Abstract
The integrated results of maximum likelihood (ML) and Bayesian inference (BI) analyses, principal component analyses (PCA), and a multiple factor analysis (MFA) recover a new, widely allopatric species of the Cyrtodactylus intermedius species group. Cyrtodactylus kulenensis sp. nov is endemic to the Phnom Kulen sandstone massif of the Phnom Kulen National Park, Siem Reap Province, in the lowlands of northwestern Cambodia. A phylogenetic analysis from a short read (275 base pairs) of the mitochondrial gene NADH dehydrogenase subunit 2 (ND2) from C. kulenensis sp. nov. was aligned with 1449 base pairs from all other species in the intermedius group. The analysis recovered C. kulenensis sp. nov. as the sister species to a lineage composed of populations from the widely separated hilly regions of Sa Keao and Sakaerat in eastern Thailand. Multivariate (PCA, DAPC, and MFA) and univariate analyses (ANOVA) using combinations of meristic (scale counts), mensural (morphometric), and categorical (color pattern and morphology) characters from 52 specimens encompassing all species of the intermedius group clearly demonstrate C. kulenensis sp. nov. is significantly different and discretely diagnosable from all other species in the intermedius group. This new discovery further highlights the herpetological diversity and high levels of range-restricted endemism in basin-habitat-island landscapes throughout Indochina and the continued need for field work in the landscapes that remain unsurveyed.
References
Acinas, S.G., Klepac-Ceraj, V., Hunt, D.E., Pharino, C., Ceraj, I., Distel, D.L. & Polz, M.F. (2004) Fine-scale phylogenetic architecture of a complex bacterial community. Nature, 430, 551–554.
https://doi.org/10.1038/nature02649
Agarwal, A., El-Ghazawi, T., El-Askary, H. & Le-Moigne, J. (2007) Efficient hierarchical-PCA dimension reduction for hyperspectral imagery. 2007 IEEE International Symposium on Signal Processing and Information Technology. Available from: https://ieeexplore.ieee.org/document/4458191 (accessed 16 February 2021)
https://doi.org/10.1109/isspit.2007.4458191
Bain, R.H. & Hurley, M.M. (2011) A biogeographic synthesis of the amphibians and reptiles of Indochina. Bulletin of the American Museum of Natural History, 360, 1–138.
Barraclough, T.G., Birky, C.W. Jr. & Burt, A. (2003) Diversification in sexual and asexual organisms. Evolution, 57, 2166–2172.
https://doi.org/10.1554/02-339
Bouckaert, R.R. & Drummond, A.J. (2017) bModelTest: Bayesian phylogenetic site model averaging and model comparison. BMC Evolutionary Biology, 17, 42.
https://doi.org/10.1186/s12862-017-0890-6
Chan-ard, T., Parr, J.W.K. & Nabhitabhata, J. (2015) A Field Guide to the Reptiles of Thailand. Oxford University Press, New York, 214 pp.
https://doi.org/10.1108/rr-03-2017-0074
De Queiroz, K. (2007) Species concepts and species delimitation. Systematic Biology, 56, 879–886.
https://doi.org/10.1080/10635150701701083
Drummond, A.J., Suchard, M.A., Xie, D. & Rambaut, A. (2012) Bayesian Phylogenetics with BEAUti and BEAST 1.7. Molecular Biology and Evolution, 29, 1969–1973.
https://doi.org/10.1093/molbev/mss075
Ellis, M. & Pauwels, O.S.G. (2012) The bent-toed geckos (Cyrtodactylus) of the caves and karst of Thailand. Cave and Karst Science, 39, 16–22.
Fujisawa, T. & Barraclough, T.G. (2013) Delimiting species using single-locus data and the Generalized Mixed Yule Coalescent approach: a revised method and evaluation on simulated data sets. Systematic Biology, 62, 707–724.
https://doi.org/10.1093/sysbio/syt033
Geissler, P., Hartmann, T., Ihlow, F., Neang, T., Seng, R., Wagner, P. & Böhme, W. (2019) Herpetofauna of the Phnom Kulen National Park, northern Cambodia—an annotated checklist. Cambodian Journal of Natural History, 2019, 40–63.
https://doi.org/10.7717/peerj.7841/fig-1
Grismer, L.L., Chan, K.O., Oaks, J.R., Neang, T., Sokun L., Murdoch, M.L., Stuart, B.L. & Grismer, J.L. (2020a) A new insular species of the Cyrtodactylus intermedius (Squamata: Gekkonidae) group from Cambodia with a discussion of habitat preference and ecomorphology. Zootaxa, 4830 (1), 75–102.
https://doi.org/10.11646/zootaxa.4830.1.3
Grismer, L.L., Chav T., Neang, T., Wood Jr., P.L., Grismer, J.L., Youmans, T.M., Ponce, A., Daltry, J.C. & Kaiser, H. (2007) The herpetofauna of the Phnom Aural Wildlife Sanctuary and checklist of the herpetofauna of the Cardamom Mountains, Cambodia. Hamadryad, 31, 216–241.
