Phylogenetic analysis and molecular dating suggest that Hemidactylus anamallensis is not a member of the Hemidactylus radiation and has an ancient Late Cretaceous origin.

UNLABELLED: BACKGROUND OF THE WORK: The phylogenetic position and evolution of Hemidactylus anamallensis (family Gekkonidae) has been much debated in recent times. In the past it has been variously assigned to genus Hoplodactylus (Diplodactylidae) as well as a monotypic genus 'Dravidogecko' (Gekkonidae). Since 1995, this species has been assigned to Hemidactylus, but there is much disagreement between authors regarding its phylogenetic position within this genus. In a recent molecular study H. anamallensis was sister to Hemidactylus but appeared distinct from it in both mitochondrial and nuclear markers. However, this study did not include genera closely allied to Hemidactylus, thus a robust evaluation of this hypothesis was not undertaken.
METHODS: The objective of this study was to investigate the phylogenetic position of H. anamallensis within the gekkonid radiation. To this end, several nuclear and mitochondrial markers were sequenced from H. anamallensis, selected members of the Hemidactylus radiation and genera closely allied to Hemidactylus. These sequences in conjunction with published sequences were subjected to multiple phylogenetic analyses. Furthermore the nuclear dataset was also subjected to molecular dating analysis to ascertain the divergence between H. anamallensis and related genera. RESULTS AND
CONCLUSION: Results showed that H. anamallensis lineage was indeed sister to Hemidactylus group but was separated from the rest of the Hemidactylus by a long branch. The divergence estimates supported a scenario wherein H. anamallensis dispersed across a marine barrier to the drifting peninsular Indian plate in the late Cretaceous whereas Hemidactylus arrived on the peninsular India after the Indian plate collided with the Eurasian plate. Based on these molecular evidence and biogeographical scenario we suggest that the genus Dravidogecko should be resurrected.

Introduction

Hemidactylus anamallensis, a gekkonid endemic to the Western Ghats of South India has undergone many taxonomic revisions, yet its phylogenetic position and taxonomic status remains unresolved. This species was originally described as a member of Hoplodactylus [1], [2], a genus in the family Diplodactylidae that is confined to New Zealand. Smith [3] assigned it to a new monotypic genus ‘Dravidogecko’ on the basis of the differences in subdigital pads and the arrangement of preanal pores, in the family Gekkonidae. Underwood [4] and Kluge [5] also demonstrated that Dravidogecko was a gekkonid gecko and not a member of the family Diplodactylidae. Russell [6], [7] on the basis of digital structure hypothesised that Dravidogecko was closely related to Hemidactylus group within family Gekkonidae. Later, Bauer and Russell [8] synonymised Dravidogecko as Hemidactylus, renaming it as Hemidactylus anamallensis, because there were no morphological features that were unique to Dravidogecko when compared with Hemidactylus. They also suggested that H. anamallensis could be a primitive Hemidactylus.

Hemidactylus is a species rich genus with 122 recognised species [9] distributed worldwide and has been identified predominantly on the basis of its phalangeal taxonomy [3], [6], [10], [11]. Russell [6] suggested that the genera Briba, Cosymbotus, Dravidogecko and Teratolepis also belong to Hemidactylus. Carranza and Arnold [12] undertook one of the most comprehensive phylogenetic studies of Hemidactylus based on mitochondrial 12S rRNA and cytochrome b sequenced from 30 species sampled from around the world. Their phylogeny retrieved five well supported clades. Three subsequent studies that included additional species (around 14) also retrieved the aforementioned clades [13]–[15]. In Carranza and Arnold [12] phylogeny Cosymbotus (distributed in Southeast Asia) and Briba (monotypic genus from Brazil) were deeply nested within the Hemidactylus group, hence they synonymised these genera with Hemidactylus. Bauer et al. [13], using molecular data from five genes, showed that Teratolepis was deeply embedded within the tropical Asian clade of Hemidactylus along with the ground dwelling geckos endemic to Indian subcontinent. Therefore, they synonymised it with Hemidactylus, renaming it as Hemidactylus imbricatus. These studies did not include H. anamallensis. Thus, its affinity to Hemidactylus based on morphological data needs to be evaluated using molecular data.

