.
chose the prior on Ne in an effort to choose a model with fewer For BPP analyses, we used the mitochondrial (ND2) data and nucle-
parameters. With only two loci, our goal was to keep the model ar (PDC) allelic data following the approach advocated by Setiadi
 Author's personal copy

R.M. Brown et al. / Molecular Phylogenetics and Evolution 65 (2012) 351–361 357

et al. (2011). We assigned samples to one of the nine inferred lin- taxa (Fig. 2). The four sampled populations of P. lionotum were
eages. Following Setiadi et al. (2011), we accommodated expected recovered as part of two divergent clades, with one clade consist-
differences between the effective population size (Ne) of mtDNA ing of populations from Peninsular Malaysia and the other clade
and autosomal DNA, and we incorporated two additional parame- consisting of populations from Thailand and Laos (Fig. 2). Members
ters into BPP analyses as follows: (1) heredity parameter with gam- of these two highly divergent clades were separated by 15.2–16.0%
ma prior G[1.39, 2.22], and (2) locusrate parameter calculated as uncorrected pairwise ND2 sequence divergence.
the largest Jukes Cantor-corrected sequence divergence from the A strongly supported clade consisting of P. horsfieldii +
outgroup sequence. P. intermedium was observed to be sister to a well supported clade
Analyses were run for 500,000 generations, sampling every 50 of P. trinotaterra + P. kuhli (Fig. 2), albeit with weak support (likeli-
generations, with a burn-in of 10,000. The ‘‘0’’ algorithm with the hood bootstrap proportion = 28%; Bayesian poster probability
fine-tuning parameter e = 15 was employed after preliminary runs 0.51). The widespread species P. kuhli was recovered as a highly
employing lower (5, 10) and greater (20, 25) values of e had no ma- structured, clade, consisting of nine well supported, divergent
jor impact on resulting inferences of species diversity. Following (Table 3) lineages (Fig. 2). Finally, our results confirm recent find-
the methods of Leaché and Fujita (2010), we explored the impact ings (Brown et al., 2012) that the genus Ptychozoon, and some spe-
of prior regime (ancestral population size [h] and root age [s]) on cies of the genus Luperosaurus are nested within Gekko (SI Fig. 1).
speciation probabilities. Three prior settings were employed: (1)
a relatively large ancestral population with shallow divergences 3.3. Phylogenetic networks of Ptychozoon kuhli
(h = 1, 10; s = 2, 2000; both prior means = 0.1 and variance = 0.01),
(2) a relatively large ancestral population with deep divergences Tests of recombination within the mitochondrial or nuclear lo-
(h = 1, 10; s = 1, 10; both prior means = 0.001 and vari- cus were not significant (ND2 PHI value = 0.746, PDC PHI
ance = 5  10 7), and (3) a relatively small ancestral population value = 1.0), and we therefore felt justified in exploring phyloge-
and shallow divergences (h = 2, 2000; s = 2, 2000). netic network analysis for each locus. The analysis of the nuclear
locus (PDC) revealed little structure (Fig. 3A); however, the analy-
sis of the mitochondrial locus (ND2) revealed nine highly diver-
3. Results
gent, well-supported, groups and a high degree of structure
(Fig. 3A) corresponding to divergent lineages (Table 3) identified
3.1. Taxon sampling, data collection, and sequence alignment
in the phylogenetic analysis of concatenated mtDNA + nDNA data
(Fig. 2).
The complete, aligned matrix contains 32 samples of Ptychozo-
on, representing six of the seven currently recognized species.
3.4. Bayesian inference of potential species boundaries within
Twenty-nine additional samples are included as outgroups from
Ptychozoon kuhli
the families Gekkonidae and Phyllodactylidae, including represen-
tative taxa of the following genera: Cyrtodactylus, Gehyra, Gekko,
Relationships inferred in our BEAST analyses mirror those of
Hemidactylus, Lepidodactylus, Luperosaurus, Pseudogekko, and
concatenated gene tree phylogenetic analyses, with support for
Tarentola. Following initial unrooted analyses, and gekkonid phylo-
the same topological relationships among the nine well-supported
genetic analyses (Gamble et al., 2011, 2012; Brown et al., 2012) we
lineages of the P. kuhli Complex (Figs. 2 and 3B); we took this
rooted the tree using the representative sample of Tarentola mau-
topology as our user-specified guide tree for subsequent BPP anal-
ritanica (Phyllodactylidae). Variable and parsimony-informative
yses. The choice of prior on Ne had no impact on the resulting rela-
characters are: 908 and 824 of 1208 (ND2); 77 and 49 of 419 (PDC).