Grismer, L.L., Neang, T., Chav, T. & Grismer, L.L. (2008a) Checklist of the amphibians and reptiles of the Cardamom Region of southwestern Cambodia. Cambodian Journal of Natural History, 1, 12–28.
Grismer, L.L., Neang, T., Chav, T., Wood Jr., P.L., Oaks, J.R., Holden, J., Grismer, J.L., Szutz, T.R. & Youmans, T.M. (2008b) Additional amphibians and reptiles from the Phnom Samkos Wildlife Sanctuary in the northwestern Cardamom Mountains, Cambodia, with comments on their taxonomy and the discovery of three new species. Raffles Bulletin of Zoology, 56, 161–175.
Grismer, L.L. & Ngo, V.T. (2017) Four new species of the Gekkonid genus Cnemaspis Strauch 1887 (Reptilia: Squamata) from southern Vietnam. Herpetologica, 63, 482–500.
https://doi.org/10.1655/0018-0831(2007)63[482:fnsotg]2.0.co;2
Grismer, L.L., Wood Jr., P.L., Grismer, M.S., Quah, E.S.H., Thura, M.K., Oaks, J.R., Lin, A. & Lim, D. (2020c) Integrative taxonomic and geographic variation analyses in Cyrtodactylus aequalis (Squamata: Gekkonidae) from southern Myanmar (Burma): one species, two different stories. Israel Journal of Ecology and Evolution, 66, 151–179.
https://doi.org/10.1163/22244662-20191082
Grismer, L.L., Wood Jr., P.L., Myint Kyaw Thura, Zin, T., Quah, E.S.H., Murdoch, M.L., Grismer, M.S., Herr, M.W., Lin A. & Kyaw, H. (2018b) Three more new species of Cyrtodactylus (Squamata: Gekkonidae) from the Salween Basin of eastern Myanmar underscores the urgent need for the conservation of karst habitats. Journal of Natural History, 52, 1243–1294.
https://doi.org/10.1080/00222933.2018.1449911
Grismer, L.L., Wood Jr., P.L., Myint Kyaw Thura, Zin, T., Quah, E.S.H., Murdoch, M.L., Grismer, M.S., Lin, A., Kyaw, H & Ngwe L. (2018a) Twelve new species of Cyrtodactylus Gray (Squamata: Gekkonidae) from isolated limestone habitats in east-central and southern Myanmar demonstrate high localized diversity and unprecedented microendemism. Zoological Journal of the Linnean Society, 182, 862–959.
https://doi.org/10.1093/zoolinnean/zlx057
Grismer, L.L., Wood Jr., P.L., Ngo, V.T. & Murdoch, M.L. (2015) The systematics and independent evolution of cave ecomorphology in distantly related clades of Bent-toed Geckos (Genus Cyrtodactylus Gray, 1827) from the Mekong Delta and islands in the Gulf of Thailand. Zootaxa, 3980 (1), 106–126.
https://doi.org/10.11646/zootaxa.3980.1.6
Grismer, L.L., Wood, P.L. Jr., Quah, E.S.H., Anuar, A., Ngadi, E.B., Izam, N.A.M. & Amad, N. (2017) Systematics, ecomorphology, cryptic speciation and biogeography of the lizard genus Tytthoscincus Linkem, Diesmos & Brown (Squamata: Scincidae) from the sky-island archipelago of Peninsular Malaysia. Zoological Journal of the Linnean Society, 179, 1–37.
https://doi.org/10.1093/zoolinnean/zlx067
Grismer, L.L., Wood, P.L. Jr., Quah, E.S.H., Grismer, M.S., Thura, M.K., Oaks, J.R. & Lin, A. (2020b) Two new species of Cyrtodactylus Gray, 1827 (Squamata: Gekkonidae) from a karstic archipelago in the Salween Basin of southern Myanmar (Burma). Zootaxa, 4718 (3), 151–183.
https://doi.org/10.11646/zootaxa.4624.3.1
Hillis, D.M. (2019) Species delimitation in herpetology. Journal of Herpetology, 53, 3–12.
https://doi.org/10.1670/18-123
Hoang, D.T., Chernomor, O., von Haeseler, A., Minh, B.Q. & Vinh, L.S. (2018) UFBoot2: Improving the ultrafast bootstrap approximation. Molecular Biology and Evolution, 35, 518–522.