Within the Hemidactylus radiation, H. anamallensis has been assigned to the H. bowringii complex in the tropical Asian clade by Zug et al. [16]. Whereas Bauer et al. [13] suspected that H. anamallensis is part of a highly derived lineage, consisting of H. albofaciatus-imbricatus-reticulatus within the H. brookii complex in the tropical Asian clade. Thus, both the above scenarios would predict H. anamallensis to be deeply nested within the Hemidactylus radiation, but differ with respect to its exact phylogenetic position. These scenarios are in sharp contrast to Bauer and Russell's [8] hypothesis, wherein they considered H. anamallensis to be a primitive Hemidactylus, thereby suggesting that phylogenetically it could be sister to all the Hemidactylus species. These putative phylogenetic positions of H. anamallensis generate very different biogeographical scenarios for the origin and spread of both H. anamallensis and other Hemidactylus species of the Indian subcontinent. Interestingly, in a recent molecular work by Bansal and Karanth [15], H. anamallensis was indeed sister to all the Hemidactylus thus supporting Bauer and Russell [8] hypothesis. Nevertheless their results also suggested that “H. anamallensis” was genetically distinct from other Hemidactylus. However, in their study genera closely allied to Hemidactylus were not included, thus a robust evaluation of the phylogenetic position of H. anamallensis with respect to the genus Hemidactylus could not be undertaken. Therefore, the authors called for a re-examination of its allocation to the genus Hemidactylus with additional molecular data from related genera.

The objective of this study was to investigate the phylogenetic position of H. anamallensis within the gekkonid radiation. To this end, several nuclear and mitochondrial markers were sequenced from multiple H. anamallensis samples and these sequences were combined with published sequences of gekkonids. These alignments were then subjected to multiple phylogenetic analyses. Results from these analyses in conjunction with molecular dating were used to understand the origin and biogeography of H. anamallensis.

Results

Phylogenetic position of H. anamallensis within Gekkonidae (C-mos and 12S rRNA dataset)

All tree building methods retrieved a strongly supported clade consisting of the genera Agamura, Crossobamon, Cyrtodactylus, Cyrtopodian, Geckoella, Hemidactylus, Stenodactylus and Tropicolotes. Members of this clade, henceforth referred to as deletion clade, also shared a 21 bp deletion in the C-mos gene (Bayesian tree shown in figure 1a and b). The relationships between members of the deletion clade were also identical across tree-building methods. Within the deletion clade, Hemidactylus (excluding H. anamallensis) formed a clade with high support. Additionally it was observed that the members of this Hemidactylus clade shared a unique 9 bp insertion in the C-mos gene (figure 1b). However, this insertion was not seen in H. anamallensis. In all the trees H. anamallensis emerged as sister to the rest of the Hemidactylus radiation. For a list of sequences used and their accession numbers see table 1.

Figure 1

(a): Bayesian tree based on combined dataset of C-mos and 12S rRNA genes showing the relationships among the members of the family Gekkoninae. The numbers at the nodes represent the maximum likelihood bootstrap/posterior probability. */* Indicates the bootstrap support ≥90%/Bayesian posterior probability of 1, -/indicates bootstrap support ≤50% and Bayesian posterior probability of <0.5. Black arrow represents the node that constitutes the members of the deletion clade and the white arrow represents the node, which separates the taxa with insertion (Hemidactylus). (b) C-mos DNA sequence alignment-showing indels among some members of the family Gekkoninae.

Table 1

List of sequences used in the current study.