tionships between species (results not shown). Taking an initial
liberal approach to identifying species boundaries, and starting
3.2. Phylogenetic analyses with the assumption that all nine genetically divergent (Table 3)
lineages could conceivably be unique species, Bayesian species
Analyses of the combined data result in topologies with high ML delimitation results for P. kuhli support five species with speciation
bootstrap support and posterior probabilities between species probabilities of 1.0 on the species guide tree (Fig. 3B). The remain-
within and between the majority of clades in the inferred phylog- ing three putative ‘‘speciation events’’ on the species guide tree re-
eny, with general topological patterns congruent across these anal- ceive low to moderate support (Fig. 3B). These results were not
yses (Fig. 2). The focal taxa from the genus Ptychozoon were affected by varying prior distributions for h or s (Fig. 3B). The five
supported to be monophyletic (Fig. 2). As observed in a recent putative entities supported in the PB&B analysis correspond to a
study (Brown et al., 2012), the genus Ptychozoon is supported to lineage sampled from the pet trade (reportedly from Java Island,
be sister to a clade consisting of some members of the genus Gekko Indonesia), a clade consisting of populations from Enggano and
(G. badenii, G. grossmanni, G. petricolus, and G. vittatus). As observed Sumatra islands, a clade consisting of samples from Sulawesi and
by Brown et al. (2012) the enigmatic species, Ptychozoon rhacopho- Borneo islands, a lineage consisting of populations from southeast
rus, is strongly supported as the sister species to all other sampled Peninsular Malaysia and the surrounding coastal islands of the

Table 3
Uncorrected pairwise sequence divergence (%) for mitochondrial data for the nine supported lineages within the Ptychozoon kuhli Complex (Fig. 1). Percentages on the diagonal
represent intraspecific genetic diversity, when sampling permits (bolded for emphasis).

A B C D E F G H I
A –
B 17.2 –
C 17.8 11.6 –
D 16.4 13.4 13.1 –
E 16.9 14.7 14.1 7.5 –
F 16.6 12.9 12.3 10.3 11.2 0.0
G 17.1 14.1 13.5 10.9 11.6 7.8 –
H 16.7 13.9–14.0 13.9–14.0 10.6 11.5 7.3–7.4 5.4 0.0
I 16.2–17.0 12.6–12.9 12.6–13.3 9.4–10.0 10.7–11.0 10.1–10.5 11.2–11.6 11.0–11.5 0.1–1.8
 Author's personal copy

358 R.M. Brown et al. / Molecular Phylogenetics and Evolution 65 (2012) 351–361

Fig. 3. SplitsTree networks (Huson and Bryant, 2006) (A) for two loci (same number and color scheme as that presented in Fig. 2) and BEAST (v1.6.2; Heled and Drummond,
2010) topology (B) with results of Bayesian lineage delimitation analyses inferred by BPP. Posterior probabilities of inferred splits are provided at each node, with ranges
representing split probabilities produced by variance in prior settings for ancestral population size and relative divergence times.

Seribuat Archipelago, and a lineage consisting of trade samples density of irregular, ornate, tuberculate scalation on dorsal surfaces
reportedly from north-central Peninsular Malaysia, together with of the body, and lacks multiple conspicuous characteristics (the nu-
samples from southwestern Peninsular Malaysia and the Langkawi chal/cephalic cutaneous expansion, expanded tail terminus, and dor-
Islands adjacent to northwest Peninsular Malaysia (Fig. 3B). sal enlarged imbricate support scales of the patagial cutaneous
expansion) shared by all other species in the genus (Russell, 1972;
Manthey, 1985; Brown et al., 1997). We are not surprised by the close
4. Discussion relationship between Bornean P. horsfieldii and southern Philippine P.
intermedium. Not only are these two species endemic to geographi-
4.1. Phylogeny of Ptychozoon and implications for morphological cally very proximate landmasses (Fig. 1), but both uniquely share
novelty in flying geckos numerous distinctive characters (posterior angling of denticulate tail
lobes, decreased distal tail lobe size, tail terminus ending in a minute
Species level relationships within the genus Ptychozoon, inferred flap, absence of caudal lobe fusion on proximate margin of tail
from concatenated Bayesian and Maximum Likelihood gene trees terminus, and separation between the femoral and precloacal
(Fig. 2) confirm several earlier character-based predictions, but also pore-bearing scales; Brown et al., 1997; Brown, 1999). In contrast,
contain a few surprises. The finding that P. rhacophorus is phylogenet- we were surprised to find the species couplet P. horsfieldii + P. inter-
ically distinct from (but sister to) the remaining known taxa is not medium recovered between P. lionotum and the clade consisting of
surprising given its distinctive morphology (Russell, 1972; Manthey, P. trinotaterra + P. kuhli (albeit with low nodal support; Fig. 2).
1985; Brown et al., 1997; Brown and Diesmos, 2000). This species is Previous authors (Boulenger, 1885; de Rooij, 1915; Smith 1930,
smaller than all other members of the genus, possesses a greater 1935; Taylor, 1963; Brown et al., 1997) have noted the close
 Author's personal copy

R.M. Brown et al. / Molecular Phylogenetics and Evolution 65 (2012) 351–361 359

morphological similarity between P. lionotum and P. kuhli (and, by bootstrap support supporting all the same groupings; Fig. 3A).
implication, P. trinotaterra; Brown, 1999), and we would find it sur- Interestingly, however, our results varied with respect to the con-
prising if the suite of morphological character states seemingly unit- clusions derived from the BPP analyses vs. more traditional tree
ing these two species and P. trinotaterra were to have evolved based species delimitation approaches.