https://doi.org/10.1093/molbev/msx281
Huelsenbeck, J.P., Ronquist, F., Nielsen, R. & Bollback, J.P. (2001) Bayesian inference of phylogeny and its impact on evolutionary biology. Science, 294, 2310–2314.
https://doi.org/10.1126/science.1065889
Jombart, T., Devillard, S. & Balloux, F. (2010) Discriminant analysis of principal components: a new method for the analysis of genetically structured populations. BMC Genetics, 11, 94.
https://doi.org/10.1186/1471-2156-11-94
Jombart, T. & Collins, C. (2015) A tutorial for discriminant analysis of principal components (DAPC) using adegenet 2.0.0. Available from: http://adegenet.r-forge.r-project.org/files/tutorial-dapc-pdf (Accessed 1 Mar. 2021)
Kalyaanamoorthy, S., Minh, B.Q., Wong, T.K., von Haeseler, A. & Jermiin, L.S. (2017) ModelFinder: fast model selection for accurate phylogenetic estimates. Nature Methods, 14, 587.
https://doi.org/10.1038/nmeth.4285
Katoh, K., Misawa, K., Kuma, K.I. & Miyata, T. (2002) MAFFT: a novel method for rapid multiple sequence alignment based on fast Fourier transform. Nucleic Acids Research, 30, 3059–3066.
https://doi.org/10.1093/nar/gkf436
Kumar, S., Stecher, G. & Tamura, K. (2016) MEGA7: Molecular evolutionary genetics analysis version 7.0 for bigger datasets. Molecular Biology and Evolution, 33, 1870–1874.
https://doi.org/10.1093/molbev/msw054
Maddison, W.P. & Maddison, D.R. (2015) Mesquite: a modular system for evolutionary analysis. Version 3.04. Available from: http://www.mesquiteproject.org (accessed 25 August 2020)
https://doi.org/10.1093/sysbio/42.2.218
Miller, M.A., Pfeiffer, W. & Schwartz, T. (2010) Creating the CIPRES Science Gateway for inference of large phylogenetic trees. Proceedings of the Gateway Computing Environments Workshop (GCE), New Orleans, Louisiana, 14 November 2010, 1–8.
https://doi.org/10.1109/GCE.2010.5676129
Minh, Q., Nguyen, M.A.T. & von Haeseler, A. (2013) Ultrafast approximation for phylogenetic bootstrap. Molecular Biology and Evolution, 30, 1188–1195.
https://doi.org/10.1093/molbev/mst024
Murdoch, M.L., Grismer, L.L., Wood Jr., P.L., Neang, T., Poyarkov, N.A., Ngo, V.T., Nazarov, R.A., Aowphol, A., Pauwels, O.S.G., Nguyen, H.N. & Grismer, J.L. (2019) Six new species of the Cyrtodactylus intermedius complex (Squamata: Gekkonidae) from the Cardamom Mountains and associated highlands of Southeast Asia. Zootaxa, 4554 (1), 1–62.
https://doi.org/10.11646/zootaxa.4554.1.1
Neang, T., Holden, J., Eastoe, T., Seng, R., Ith, S. & Grismer, L.L. (2011) A new species of Dibamus (Squamata: Dibamidae) from Phnom Samkos Wildlife Sanctuary, southwestern Cardamom Mountains, Cambodia. Zootaxa, 2828 (1), 58–68.
https://doi.org/10.11646/zootaxa.2828.1.5
Neang, T., Henson, A. & Stuart, B.L. (2020) A new species of Cyrtodactylus (Squamata, Gekkonidae) from Cambodia’s Prey Lang Wildlife Sanctuary. ZooKeys, 926, 133–158.
https://doi.org/10.3897/zookeys.926.48671.figure7
Ngo, V.T. & Grismer, L.L. (2008) The first record of Cardamom slender-toed gecko – Cyrtodactylus intermedius Smith, 1917 in Vietnam. Proceedings of the 5th Scientific Conference of University of Natural Science, Ho Chi Minh City, 30 November 2006, 328.
