Zootaxa 4107 (3): 367–380 ISSN 1175-5326 (print edition) http://www.mapress.com/j/zt/ Article ZOOTAXA Copyright © 2016 Magnolia Press ISSN 1175-5334 (online edition) http://doi.org/10.11646/zootaxa.4107.3.5 http://zoobank.org/urn:lsid:zoobank.org:pub:7791AAD7-2F1D-47EB-88A7-1B97C765BFFC Phylogenetic relationships of geckos of the Hemiphyllodactylus harterti group, a new species from Penang Island, Peninsular Malaysia, and a likely case of true cryptic speciation ANTHONY COBOS1, L. LEE GRISMER1, PERRY L. WOOD, JR.2, EVAN S. H. QUAH3, SHAHRUL ANUAR3,4 & MOHD ABDUL MUIN3 1Department of Biology, La Sierra University, 4500 Riverwalk Parkway, Riverside, California 92515 USA. E-mail: [email protected], [email protected] 2Department of Biology, Brigham Young University, 150 East Bulldog Boulevard, Provo, Utah 84602 USA. E-mail: [email protected] 3School of Biological Sciences, Universiti Sains Malaysia, 11800 USM, Pulau Pinang, Penang, Malaysia. E-mail: [email protected], [email protected], [email protected] 4Center for Marine and Coastal Studies, Universiti Sains Malaysia, 11800 USM, Pulau Pinang, Malaysia Abstract An integrative taxonomic analysis based on the mitochondrial gene ND2 and its flanking tRNAs, morphology, and color pattern indicates that a newly discovered gecko described herein as Hemiphyllodactylus cicak sp. nov. from Penang Hill on the Island of Penang, Peninsular Malaysia is a member of the H. harterti group. Hemiphyllodactylus cicak sp. nov. is most closely related to the clade composed of the sister species H. harterti from Bukit Larut, Perak in the Bintang Moun- tain Range and H. bintik from Gunung Tebu, Terengganu from the Timur Mountain Range. These three allopatric species form a monophyletic group that extends approximately 270 km across three isolated mountain ranges in northern Penin- sular Malaysia. The molecular analysis also indicates that H. titiwangsaensis from the Titiwangsa Mountain Range is com- posed of three genetically distinct allopatric populations. The southern two populations from Fraser’s Hill and Genting Highlands, Pahang have an uncorrected pairwise sequence divergence of 3.5% whereas these two populations have 12.4 and 12.8 % sequence divergences, respectively, from the northern population at Cameron Highlands, Pahang. Although the high sequence divergence clearly distinguishes the southern two populations from the former as a different species, all three populations are morphologically indistinguishable, leading to the hypothesis of a true, cryptic speciation event. Key words: Hemiphyllodactylus, Malaysia, Penang, phylogeny, new species, cryptic speciation Introduction The gekkonid genus Hemiphyllodactylus Bleeker currently comprised of 25 species (Grismer et al. 2013, 2014,a,b, 2015; Ngo et al. 2014; Nguyen et al. 2013, 2014). Although the genus extends across a broad geographic range from the Mascarene Islands in the western Indian Ocean, eastward through southern Asia, Indochina, the Philippines, and the Indo-Australian Archipelago into much of Oceania as far east as Hawaii (Zug 2010), most species are geographically restricted to upland areas or islands (Grismer et al. 2013). Grismer et al. (2013) divided the genus into two monophyletic groups, the harterti group and the typus group. The typus group contains species that span the entire geographic distribution of the genus, whereas the harterti group contains only species endemic to the uplands of Peninsular Malaysia. The harterti group currently contains six species known from three major mountain ranges in Peninsular Malaysia: the Bintang Mountain Range in the west, the central Titiwangsa Mountain Range, and the Timur Mountain Range in the east (Fig. 1). During a recent expedition to Penang Island, Penang, Peninsular Malaysia, four individuals from a new population of Hemiphyllodactylus were collected from an upland region on Penang Hill. Although all have the diagnostic characters of a vestigial first digit on both the fore- and hind limbs, as well as a long slender body with widely splayed limbs that place them in the genus Hemiphyllodactylus, they also share a suite of morphological characters that differentiate them from all other Accepted by A. Bauer: 29 Mar. 2016; published: 3 May 2016 367 congeners. Additionally, all were found to be genetically similar to one another but distinct from all other congeners and phylogenetically embedded within the harterti group. We, therefore, describe this population of Hemiphyllodactylus as a new species. Grismer et al. (2013) demonstrated that Hemiphyllodactylus titiwangsaensis was composed of two reciprocally monophyletic lineages and noted that it formed a species complex. Bickford et al. (2007) considered two or more species that are morphologically indistinguishable