convergently. Shared character states include denticulate tail lobes NeighborNet bootstrapping analysis implemented in Splits-
that do not diminish in size posteriorly, presence of a greatly ex- Tree phylogenetic networks provided significant support for deep
panded terminal tail flap, lobe fusion at the proximate margin of divergences between nine Ptychozoon kuhli lineages, even be-
the tail terminus, and continuous precloacofemoral pore bearing tween those not supported with high speciation probabilities
scales (Brown et al., 1997; Brown, 1999). The nodal support for the in BPP analyses. Thus, the genetic distinctiveness of each highly
placement of the P. horsfieldii + P. intermedium clade (Fig. 2) suggests divergent lineage apparent in our concatenated gene tree (Fig. 2),
to us that a conservative interpretation might be to consider these examination of uncorrected p-distances between taxa, (Table 3)
well supported clades (1: P. lionotum; 2: P. horsfieldii + P. intermedi- and confirmed by phylogenetic networks (Fig. 3A) is not suffi-
um; 3: P. trinotaterra + P. kuhli) to be an unresolved trichotomy. We cient for our Bayesian species delimitation analysis to infer sig-
would not be surprised to find with future analyses (preferably nificant splits between some terminals (B + C; D + E; F + G + H)
involving additional unlinked loci) that actual relationships among included in our study. Confirmation or refutation of the hypoth-
species of Ptychozoon are (P. rhacophorus, ((P. horsfieldii + P. intermedi- esis of taxonomic distinctiveness of the relevant populations
um), (P. lionotum, (P. trinotaterra + P. kuhli)))). The unsampled species (Sumatra vs. Enggano islands; Borneo vs. Sulawesi islands; and
P. nicobarensis is most likely closely related to P. kuhli, from which it is Langkawi vs. central Malaysian Peninsula) will necessarily in-
only slightly distinguishable on the basis of phenotypic traits dis- volve examination of other sources of data (additional loci, mor-
cussed by Das and Vijayakumar (2009). phology, ecology, behavior) before firm conclusions can be
drawn.
4.2. Taxonomic implications of genetic divergences, phylogenetic The presence of substantial genetic structure and numerous
network analyses and Bayesian lineage delimitation in P. kuhli unrecognized putative species masquerading within the wide-
spread and morphologically variable (Fig. 3B) P. kuhli is not surpris-
The results of our Bayesian species delimitation analysis re- ing. Ptychozoon kuhli, as currently recognized, has a geographic
vealed strong support for subdivision of the widespread, morpho- range encompassing nearly that of all remaining Ptychozoon spe-
logically variable Ptychozoon kuhli into five genetically distinct cies combined, and we find it extremely unlikely that many single
units, or putative species, with high ‘‘speciation’’ probabilities, native terrestrial vertebrate species have actual ranges this broad
some of which contain additional highly divergent gene lineages. (Dickerson, 1928; Corbet and Hill, 1992; Inger, 1999; Inger and
Although the same nine genetically distinct lineages were identi- Voris, 2001; Brown and Diesmos, 2009; Brown and Stuart, 2012),
fied in phylogenetic analyses (Fig. 2), phylogenetic networks given the ecologically heterogeneous and geographically parti-
(Fig. 3A), and examination of genetic distances (Table 3), Bayesian tioned nature of the Southeast Asian and Indo-Australian island
species delimitation analyses inferred low to moderate speciation archipelagos (Whitmore and Sayer, 1992; Whitmore, 1975, 1987;
probabilities between four pairs of terminals (Fig. 3B). Although Woodruff, 2010; Lomolino et al., 2010). Previous workers have
we would not advocate the recognition of new putative species noted considerable morphological variation in this species (Brown
on the basis of genetic divergences, we do note that the nine genet- et al., 1997), and recently, the Nicobar Island population, previ-
ic lineages identified by our analyses differ by substantially more ously referred to this species, was described as a distinct species
mitochondrial sequence divergence (Table 3) than most diver- of Ptychozoon on the basis of morphological characters and color
gences identified in numerous recent studies of cryptic gekkonid pattern (Das and Vijayakumar, 2009). Thus, we find the results of
lizard diversification (see Gamble et al., 2012, for discussion) and, phylogenetic networks and BPP analyses compelling evidence for
thus, may warrant further taxonomic scrutiny with other sources the possibility of additional species diversity, albeit with the caveat
of data. that before species can be formally recognized, a comprehensive
Five highly significant putative species splits were favored, pri- review of the taxon P. kuhli must be performed such that
marily involving geographically circumscribed or isolated popula- character-based diagnoses can be formulated to identify distinct
tions. These correspond to (letters referring to those in Figs. 2, evolutionary lineage segments and define these as formal, line-
3A): A: a sample of unknown provenance (pet trade, reportedly age-based species (Simpson, 1961; Wiley, 1978; Frost and Hillis,
shipped to the US from Jakarta, Indonesia and therefore, possibly 1990; de Queiroz, 1998; Brown and Diesmos, 2001; Bauer et al.,
from Java Island); Clade B + C: two divergent lineages, together 2010), hopefully using a variety of different types of evidence.