	GenBank Accession numbers
Sample name	Voucher Number	Locality	C-mos	12S rRNA	RAG-1	PDC
Aristelliger lar	JB 01	Dominican Republic	-	-	EF534805	EF534847
Bavayia cyclura	AMB 7683	New Caledonia	-	-	HQ 426264	HQ 426176
Blaesodactylus antongilensis	ZCMV 2187	Nosy Mangabe, Madagascar	-	-	EU054229	EU054205
Blaesodactylus sakalava	WRBM 18	Will's Track, Toliara District, Madagascar	-	-	EU054227	EU054203
Carphodactylus laevis	AMS 143258	Queensland, Australia	-	-	EF534781	EF534821
Cnemaspis limi	LLG 6267	Pulau Tioman, Malaysia	-	-	EF534809	EF534851
Coleonyx variegatus	CAS 205334	California, USA	-	-	EF 534777	EF 534817
Cyrtodactylus ayeyawardyensis	CAS 216446	vic. Kanthaya Beach, Rakhine State, Myanmar	-	-	EU268287	EU268317
Cyrtodactylus consobrinus	LLG 4062	Niah Cave, Sarawak, Malaysia	-	-	EU268288	EU268318
Cyrtodactylus fasciotlatus	CES 091196	Kempty road, Mussoorie, Uttarakhand, India	KC735108	KC735096	HM622351	HM622366
Cyrtodactylus gubernatoris	CES 1197	Singhtum, Sikkim	-	-	KC735086	KC735091
Cyrtodactylus khasiensis	CES 1101	Northeast India	KC735109	KC735097	-	-
Cyrtodactylus loriae	FK 7709	N slope of Mt. Simpson, Bunisi, Milne Bay Province, Papua New Guinea	-	-	EU268289	EU268319
Cyrtopdian scrabum	CES1104	Sam, Rajasthan	KC735110	KC735098	-	-
Cyrtopodian kacchensis	CES1146	Kutch, Gujarat	KC735111	KC735099	-	-
Cyrtopodian species	CES1107	Kuno, Madhya Pradesh	KC735112	KC735100	-	-
Delma Tincta	AMS 151607	Sturt Natl. Pk., NSW, Australia	-	-	HQ 426277	HQ 426188
Diplodactylus conspicillatus	AMS 158426	Sturt Natl. Park, NSW, Australia	-	-	HQ 426278	HQ 426189
Elgaria kingii	TG 00065	Navajo County, Arizona, USA	-	-	AY662603	HQ426252
Eublepharis macularius	JS 2	Pakistan	-	-	EF 534776	EF 534816
Euleptes europea	-	Liguria, Italy	-	-	EF534806	EF534848
Geckoella collagensis	CES 1136	Mumbai, Maharashtra	-	-	KC735087	KC735092
Gekko gecko	No ID	unknown	-	-	EF534813	EF534854
Goniurosaurus araneus	JFBM 15830	Vietnam	-	-	HQ 426286	HQ 426197
Gymnodactlus amarali	CHUNB 38646	Cocalzinho, Goiás, Brazil	-	-	HQ 426288	HQ 426199
Heloderma suspectum	TG 00068	Arizona, USA	-	-	AY662606	HQ426254
Hemidactylus anamallensis 1	CES 08029	Vadiyoor, Eravikulam, Tamil Nadu, India	KC735113	HM595680	HM622353	HM622368
Hemidactylus anamallensis 2	CES 08030	Vadiyoor, Eravikulam, Tamil Nadu, India	KC735114	KC735101	KC735088	KC735093
Hemidactylus anamallensis 3	CES 10002	Wayanad, Tamil Nadu, India	KC735115	KC735102	KC735089	KC735094
Hemidactylus anamallensis 4	CES 10003	Wayanad, Tamil Nadu, India	KC735116	KC735103	-	-
Hemidactylus anamallensis 5	CES 10004	Wayanad, Tamil Nadu, India	KC735117	KC735104	KC735090	KC735095
Hemidactylus angulatus	MVZ 245438	Nigeria, Togo Hills, Nkwanta	HQ426540	-	EU268306	EU268336
Hemidactylus angulatus 1	E1708.15	Kajiado District, Rift valley, Kenya	-	DQ120412	-	-
Hemidactylus bowringii	CES 08008	Sikkim, India	-	-	HM622354	HM622369
Hemidactylus brookii 2	CES 06080	Palakkad, Kerala, India	KC735118	HM595685	HM622355	HM622370
Hemidactylus fasciatus 2	-	Rabi, Gabon	-	-	EU268309	EU268339