and classified as a single nominal species as cryptic species. The pluralistic approach used in this study where both morphology and molecular genetics are taken into account reveals that genetically distinct populations cannot be separated on the basis of morphology and may represent true cryptic species. Grismer et al. (2014) noted that the numerous papers purporting to demonstrate that integrative taxonomic studies are revealing cryptic species are doing nothing of the sort. They are only revealing that the last papers dealing with the taxon in question did not look closely enough at the morphology. This is not the case with Hemiphyllodactylus titiwangsaensis. FIGURE 1. Distribution of the species of the Hemiphyllodactylus harterti group in Peninsular Malaysia. Materials and methods Phylogenetic analysis. A 1446 base pair fragment of the NADH dehydrogenase subunit 2 gene (ND2), including the flanking tRNA’s (tRNAmet, tRNAtrp, tRNAala, tRNAsn, tRNAcys, tRNAtyr), was analyzed from 20 sequenced individuals. Three new samples of the new population from Penang Hill were sequenced for the same fragment along with three taxa used as outgroups (Table 1). Total genomic DNA was isolated from liver or skeletal muscle tissues stored in 95% ethanol using the Qiagen DNeasyTM tissue kit (Valencia, CA, USA). ND2 was amplified using a double-stranded Polymerase Chain Reaction (PCR) under the following conditions: 1.0 µl genomic DNA (~10–30 ng), 1.0 µl light strand primer (10 µM), 1.0 µl heavy strand primer (10 µM), 1.0 µl deoxynucleotide solution (1.5 µM), 2.0 µl 5x buffer (1.5 µM), 1.0 10x PCR buffer withMgCL2 (1.5 µM), 0.18 µl 368 · Zootaxa 4107 (3) © 2016 Magnolia Press COBOS ET AL. Taq polymerase (5u/µl), and 7.5 µl H2O. PCR reactions were executed on an Eppendorf Mastercycler gradient thermocycler under the following conditions: initial denaturation at 95ºC for 2 min, followed by 31 cycles of denaturation at 95ºC for 35 s, annealing at 52ºC for 35 s, followed by a cycle extension at 72ºC for 35 s. All PCR products were visualized on a 1% agarose gel electrophoresis. Successful targeted PCR products were vacuum purified using MANU 30 PCR plates Millipore plates and purified products were re-suspended in sterile water. Cycle sequencing was performed on the purified PCR products using the ABI Big-Dye Terminator v3.1 Cycle Sequencing Kit in an ABI GeneAmp PCR 9700 thermal cycler. Cycle sequencing reactions were purified with Sephadex G-50 Fine (GE Healthcare) and analyzed on an ABI 3730xl DNA Analyzer at the BYU DNA Sequencing center. TABLE 1. Taxon sampling for ingroup and outgroup, locality data, and Genbank accession numbers. Voucher Genus and species Locality GenBank accession numbers AMB (n/a) Hemiphyllodactylus aurantiacus Tamil Nadu, Yercaud, India JN393933 LSUHC 11216 Hemiphyllodactylus bintik Gunung Tebu, Terengganu, Malaysia KJ663757 LSUCH 9504 Hemiphyllodactylus chiangmaiensis Chang Mai, Thailand KF219782 LSUHC 11762 Hemiphyllodactylus cicak sp. nov. Penang Hill, Penang, Malaysia KU845548 LSUHC 11763 Hemiphyllodactylus cicak sp. nov. Penang Hill, Penang, Malaysia KU845549 LSUHC 11764 Hemiphyllodactylus cicak sp. nov. Penang Hill, Penang, Malaysia KU845550 LSUHC10384 Hemiphyllodactylus harterti Bukit Larut, Perak, Malaysia KF219761 LSUHC10383 Hemiphyllodactylus harterti Bukit Larut, Perak, Malaysia KF219760 LSUHC 11295 Hemiphyllodactylus larutensis Bukit Larut, Perak, Malaysia KJ663758 MVZ 239346 Hemiphyllodactylus engganoensis Pulau Enggano, Sumatra KF219776 LSUHC 6489 Hemiphyllodactylus cf. titiwangsaensis Fraser's Hill, Pahang, Malaysia KF219769 LSUHC 8092 Hemiphyllodactylus cf. titiwangsaensis Fraser's Hill, Pahang, Malaysia KF219774 LSUHC 10693 Hemiphyllodactylus cf. titiwangsaensis Genting Highlands, Pahang, Malaysia KF219763 LSUHC 10700 Hemiphyllodactylus cf. titiwangsaensis Genting Highlands, Pahang, Malaysia KF219764 LSUHC 10699 Hemiphyllodactylus cf. titiwangsaensis Genting Highlands, Pahang, Malaysia KF219765 LSUHC 10694 Hemiphyllodactylus cf. titiwangsaensis Genting Highlands, Pahang, Malaysia KF219766 LSUHC 10904 Hemiphyllodactylus tehtarik Gunung Tebu, Malaysia KF219784 LSUHC 7208 Hemiphyllodactylus titiwangsaensis Cameron Highlands, Malaysia JN393934 LSUHC 10717 Hemiphyllodactylus titiwangsaensis Cameron Highlands, Malaysia KF219785 LSUHC 10718 Hemiphyllodactylus titiwangsaensis Cameron Highlands, Malaysia KF219790 TABLE 2. Primers used for PCR amplification and sequencing reactions. Specific amplification conditions are presented in the materials and methods. Primer name Primer citation Sequence L4437b (Macey & Schulte 1999) External 5’ –AAGCAGTTGGGCCCATACC –3’ CyrtintF1 (Siler et al. 2010) Internal 5’ –TAGCCYTCTCYTCYATYGCCC –3’ CyrtintR1 (Siler et al. 2010) Internal 5’ –ATTGTKAGDGTRGCYAGGSTKGG