most likely representing a single putative taxonomic entity (with
a weakly supported split between them) from Sumatra and nearby 4.3. Conservation implications
Enggano islands, respectively; Clade D + E: two additional diver-
gent lineages (possibly representing a single putative taxonomic Although its formal conservation status currently remains unas-
entity) with a weakly supported split between them, from Borneo sessed (IUCN, 2011), herpetologists have long considered Ptychozo-
and adjacent Sulawesi islands, respectively; F: a possibly distinct on kuhli to be the most widespread, commonly encountered species
species from central Peninsular Malaysia (two pet trade samples, of Ptychozoon (Manthey and Grossmann, 1997; Das, 2004; Malkmus
reportedly from the vicinity of Gua Musang, central Peninsular et al., 2002; Nguyen et al., 2009; Grismer, 2011). Our study demon-
Malaysia) and Langkawi Island (G), northwestern Peninsular strates that this single species is composed of at least nine distinct,
Malaysia and of south-central Peninsular Malaysia (H) and I: a genetically divergent lineages (Fig. 2; Table 3), some of which are
hypothetically distinct species from the lowlands of southern Pen- endemic or range-restricted to one or two nearby landmasses (is-
insular Malaysia and nearby islands of the Seribuat Archipelago. lands) or habitat types (lowland coastal areas, highland forests, tree
In general, our varied approaches to inferring the genealogical canopies), and several of which might soon be defined as distinct
relationships among populations of Ptychozoon kuhli provided very evolutionary species. Although we did not perform Bayesian spe-
similar results. Bayesian and Maximum Likelihood concatenated cies delimitation analyses involving P. lionotum, we suspect the
gene tree analyses generated the same topology inferred in our same taxonomic implications may apply to the highly divergent

BEAST species tree estimation procedures, which were again (15.2–16.0% uncorrected pairwise sequence divergence) north–
mirrored in Splits Tree phylogenetic networks (with strong south split detected in this species, roughly spanning the Isthmus
 Author's personal copy

360 R.M. Brown et al. / Molecular Phylogenetics and Evolution 65 (2012) 351–361

of Kra and the Kangar-Pattani Line, a recognized, and well charac- Bobrov, V.V., 1995. Checklist and bibliography of the lizards of Vietnam.
Smithsonian Herpetol. Infor. Ser. 105, 1–28.
terized, biogeographic boundaries (Whitmore, 1987; Voris, 2000;
Bauer, A.M., Giri, V.B., Greenbaum, E., Jackman, T.R., Dharne, M.S., Shouche, Y.S.,
Woodruff, 2010). Thus, the general results of this study suggest 2008. On the systematics of the gekkonid genus Teratolepis Günther, 1869:
that, in addition to a taxonomic review, conservation status assess- another one bites the dust. Hamadryad 33, 13–27.
ments (sensu IUCN, 2011) should be performed to the best ability of Boulenger, G.A., 1885. Catalogue of the Lizards in the British Museum (Nat. Hist.) I.
Geckonidae, Eublepharidae, Uroplatidae, Pygopodidae, Agamidae. British
the available field-based data, soon after formal taxonomic revision Museum (Natural History), London.
of P. kuhli becomes available. As increasing numbers of ‘‘wide- Brown, R.M., 1999. New species of parachute gecko (Squamata; Gekkonidae; genus
spread’’ Southeast Asian species turn out to be complexes of Ptychozoon) from northeastern Thailand and Central Vietnam. Copeia 1999,
990–1001.
range-restricted microendemics (Stuart et al., 2006; Brown et al., Brown, R.M., Diesmos, A.C., 2000. The lizard genus Luperosaurus: taxonomy, history,
2009b; Grismer et al., 2010; McLeod, 2010; Brown and Stuart, and conservation prospects for some of the world’s rarest lizards. Sylvatrop.:
2012), it becomes increasingly difficult—yet essential—to protect Tech. J. Philipp. Eco. Nat. Res. 10, 107–124.
Brown, R.M., Diesmos, A.C., 2001. Application of lineage-based species concepts to
this diversity by mitigating habitat destruction and forest loss oceanic island frog populations: the effects of differing taxonomic philosophies
throughout the Asian mainland and adjacent archipelagos (Sodhi on the estimation of Philippine biodiversity. Silliman J. 42, 133–162.
et al., 2004). Brown, R.M., Diesmos, A.C., 2009. Philippines, biology. In: Gillespie, R., Clague, D.
(Eds.), Encyclopedia of Islands. University of California Press, Berkeley, pp. 723–
732.
Acknowledgments Brown, R.M., Stuart, B.L., 2012. Patterns of biodiversity discovery through time: an
historical analysis of amphibian species discoveries in the Southeast Asian
mainland and island archipelagos. In: Gower, D.J., Johnson, K.G., Richardson, J.E.,
We thank the Protected Areas and Wildlife Bureau (PAWB) of Rosen, B.R., Rüber, L., Williams, S.T. (Eds.), Biotic Evolution and Environmental
the Philippine Department of Environment and Natural Resources Change in Southeast Asia. Cambridge University Press, pp. 348–389.
Brown, R.M., Ferner, J.W., Diesmos, A.C., 1997. Definition of the Philippine parachute
(DENR) and the Economic Planning Unit of Malaysia (EPU) as well gecko, Ptychozoon intermedium Taylor 1915 (Reptilia: Squamata: Gekkonidae):
as the Sarawak Forest Department for facilitating collecting and ex- redescription, designation of a neotype, and comparisons with related species.
port permits (No. NCCD.907.4.4.[Jld.7]-38 and Park Permit 173/ Herpetologica 53, 357–373.
Brown, R.M., Oliveros, C., Siler, C.D., Diesmos, A.C., 2009a. Phylogeny of Gekko from
2011; export form of license/permit No. 09861) necessary for this the northern Philippines, and description of a new species from Calayan Island.
and related studies. We thank the Indonesian Institute of Sciences J. Herpetol. 43, 620–635.
(LIPI) for permission to conduct research in Indonesia, our col- Brown, R.M., Siler, C.D., Das, I., Pui, Y.M., 2012. Testing the phylogenetic affinities of
Southeast Asia’s rarest geckos: flap-legged geckos (Luperosaurus), flying geckos
leagues J. Supriatna, Y. Andayani (University of Indonesia) and D.