Hemidactylus frenatus 2	CES 07035	Athirapalli, Valparai, Tamil Nadu, India	KC735119	KC735105	HM622371	HM622356
Hemidactylus giganteus	CES 07013	Nandi Hills, near Bangalore, Karnataka, India	KC735120	KC735106	-	-
Hemidactylus giganteus	CES 08013	Hampi, Karnataka, India	-	-	HM622357	HM622372
Hemidactylus graniticolous	CES 08028	Nilgiri Hills, Tamil Nadu, India	-	-	HM622361	HM622375
Hemidactylus greefii	CAS 219044	Praia da Mutamba, São Tome Island, São Tome and Principe	HQ426542	-	EU268308	EU268338
Hemidactylus greefii	E7014.4	Principe, Sao Tome and Principe	-	DQ120414	-	-
Hemidactylus haitianus	AMB 4189	Dominican Republic (1), Santo Domingo	HQ426543	-	-	-
Hemidactylus haitianus 1	HhaitiS	Matanzas, Matanzas province, Cuba	-	DQ120388	-	-
Hemidactylus haitianus 2	CAS 198442	near Santo Domingo, Nacional Dist., Dominican Republic	-	-	EU268311	EU268341
Hemidactylus mabouia	E609.20	Lake Nabugabo, Masaka District, Uganda	-	DQ120377	-	-
Hemidactylus mabouia	MCZ R-184446	Limpopo Province, South Africa	-	-	EU268300	EU268330
Hemidactylus mabouia	JME 1864	Wundanyi, Kenya	HQ426546	-	-	-
Hemidactylus maculatus	BNHS 1516	Zirad, Raigadh dist., Maharashtra, India	-	-	HM559707	HM559674
Hemidactylus palaichthus	LSUMZ H-12421	Roraima State, Brazil	-	-	EU268307	EU268337
Hemidactylus persicus 2	CES 08027	Nabh Dongar, Jaisalmer, Rajasthan, India	KC735121	KC735107	HM622362	HM622376
Hemidactylus platyurus 2	CES 08025	Kalimpong, West Bengal, India	-	-	HM622363	HM622377
Hemidactylus robustus	MVZ 248437	40 km South of Mipur Sakro, Thatta District, Pakistan	-	-	EU268315	EU268345
Hemidactylus turcicus	LSUMZ H-1981	Baton Rouge, Louisiana, USA	-	-	EU268299	EU268329
Homonota fasciata	TG 00085	Paraguay	-	-	EU 293629	EU 293697
Lepidodactylus lugubris	AMB 4111	Kirimati, Kiribati	-	-	EF534812	EF534853
Lialis burtonis	TG 00078	Provinsi Papua, Indonesia	-	-	EF 534782	EF 534822
Narudasia festiva	AMB 3243	Narudas, Namibia	-	-	EF534808	EF534850
Nephrurus milii	AMB 499	Western Australia, Australia	-	-	EF534780	EF534820
Oedura marmorata	AMS 143861	Queensland, Australia	-	-	EF 534779	EF 534819
Paradelma orientalis	QM-J56089	20 km N Capella, Queensland, Australia	-	-	HQ 426304	HQ 426215
Phelsuma madagascariensis	FG/MV 2002.797	Manongarivo, Madagascar	-	-	EF534811	AB081507
Phyllodactylus xantii	ROM 38490	Baja California Sur, Mexico	-	-	EF 534807	EF 534849
Phyllodactylus xantii	ROM 38490	Baja California Sur, Mexico	-	-	EF534807	EF534849
Pristurus carteri	TG 00083	Yemen	-	-	EF534803	EF534845
Pygopus nigriceps	AMB 53	Northern Territory, Australia	-	-	EF 534783	EF 534823
Rhoptropus boultoni	CAS 214713	Twyfelfontein, Namibia	-	-	EF534810	EF534852
Sphaerodactylus elegans	YPM 14795	Florida, USA	-	-	EF534787	EF534828
Tarentola Americana	MVZ 241223	13 km E of Pilon, Granma Province, Cuba	-	-	HQ 426332	HQ 426243
Teratoscincus roborowskii	TG 00070	China	-	-	EF534799	EF534841
Thecadactylus solimoensis	KU 214929	Cuzco Amazonico, Madre de Dios, Peru	-	-	EU 293644	EU 293711