Ngo, V.T., Grismer, L.L. & Grismer, J.L. (2010) A new species of Cyrtodactylus Gray, 1827 (Squamata: Gekkonidae) in Phu Quoc National Park, Kien Giang Biosphere Reserve, Southwestern Vietnam, Zootaxa, 2604 (1), 37–51.
https://doi.org/10.11646/zootaxa.2604.1.3
Nguyen, L.T., Schmidt, H.A., von Haeseler, A. & Minh, B.Q. (2015) IQ-TREE: A fast and effective stochastic algorithm for estimating maximum likelihood phylogenies. Molecular Biology and Evolution, 32, 268–274.
https://doi.org/10.1093/molbev/msu300
Pons, J., Barraclough, T.G., Gomez-Zurita, J., Cardoso, A., Duran, D.P., Hazell, S., Kamoun, S., Sumlin, W.D. & Vogler, A.P. (2006) Sequence-Based Species Delimitation for the DNA Taxonomy of Undescribed Insects. Systematic Biology, 55, 595–609.
https://doi.org/10.1080/10635150600852011
R Core Team (2018) R: A language and environment for statistical computing. R Foundation for Statistical Computing. Vienna. Available from: http://www.R-project.org (accessed 29 December 2018)
Rambaut, A. & Drummond, A.J. (2013) TreeAnnotator. Version 1.8.0 MCMC Output Analysis. Available from: https://beast.community/treeannotator (accessed 16 February 2021)
Rambaut, A., Suchard, M.A., Xie, D. & Drummond, A.J. (2014) Tracer. Version 1.6. Available from: http://tree.bio.ed.ac.uk/software/tracer/ (accessed 16 February 2021)
Reist, J.D. (1986) A empirical evaluation of coefficients used in residual and allometric adjustment of size covariation. Canadian Journal of Zoology, 64, 1363–1368.
https://doi.org/10.1139/z86-203
Rujirawan, A. Fong, J.J., Ampai, N., Yodthong, S., Termprayoon, K. & Aowphol, A. (2019) A new karst-dwelling gecko of the Gekko petricolus group (Reptilia: Gekkonidae) from Phitsanulok Province, central Thailand. Journal of Natural History, 53, 557–576.
https://doi.org/10.1080/00222933.2019.1597937
Siler, C.D., Oaks, J.R., Welton, L.J., Linkem, C.W., Swab, J.C., Diesmos, A.C. & Brown, R.M. (2012) Did geckos ride the Palawan raft to the Philippines? Journal of Biogeography, 37, 1217–1234.
https://doi.org/10.1111/j.1365-2699.2011.02680.x
Smith, M.A. (1917) Descriptions of new reptiles and a new batrachian from Siam. The Journal of the Natural History Society of Siam, 2, 221–225.
Smith, M.A. (1935) s.n. In: The Fauna of British India, including Ceylon and Burma. Reptilia and Amphibia. Sauria. Vol II. Taylor and Francis, London, pp. 20–408.
Stuart, B.L. & Emmet, D.A. (2006) A Collection of amphibians and reptiles from the Cardamom Mountains, Southwestern Cambodia. Zoology, 109, 1–17.
https://doi.org/10.5962/bhl.title.3421
Sukumaran, J. & Knowles, L.L. (2017) Multispecies coalescent delimits structure, not species. Proceedings of the National Academy of Sciences, 114, 1607–1612.
https://doi.org/10.1073/pnas.1607921114
Taylor, E.H. (1963) The Lizards of Thailand. The University of Kansas Science Bulletin, 44, 687–1077.
Thorpe, R.S. (1975) Quantitative handling of characters useful in snake systematics with particular reference to interspecific variation in the Ringed Snake Natrix natrix (L.). Biological Journal of the Linnean Society, 7, 27–43.
https://doi.org/10.1111/j.1095-8312.1975.tb00732.x
Thorpe, R.S. (1983) A review of the numerical methods for recognizing and analyzing racial differentiation. In: Felsenstein, J. (Ed.), Numerical Taxonomy. NATO ASI Series (Series G: Ecological Sciences). Vol. 1. Springer-Verlag, Berlin, pp. 404–423.
https://doi.org/10.1007/978-3-642-69024-2_43
Turan, C. (1999) A note on the examination of morphometric differentiation among fish populations: The Truss System. Turkish Journal of Zoology, 23, 259–263.
Trifinopoulos, J., Nguyen, L.-T., von Haeseler, A. & Minh, B.Q. (2016) W-IQ-TREE: a fast online phylogenetic tool for maximum likelihood analysis. Nucleic Acids Research, 44, W232–W235.
https://doi.org/10.1093/nar/gkw256
Wilcox, T.P., Zwickl, D.J., Heath, T.A. & Hillis, D.M. (2002) Phylogenetic relationships of the Dwarf Boas and a comparison of Bayesian and bootstrap measures of phylogenetic support. Molecular Phylogenetics and Evolution, 25, 361–371.
https://doi.org/10.1016/s1055-7903(02)00244-0
Wood Jr., P.L., Heinicke, M.P., Jackman, T.R. & Bauer, A.M. (2012) Phylogeny of bent-toed geckos (Cyrtodactylus) reveals a west to east pattern of diversification. Molecular Phylogenetics and Evolution, 65, 992–1003.
https://doi.org/10.1016/j.ympev.2012.08.025