(Ptychozoon) and their relationship to the pan-Asian genus Gekko. Mol.
Iskandar (Bandung Institute of Technology) for their hospitality, Phylogenet., Evol. 63, 915–921.
and the Museum Zoologicum Bogoriense for assistance with Brown, R.M., Siler, C.D., Diesmos, D.C., Alcala, A.C., 2009b. The Philippine frogs of the
permits. Sampling that contributed to this work resulted from genus Leptobrachium (Anura; Megophryidae): phylogeny-based species
delimitation, taxonomic revision, and descriptions of three new species.
fieldwork funded with National Science Foundation Grants DEB Herpetol. Monogr. 23, 1–44.
0743491 to RMB, DEB 0804115 to CDS, and DEB 0328700 to Brown, R.M., Supriatna, J., Ota, H., 2000b. Discovery of a new species of Luperosaurus
J. McGuire. Field work on Borneo was funded by a Fundamental (Squamata; Gekkonidae) from Sulawesi, with a phylogenetic analysis of the
genus and comments on the status of L. serraticaudus. Copeia 2000, 191–
Research Grant from the Ministry of Higher Education, Malaysia 209.
(FRGS/07(04)787/2010[68]) and a Shell Chair (SRC/05/2010[01]) Brown, W.C., Alcala, A.C., 1978. Philippine Lizards of the family Gekkonidae.
grant, administered by the Institute of Biodiversity and Environ- Silliman Univ. Nat. Sci. Monogr. Ser. 1, Dumaguete.
Bruen, T.C., Philippe, H., Bryant, D., 2006. A simple and robust statistical test for
mental Conservation, Universiti Malaysia Sarawak (to ID). For detecting the presence of recombination. Genetics 172, 2665–2681.
provision of additional genetic samples, we thank B. Stuart, H. Bryant, D., Moulton, V., 2004. Neighbor-Net: an agglomerative method for the
Ota, D. McLeod, Y.M. Pui, A. Lathrop, and R. Murphy. Thanks are construction of phylogenetic networks. Mol. Biol. Evol. 21, 255–265.
Cantor, T., 1847. Catalogue of the reptiles inhabiting the Malayan Peninsula and
due to C. Lee, A. Lathrop, R. Murphy, and D. Wechsler for use of islands, collected or observed by Theodore Cantor, Esqr., M. D. J. Asiat. Soc.
their photographs in Fig. 2. We thank two anonymous reviewers Bengal 16, 607–656 (part).
for comments on an earlier draft of this manuscript. Chan-ard, T., Grossmann, W., Gumprecht, A., Schulz, K.D., 1999. Amphibians and
Reptiles of Peninsular Malaysia and Thailand – An Illustrated Checklist
(bilingual English and German). Bushmaster Publications, Würselen.
Colbert, E.H., 1967. Adaptations for gliding in the lizard Draco. Am. Mus. Novitates
Appendix A. Supplementary material
2283, 1–20.
Corbet, G.B., Hill, J.E., 1992. The Mammals of the Indomalayan Region. Oxford
Supplementary data associated with this article can be found, in University Press, New York.
Cox, J., van Dijk, P., Nabhitabhata, J., Thirakhupt, K., 1998. A Photographic Guide to
the online version, at http://dx.doi.org/10.1016/j.ympev.2012.
Snakes and Other Reptiles of Peninsular Malaysia, Singapore and Thailand. New
06.009. Holland Publishers Ltd., UK, London.
Das, I., 1994a. A check-list of the amphibians and reptiles of Andaman and Nicobar
Islands. J. Andaman Sci. Assoc. 10, 44–49.
References Das, I., 1994b. The reptiles of south Asia: a checklist and distributional summary.
Hamadryad 19, 15–40.
Albert, E.M., San Mauro, D., Garcia-Paris, M., Ruber, L., Zardoya, R., 2009. Effect of Das, I., 2004. Lizards of Borneo. Natural History Publications (Borneo) Sdn Bhd., Kota
taxon sampling on recovering the phylogeny of squamate reptiles based on Kinabalu.
complete mitochondrial genome and nuclear gene sequence data. Gene 441, Das, I., Vijayakumar, S.P., 2009. New species of Ptychozoon (Sauria: Gekkonidae)
12–21. from the Nicobar Archipelago, Indian Ocean. Zootaxa 2095, 8–20.
Annandale, N., 1904. Contribution to Oriental Herpetology I: the lizards of the de Queiroz, K., 1998. The general lineage concept of species. Species criteria, and the
Andamans, with the description of a gecko and a note on the reproduced tail in process of speciation. In: Howard, D.J., Berlocher, S.H. (Eds.), Endless Forms:
Ptychozoon homalocephalum. J. Asiatic Soc. Bengal 73, 12–22. Species and Speciation. Oxford University Press, New York, pp. 57–75.
Annandale, N., 1905. Notes on some oriental geckos in the Indian Museum, Calcutta, Dickerson, R.E., 1928. Distribution of Life in the Philippines. Philipp. Bur. Sci.
with descriptions of new forms. Ann. Mag. Nat. Hist., Lond. 15, 26–32. Monogr. 21, Manila.