Sequences generated by the authors have accession numbers starting with KC. For a complete list of C-mos and 12S rRNA sequences see Feng et al. [25].

Clarifying the position of H. anamallensis within the clade consisting of Hemidactylus and other closely related genera (RAG-1 and PDC dataset)

In all the methods of phylogenetic inference, H. anamallensis emerged as sister to Hemidactylus and was separated from Hemidactylus by a long branch (Bayesian tree shown in figure 2). Genera Cyrtodactylus and Geckoella were sister to Hemidactylus-H. anamallensis clade. The overall topology of the Bayesian, ML and MP trees were similar with respect to the relationships among Cyrtodactylus, Geckoella, Hemidactylus and H. anamallensis. For a list of sequences used and their accession numbers see table 1.

Figure 2

Bayesian estimates of dates based on RAG-1 and PDC dataset.

Bayesian posterior probabilities and maximum likelihood bootstrap supports are shown at the base of the nodes. Grey bars indicate the credible intervals. Black circle on the node represents the fixed date node and the hollow circle represents minimum age constraint node. Black arrow represents the node at which H. anamallensis split from the Hemidactylus lineage (68.9 mya) and white arrow represents the node at which Indian Hemidactylus lineage started radiating (36.47 mya). */* indicates the bootstrap support ≥90% and Bayesian posterior probability of 1, −/− indicates the bootstrap support ≤50% and Bayesian posterior probability of <0.5. K-T indicates Cretaceous-Tertiary boundary and I/A indicates the date of collision of India with Asian plate. For the complete tree see figure S1 and table S4.

Divergence dates estimates

Bayesian estimation of divergence dates suggests that the ancestral lineage leading to H. anamallensis and the remaining Hemidactylus (node C) diverged from each other around 68.9 million years ago (mya) (95% HPD 45.15–92.65 mya) (figure 2, table 2). Additionally the lineage leading to the remaining Hemidactylus underwent radiation much later around 49.62 mya (Node D, 95% HPD 32.12–67.12 mya) (figure 2, table 2). The divergence dates estimated at the other nodes in this analysis were concordant with the divergence dates from previous studies [17]–[19].

Table 2

Estimated ages in Myr and in the corresponding 95% HPD for the nodes labelled in fig. 2.

Node	Age	95% HPD
A	49.79	30.19–69.39
B	16.12	7.6–24.64
C	68.9	45.15–92.65
D	49.62	32.12–67.12
E	36.47	19.89–53.05

The ages were obtained using uncorrelated lognormal clock in Bayesian estimation method BEAST.

Discussion

The molecular data presented in the current study provided interesting insights into the phylogenetic position of H. anamallensis within Gekkonidae. The C-mos and 12S rRNA dataset suggested that H. anamallensis was part of a large clade consisting of genera such as Agamura, Cyrtodactylus, Cyrtopodian, Geckoella, Hemidactylus, Stenodactylus, and Tropiocolotes (figure 1). This clade received high posterior probability and bootstrap support and, more importantly the members of this clade shared a 21 bp deletion that was not seen in any other gekkonid. Within the deletion clade H. anamallensis was sister to Hemidactylus. H. anamallensis and Hemidactylus were also retrieved as sister to each other by RAG-1 and PDC dataset. Thus the nuclear markers support Bauer and Russell's [8] hypothesis that H. anamallensis might be a primitive Hemidactylus.

Interestingly in the C-mos gene, a 9 bp insertion was observed among Hemidactylus (figure 1b). This insertion was unique to the Hemidactylus lineage and was not shared with any other Gekkonid including H. anamallensis. Furthermore in the RAG-1 + PDC tree H. anamallensis was separated form the rest of the Hemidactylus by a long branch. Thus among nuclear markers H. anamallensis appeared distinct from the remaining Hemidactylus.

Our divergence date estimates based on both fossils as well as biogeographical events suggested that the divergence between the lineage leading to H. anamallensis and the rest of the Hemidactylus lineage occurred around 68.9 mya (95% HPD 48.15–89.65) (figure 2, table 2) in the late Cretaceous. However, the remaining members of the Hemidactylus lineage radiated much later around 49.62 mya (95% HPD 36.12–63.12) (figure 2, table 2) in the Eocene. During the late Cretaceous period peninsular Indian landmass was isolated from all other landmasses having separated from Madagascar around 80 mya. Nevertheless fossil evidence suggested that peninsular India, during its northward journey, remained close to Africa and Eurasia until it collided with the Asian plate around 55 mya [20], [21]. Thus faunal links between peninsular Indian and these landmasses were maintained by vagile animals, which were able to surmount minor marine barriers [20]. Interestingly members of the deletion clade (figure 1a), which consisted of genera closely related to H. anamallensis, are distributed predominantly in Northern Africa and Asia. This distribution pattern suggested that basal radiation within this clade might have occurred on these landmasses. Furthermore during the early stages of this radiation one of the lineages might have dispersed on to the drifting peninsular Indian plate where it eventually evolved into H. anamallensis. Much later, around 49.62 mya, the genus Hemidactylus underwent radiation (figure 2, table 2) probably on the Asian plate [12] and dispersed to other parts of the world including peninsular India. Recent molecular studies on Hemidactylus revealed that India harboured an endemic radiation [14], [15]. According to our dating estimate, this Indian radiation occurred around 36.47 mya (Node E) (95% HPD 19.89–53.05 mya) (figure 2, table 2). Taken together these dates suggested that Hemidactylus arrived on the Indian plate after peninsular India collided with Asia. During this time H. anamallensis was already present in India, having dispersed on to drifting peninsular India before collision. In a recent molecular study a similar late Cretaceous dispersal of frogs on to drifting peninsular India has been reported [22].