Bauer, A.M., Parham, J.F., Brown, R.M., Stuart, B.L., Grismer, L.L., Papenfuss, T.J., Dring, J.C.M., 1979. Amphibians and reptiles from northern Trengganu, Malaysia,
Bohme, W., Savage, J., Carranza, S., Grismer, J., Wagner, P., Ananjeva, N., Inger, with descriptions of two new geckos: Cnemaspis and Cyrtodactylus. Bull. British
R.F., 2010. On the availability of new Bayesian-delimited gecko names and the Mus. (Natur. Hist.) Zool. Ser. 34, 181–241.
importance of character based definitions of species. Proc. Roy. Soc. Lond. B 278, Drummond, A.J., Rambaut, A., 2007. BEAST: Bayesian evolutionary analysis by
490–492. sampling trees. BMC Evol. Biol. 7, 214.
Barbour, T., 1912. A contribution to the zoogeography of the East Indian Islands. Dudley, R., Byrnes, G., Yanoviak, S., Borrell, B., Brown, R.M., McGuire, J., 2007. Gliding
Mem. Mus. Comp. Zool. 44, 1–203. and the functional origins of flight: biomechanical novelty or necessity? Ann.
Biswas, S., Sanyal, D.P., 1980. A report on the Reptilia fauna of Andaman and Nicobar Rev. Ecol. Syst. 38, 179–201.
Islands in the collection of Zoological Survey of India. Rec. Zool. Surv. India 77, Edgar, R.C., 2004. MUSCLE: multiple sequence alignment with high accuracy and
255–292. high throughput. Nucl. Acids Res. 32, 1792–1797.
 Author's personal copy

R.M. Brown et al. / Molecular Phylogenetics and Evolution 65 (2012) 351–361 361

Emerson, S.B., Koehl, M.A.R., 1990. The interaction of behavioral and morphological change Ronquist, F., Huelsenbeck, J.P., 2003. MRBAYES 3: Bayesian phylogenetic inference
in the evolution of a novel locomotor type—flying frogs. Evolution 44, 1931–1946. under mixed models. Bioinformatics 19, 1572–1574.
Esselstyn, J.A., Garcia, H.J.D., Saulog, M.G., Heaney, L.R., 2008. A new species of Ronquist, F., Huelsenbeck, J.P., 2003. MRBAYES 3: Bayesian phylogenetic inference
Desmalopex (Pteropodidae) from the Philippines, with a phylogenetic analysis of under mixed models. Bioinformatics 19, 1572–1574.
the Pteropodini. J. Mammal. 89, 815–825. de Rooij, N., 1915. The Reptiles of the Indo-Australian Archipelago, I: Lacertilia,
Flower, S.S., 1896. Notes on a collection of reptiles and batrachians made in the Chelonia. Emydosauria. E. J. Brill, Ltd., Leiden.
Malay Peninsula in 1895–96; with a list of the species recorded from that Russell, A.P., 1972. The Foot of Gekkonid Lizards: A Study in Comparative and
region. Proc. Zool. Soc. Lond. 1896, 856–914. Functional Anatomy. Unpubl. Ph.D. Diss, Univ. of London, London.
Frost, D.R., Hillis, D.M., 1990. Species in concept and practice: herpetological Russell, A.P., 1979a. The origin of parachuting locomotion in gekkonid lizards
applications. Herpetologica 46, 87–104. (Reptilia: Gekkonidae). Zool. J. Linn. Soc. 65, 233–249.
Gadow, H., 1901. Amphibia and Reptiles. Macmillan and Co., London. Russell, A.P., 1979b. A new species of Luperosaurus (Gekkonidae) with comments on
Gamble, T., Bauer, A.M., Greenbaum, E., Jackman, T.R., 2008. Evidence of Gondwanan the genus. Herpetologica 35, 282–288.
vicariance in an ancient clade of gecko lizards. J. Biogeogr. 35, 88–104. Russell, A.P., Dijkstra, L.D., Powell, G.L., 2001. Structural characteristics of the
Gamble, T., Bauer, A.M., Colli, G.R., Greenbaum, E., Jackman, T.R., Vitt, L.J., Simons, patagium of Ptychozoon kuhli (Reptilia:Gekkonidae) in relation to parachuting
A.M., 2011. Coming to America: multiple origins of New World geckos. J. Evol. locomotion. J. Morphol. 247, 252–263.
24, 231–244. Setiadi, M.I., McGuire, J.A., Brown, R.M., Zubairi, M., Iskandar, D.T., Andayani, N.,
Gamble, T., Colli, G.R., Rodriques, M.T., Werneck, F.P., Simmons, A.M., 2012. Phylogeny Supriatna, J., Evans, B.J., 2011. Adaptive radiation and ecological opportunity in
and cryptic diversity in geckos (Phyllopezus; Phyllodactylidae; Gekkota) from Sulawesi and Philippine fanged frog (Limnonectes) communities. Amer. Natur.
South America’s open biomes. Mol. Phylogenet. Evol. 62, 943–953. 178, 221–240.
Goldingay, R., Scheibe, J., 2000. Biology of Gliding Mammals. Filander Press, Fürth. Siler, C.D., Oaks, J.R., Linkem, C.W., Swab, J., Diesmos, A.C., Brown, R.M., 2012. Did
Grismer, L.L., 2011. Lizards of Peninsular Malaysia, Singapore and Their Adjacent geckos ride the Palawan raft to the Philippines? J. Biogeogr. 39, 1217–1234.
Archipelagos. Edition Chimaira, Frankfurt. Simpson, G.G., 1961. Principles of Animal Taxonomy. Columbia University Press,
Grismer, L.L., Sumontha, M., Cota, M., Grismer, J.L., Wood Jr., P.L., Pauwels, O.S.G., New York.
Kunya, K., 2010. A revision and redescription of the rock gecko Cnemaspis Smith, M.A., 1930. The Reptilia and Amphibia of the Malay Peninsula. Bull. Raff.
siamensis (Taylor 1925) (Squamata: Gekkonidae) from Peninsular Thailand with Mus. 3, 1–149.
descriptions of six new species. Zootaxa 2576, 1–55. Smith, M.A., 1935. The Fauna of British India, including Ceylon and Burma. Reptilia
Grismer, L.L., Thy, N., Thou, C., Grismer, J.L., 2008. Checklist of the amphibians and and Amphibia. II.-Sauria. Taylor and Francis, London.
reptiles of southwestern Cambodia. Cambodian J. Nat. Hist. 2008, 12–28. Sodhi, N.S., Koh, L.P., Brook, B.W., Ng, P.K., 2004. Southeast Asian biodiversity: an
Günther, A.C.L.G., 1864. The Reptiles of British India. Ray Society, London. Reprinted impending disaster. Trends Ecol. Evol. 19, 654–660.
1982, Oxford and IBH Publishing Co., New Delhi. Stephens, M., Donnelly, P., 2003. A comparison of Bayesian methods for haplotype
Günther, A., 1885. Catalogue of Lizards in the British Museum. Geckonidae, reconstruction from population genotype data. Amer. J. Human Gen. 73, 1162–
Eublepharidae, Uroplatidae, Pygopodidae, Agamidae, vol. I, second ed. British 1169.
Museum, London. Stamatakis, A., 2006. RAxML-VI-HPC: maximum likelihood-based phylogenetic
Heled, J., Drummond, A., 2010. Bayesian inference of species trees from multilocus analyses with thousands of taxa and mixed models. Bioinformatics 22, 2688–
data. Mol. Biol. Evol. 27, 570–580. 2690.
Heyer, W.R., Pongsapipatana, S., 1970. Gliding speeds of Ptychozoon lionotum Stamatakis, A., Hoover, P., Rougemont, J., 2008. A rapid bootstrap algorithm for the
(Reptilia: Gekkonidae) and Chrysopelea ornata (Reptilia: Colubridae). RAxML Web Servers. Syst. Biol. 57, 758–771.
Herpetologica 26, 317–319. Stejneger, L.H., 1907. Herpetology of Japan and adjacent territory. Bull. US Natl.
Huson, D., 1998. Splitstree—a program for analyzing and visualizing evolutionary Mus. 58, 1–577.
data. Bioinformatics 14, 68. Stuart, B.L., Emmett, D.A., 2006. A collection of amphibians and reptiles from the
Huson, D.H., Bryant, D., 2006. Application of phylogenetic networks in evolutionary Cardamom Mountains, southwestern Cambodia. Fieldiana Zool. N.S. 109, 1–27.
studies. Mol. Biol. Evol. 23, 245–267. Stuart, B.L., Inger, R.F., Voris, H.K., 2006. High level of cryptic species diversity revealed
Inger, R.F., 1999. Distributions of amphibians in southern Asia and adjacent islands. by sympatric lineages of Southeast Asian forest frogs. Biol. Lett. 2, 470–474.
In: Duellman, W.E. (Ed.), Patterns of Distribution of Amphibians: A Global Taylor, E.H., 1915. New species of Philippine lizards. Philipp. J. Sci. 10, 89–108.
Perspective. John Hopkins University Press, Baltimore, pp. 445–482. Taylor, E.H., 1922a. The lizards of the Philippine Islands. Philipp. Bur. Sci., Monogr.
Inger, R.F., Colwell, R.K., 1977. Organization of contiguous communities of 17, Manila.
amphibians and reptiles in Thailand. Ecol. Monogr. 47, 229–253. Taylor, E.H., 1922b. Additions to the herpetological fauna of the Philippine Islands, I.
Inger, R.F., Voris, H.K., 2001. The biogeographical relations of the frogs and snakes of Philipp. J. Sci. 21, 161–206.
Sundaland. J. Biogeogr 28, 863–891. Taylor, E.H., 1928. Amphibians, lizards, and snakes of the Philippines. In: Dickerson,
IUCN, 2011. IUCN Red List of Threatened Species. Version 2011.2. <http:// R. (Ed.), Distribution of Life in the Philippines, pp. 214–241. Philippine Bur. Sci.,
www.iucnredlist.org> (downloaded 13.01.12). Monogr. 21, Manila.
Kiew, B.H., 1987. The flying gecko – Ptychozoon. Nat. Malaysiana 12, 18–19. Taylor, E.H., 1963. The lizards of Thailand. Univ. Kansas Sci. Bull. 44, 687–1077.
Leaché, A.D., Fujita, M.K., 2010. Bayesian species delimitation in West Aftrican forest Taylor, E.H., 1975. Philippine adventures: an autobiographical memoir. In: Taylor,
geckos (Hemidactylus fasciatus). Proc. Roy. Soc. Lond. B 277, 3071–3077. E.H., Leonard, A.B., Smith, H.M., Pisani, G.R. (Eds.), Recollections of an
Lomolino, M.V., Riddle, B.R., Whittaker, R.J., Brown, J.H., 2010. Biogeography, fourth Herpetologist. University of Kansas Museum of Natural History (Monogr. 4),
ed. Sinauer Associates, Sunderland. Lawrence, pp. 1–105.