Thus, the dating estimates suggests that H. anamallensis has a unique biogeographical history that appears to be very different from that of the remaining Hemidactylus. Additionally H. anamallensis also appears to be genetically distinct from the remaining Hemidactylus. Taken together, these results support the reassignment of H. anamallensis to a separate genus by resurrection of Dravidogecko, the genus to which H. anamallensis was previously assigned. In the past, authors have sunk Dravidogecko into Hemidactylus, as there were no morphological features that were unique to Dravidogecko [7], [8], [23]. According to Bauer et al. [8] the characteristic undivided lamellae seen in H. anamallensis is not unique to this species as it is shared with a highly derived lineage of ground dwelling Hemidactylus spp. of South Asia. They suggested that H. anamallensis was part of this highly derived lineage within the H. brookii complex. However the present study does not support this relationship as in both the phylogenies H. anamallensis is not sister to H. brookii within the Hemidactylus radiation. Thus this character (undivided lamellae) appears to have been secondarily derived in one of the lineages of Hemidactylus.

Materials and Methods

Sample collection and DNA sequencing

Genera that are purported to be closely related to Hemidactylus such as Cyrtodactylus, Cyrtopodian, Geckoella as well as H. anamallensis were collected opportunistically from across India (table 1). Total DNA was extracted from the tail clippings stored in absolute alcohol following standard proteinase K protocol [24]. Three nuclear, C-mos, recombination activation gene (RAG-1) and phosducin (PDC), and one mitochondrial marker, 12S ribosomal RNA (12S rRNA), were PCR amplified from the above samples. All PCR amplifications were carried out in 25 µl reaction volume, with 1.5 unit of Taq DNA polymerase (Bangalore Genei, Bangalore, India), 0.25 mM of dNTP's (Bangalore Genei), 2.0 mM of MgCl2, 1 ul of 0.5 mg/ml of BSA, 0.1 µM (Sigma) of each primer and 40 ng of DNA. Primer combinations and thermocycler conditions are given in supporting information (tables S1 & S2). PCR products were purified using QIAquick PCR Purification kit (Qiagen) and sequences were obtained commercially from Eurofins Biotech Pvt. Ltd. (Bangalore, India). For the remaining genera of the family Gekkonidae, sequences were downloaded from GenBank (table 1). Percent sequence generated for this study: C-mos 30%, 12S rRNA 20%, RAG-18%, PDC 8%.

Phylogenetic analyses

The sequences generated here were combined with published sequences to derive two different datasets. First, to determine the phylogenetic position of H. anamallensis within Gekkonidae, the sequences generated by us were added to a combined dataset of the nuclear C-mos and mitochondrial 12S rRNA genes generated by Feng et al. [25]. To clarify the position H. anamallensis within the clade consisting of Hemidactylus and other closely related genera: RAG-1 and PDC datasets generated by Bauer et al. [13], Gamble et al. [17] and Bansal and Karanth [15] were used. In both the above datasets representatives from all the five clades of the Hemidactylus radiation were included. These sequences were aligned using ClustalW 1.6 [26] in the software MEGA v. 4.1 [27], using default parameters. These two datasets were then subjected to maximum parsimony (MP), maximum likelihood (ML) and Bayesian analyses. The two datasets could not be combined because there was a lack of overlap in sequence data between them. The C-mos+12S rRNA dataset generated by Feng et al. [25] had sequences largely for family Gekkonidae, thus this dataset was useful in inferring the position of H. anamalensis within Gekkonidae radiation. However RAG-1 + PDC dataset generated by Gamble et al. [17] had representatives of all the closely related families of Gekkonidae and therefore was useful in molecular dating (see below). Furthermore, in the case of RAG-1+ PDC extensive sequence data was available for Hemidactylus from previous works by Bauer et al. [13], and Bansal and Karanth [15]. Thus this dataset was also useful in clarifying the position of H. anamalensis within the clade consisting of Hemidactylus and other closely related genera.

The MP tree was derived through a heuristic search in in PAUP* version 4.0b10 [28] with tree bisection–reconnection branch swapping and 10 replicates of random addition options. Here transversions were weighted based on empirically determined transition/transversion ratios. Supports for various nodes were evaluated through 1000 replicates of bootstrapping in parsimony analysis. Phylogenetic inference using ML algorithm was also performed in PAUP with the substitution model chosen by MODELTEST [29] and tree bisection–reconnection branch swapping and 10 replicates of random addition options. Since PAUP does not allow for partitioning the dataset for ML search, another ML tree was derived in RAxML [30] wherein the dataset was partitioned. Bayesian analysis was run in Mr. Bayes version 3.1 [31] using the mixed model (see supporting information for partitioning scheme) with variable priors for 107 generations with four chains, wherein sampling was undertaken for every 100 generations. All sample points before the stage when the Markov chain reached a stable likelihood value were discarded as burn-in determined in Tracer v 1.4.1 [32]. The remaining trees were imported into PAUP* to generate a majority-rule consensus tree and to derive posterior probabilities for each node. Gaps were treated as missing data for all analyses.

Analysis of insertions and deletions (indels) in C-mos gene

C-mos is a proto-oncogene that encodes the protein serine/threonine kinase that regulates meiotic maturation in germ cells [33]. It is a single-copy gene that lacks introns and repetitive elements. Insertions and deletions in C-mos have been reported to be uncommon [34]. However, Han et al. [35] reported a 21 bp deletion in C-mos that was shared by some gekkonids. Additionally, our preliminary analysis suggested that members of the Hemidactylus radiation shared a 9 bp insertion. Given that indels are quite rare in coding regions, such changes could be used as phylogenetically informative characters for determine the position of H. anamallensis. Thus we checked the C-mos alignment for the presence of these indels in Hemidactylus (including H. anamallensis) and other related genera.

Molecular dating

The RAG-1 and PDC dataset (1439 characters) was also used to determine the divergence dates among H. anamallensis, Hemidactylus and other closely related genera. Independent calibrations from previously published studies [17]–[19] were used to constrain nodes in the divergence date analyses. Two out of five calibrations used in the previous studies were excluded from further analysis by the fossil cross- validation method used by Gamble et al. [17]. The excluded calibrations were (i) the minimum age of Paradelma orientalis/Pygopus nigriceps split, using the fossil Pygopus hortulanus, (ii) the maximum age of squamates, using the oldest known squamate fossil. The calibration points included and used to infer the divergence dates were: (i) Fossil Primaderma nessovi [36] was used to constrain the Helodermatidae/Anguidae split (exponential distribution, mean 3.0, offset 99.0). (ii) Two amber preserved specimens of Sphaerodactylus spp. [37], [38] were used to constrain the node constituting Sphaerodactylus species (exponential distribution, mean 5.0, offset 23.0). (iii) The split of Teratoscincus scincus- Teratoscincus roborowskii [39] which was purported to have occurred due to Tein Shan-Pamir uplift in western China, 10 Ma [40], [41] (Normal distribution, mean 10.0, SD 0.5)

The dataset was partitioned into two genes (RAG-1 1044 bp, PDC 395 bp) and the model of sequence evolution as mentioned in supporting information (table S3) was applied to both the partitions. Given that a strict clock model of molecular evolution is purported to be biologically unrealistic [42] a relaxed molecular clock model with uncorrelated lognormal distribution and Yule process tree prior (as recommended for species level phylogenies) were used. These analyses were undertaken in the program BEAST v 1.6.1 [43]. Base frequencies were estimated in BEAST, and gamma distribution categories were set to four. A default setting for substitution rate was used. The program was run for 5×107 generations. Tracer v 1.4.1 [32] was used to determine convergence and effective sample sizes for the run.

Bayesian estimates of dates based on and dataset. Bootstrap supports and Bayesian posterior probabilities are shown at the base of the nodes. Grey bars indicate the credible intervals. K-T indicates Cretaceous-Tertiary boundary and I/A indicates the date of collision of India with Asian plate.

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List of Primers used.

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Thermo cycler profile used for amplification of genes.

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Partitioning scheme and model of sequence evolution for the genes in the datasets. The datasets were partitioned according to the genes in both MrBayes and RAxML.

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Estimated ages (in Myr) of the nodes and the corresponding 95% CI for the nodes labelled in

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