Malkmus, R., Manthey, U., Vogel, G., Hoffmann, P., Kosuch, J., 2002. Amphibians and Tho, Y.P., 1974. Camouflage in the flying gecko, Ptychozoon kuhli Stejn. Malayan Nat.
Reptiles of Mount Kinabalu (North Borneo). A.R.G. Ganter Verlag, K.G., Rugell. J. 28, 36.
Manthey, U., 1985. Die Gattung Ptychozoon Kuhl, 1822 (Faltengecko) Teil I mit Tiwari, K.K., 1961. The eggs and flight of the gecko Ptychozoon kuhli Stejneger from
einem Bestimmungsschlüssel für die fünf Arten. Sauria, Berlin 4, 5–12. Car Nicobar. J. Bombay Nat. Hist. Soc. 58, 523–527.
Manthey, U., Grossmann, W., 1997. Amphibien und Reptilien Südostasiens. Natur Tweedie, M.W.F., 1954. Notes on Malayan reptiles, no. 3. Bull. Raff. Mus. 25, 107–117.
und Tier-Verlag, Münster. Venugopal, P.D., 2010. An updated and annotated list of Indian lizards (Reptilia:
Marcellini, D.L., Keefer, T.E., 1976. Analysis of the gliding behavior of Ptychozoon Sauria) based on a review of distribution records and checklists of Indian
lionotum (Reptilia: Gekkonidae). Herpetologica 32, 362–366. reptiles. J. Threatened Taxa 2, 725–738.
Marshall, D.C., 2010. Cryptic failure of partitioned Bayesian phylogenetic analyses: Vetter, R.S., Brodie, E.D., 1977. Background color selection and antipredator
lost in the land of long trees. Syst. Biol. 59, 108–117. behavior of the flying gecko, Ptychozoon kuhli. Herpetologica 33, 464–467.
McLeod, D.S., 2010. Of Least Concern? Systematics of a cryptic species complex: Voris, H., 2000. Maps of Pleistocene sea levels in Southeast Asia: shorelines, river
Limnonectes kuhlii (Amphiba; Anura: Dicroglossidae). Mol. Phylogenet. Evol. 56, systems and time durations. J. Biogeogr. 27, 1153–1167.
991–1000. Wiley, E.O., 1978. The evolutionary species concept reconsidered. Syst. Zool. 21, 17–
Medway, L., 1975. The function of the membranes of Ptychozoon spp. Malayan 26.
Natur. J. 29, 28–30. Wilgenbusch, J.C., Warren, D.L., Swofford, D.L., 2004. AWTY: A System for Graphical
Nabhitabhata, J., Chan-ard, T., Chuaynkern, Y., 2000. Checklist of Amphibians and Exploration of MCMC Convergence in Bayesian Phylogenetic Inference. <http://
Reptiles in Thailand. Office of Environmental Policy and Planning, Bankok. ceb.csit.fsu.edu/awty>.
Nguyen, V.S., Ho, Thu Cuc, Nguyen, Q.T., 2009. Herpetofauna of Vietnam. Chimaira, Wermuth, H., 1965. Gekkonidae, Pygopodidae, Xantusidae. In: Mertens, R., Hennig,
Frankfurt am Main. W., Wermuth, H. (Eds.), Das Tierreich, vol. 80. Walter de Gruyter & Co., Berlin.
Pauwels, O.S.G., Laohawat, O.-A., Naaktae, W., Puangjit, C., Wisutharom, T., Whitmore, T.C., 1975. Tropical Rain Forests of the Far East. Clarendon Press, Oxford.
Chimsunchart, C., David, P., 2000. Herpetological investigations in Phang-Nga Whitmore, T.C. (Ed.), 1987. Biogeographical Evolution of the Malay Archipelago.
Province, southern Peninsular Thailand, with a list of reptile species and notes Clarendon Press, Oxford.
on their biology. Dumerilia 4, 123–154. Whitmore, T.C., Sayer, J.A., 1992. Tropical Deforestation and Species Extinction.
Pawar, S., Biswas, S., 2001. First record of the Smooth-Backed Parachute Gecko Chapman and Hall, London.
Ptychozoon lionotum Annandale 1905 from the Indian mainland. Asiatic Woodruff, D.S., 2010. Biogeography and conservation in Southeast Asia: how 2.7
Herpetol. Res. 9, 101–106. million years of repeated environmental fluctuations affect today’s patterns and
Pong, T.Y., 1974. Camouflage in the flying gecko, Ptychozoon kuhli Stj. Malayan Nat. J. the future of the remaining refugial-phase biodiversity. Biodivers. Conserv. 19,
28, 36–37. 919–941.
Posada, D., 2008. JModelTest: phylogenetic model averaging. Mol. Biol. Evol. 25, 1253–1256. Yang, Z., Rannala, B., 2010. Bayesian species delimitation using multilocus sequence
Rambaut, A., Drummond, A.J., 2005. Tracer v1.4. <http://beast.bio.ed.ac.uk/Tracer>. data. Proc. Natl. Acad. Sci. 107, 9264–9269.