Genetic and Morphometric Variation of the Genus Nyctixalus in Indonesia Based on mtDNA 16S rRNA Gene

Rouland  Ibnudarda; Ahmad Muammar  Kadafi; Bagus  Priambodo; Achmad  Farajallah; Dyah  Perwitasari-Farajallah; Amir  Hamidy

doi:10.22146/jtbb.13642

Rouland Ibnudarda Animal Bioscience Study Program, Department of Biology, Faculty of Mathematics and Natural Sciences, IPB University, Bogor, 16680, West Java, Indonesia
Ahmad Muammar Kadafi Department of Biology, Faculty of Mathematics and Natural Sciences, Universitas Palangka Raya, Palangka Raya, Central Kalimantan, Indonesia. https://orcid.org/0000-0003-4646-687X
Bagus Priambodo Department of Biology, Faculty of Mathematics and Natural Sciences, Universitas Negeri Malang, Malang, Indonesia https://orcid.org/0000-0002-0195-0177
Achmad Farajallah Department of Biology, Faculty of Mathematics and Natural Sciences, IPB University, Bogor, 16680, West Java, Indonesia https://orcid.org/0000-0002-7733-6528
Dyah Perwitasari-Farajallah Department of Biology, Faculty of Mathematics and Natural Sciences, IPB University, Bogor, 16680, West Java, Indonesia https://orcid.org/0000-0001-6206-1675
Amir Hamidy Laboratory of Herpetology, Research Center for Biosystematics and Evolution, National Research and Innovation Agency (BRIN), Widyasatwaloka Building, Jl. Raya Jakarta Bogor Km. 46 Cibinong 16911, West Java, Indonesia

DOI: https://doi.org/10.22146/jtbb.13642

Keywords: Anura, Molecular phylogeny, Nyctixalus, Rhacophorid, Southeast Asia

Abstract

The genus Nyctixalus was first described by Boulenger in 1882. Previous systematic studies of this group primarily focused on its relationships with other rhacophorid frogs and did not explore inter- and intraspecific variation in detail. This study aimed to determine the position of Indonesian species within their genus. We used DNA barcoding of the mitochondrial 16S rRNA gene and conducted a morphological analysis. Mitochondrial genealogies were constructed based on NJ, ML, and BI analyses, while uncorrected p-distance was also calculated. The Mann-Whitney U-test and PCA were used to analyse variations among species for each character component. Dissimilarity may contribute to measurable group structure. DNA barcoding revealed that Nyctixalus forms a monophyletic group. The PCA analysis revealed that N. margaritifer population from Java formed a group, while N. pictus could not be separated on the scatter plot. DNA barcoding revealed that Nyctixalus forms a monophyletic group. The genetic distance within the population of N. margaritifer (0–1.52 %) and N. spinosus (0–0.2 %) showed a variation of intraspecific genetic distance. However, in the two allopatric populations of N. pictus Kalimantan and Sumatra (p-distance = 4.3–4.6 %), there are high variations (6.9–9 %) between populations from Sumatra and Malaysia. These findings suggest that N. anodon van Kampen, 1907, from Kayu Tanam, Sumatra Barat, Indonesia, represents a distinct species from N. pictus Peters, 1871, originally described from Sarawak, Malaysia.

References

Annandale, N., 1917. XIV. Report on a collection of reptiles and batrachians from Java. Journal of the Federated Malay States Museums, 7, pp.107–111.

Boettger, O., 1893. Neue Reptilien und Batrachier ausWest-Java. Zoologischer Anzeiger, 16, pp.334–340.

Boulenger, G.A., 1888. Note on the Classification of the Ranidae. Proceedings of the Zoological Society of London, 56(1), pp.204–206. doi: 10.1111/j.1469-7998.1888.tb06694.x.

Boulenger, G.A., 1882. V.—Description of a new genus and species of frogs of the family Ranidæ. Annals and Magazine of Natural History, 10(55), 35. doi: 10.1080/00222938209459662.

Boulenger, G.A. & Robinson, H.C., 1912. A vertebrate fauna of the Malay Peninsula from the Isthmus of Kra to Singapore including the adjacent islands Reptilia and Batrachia, London: Taylor and Francis.

Chen, W. et al., 2019. First record of Theloderma lateriticum Bain, Nguyen et Doan, 2009 (Anura Rhacophoridae) from China with redescribed morphology. Biodiversity Journal, 10(1), pp.25–36. doi: 10.31396/biodiv.jour.2019.10.1.25.36.

Chunskul, J. et al., 2021. Molecular identification and morphological description of theloderma albopunctatum tadpoles from the phu khiao-nam nao forest complex, northeastern thailand. Biodiversitas, 22(11), pp.5145–5161. doi: 10.13057/biodiv/d221153.

Cope, E.D., 1889. The Batrachia of North America, Washington: G.P.O.

Darriba, D. et al., 2014. High-performance computing selection of models of DNA substitution for multicore clusters. International Journal of High Performance Computing Applications, 28(1), pp.112–125. doi: 10.1177/1094342013495095.

Das, I. & Haas, A., 2010. New species of microhyla from sarawak: Old World’s smallest frogs crawl out of miniature pitcher plants on borneo (amphibia: Anura: Microhylidae). Zootaxa, (2571), pp.37–52. doi: 10.11646/zootaxa.2571.1.2.

Dever, J.A., 2017. A new cryptic species of the theloderma asperum complex (Anura: Rhacophoridae) from Myanmar. Journal of Herpetology, 51(3), pp.425–436. doi: 10.1670/17-026.

Dubeux, M.J.M. et al., 2022. DNA barcoding in Neotropical tadpoles: evaluation of 16S rRNA gene for the identification of anuran larvae from northeastern Brazil. Cuadernos de Herpetología, 36(2), pp.169-183. doi: 10.31017/CdH.2022.(2021-030)

Dubois, A., 1981. Liste des genres et sous-genres nominaux de ranoidea (Amphibiens anoures) du monde, avec identification de leurs espÈces-types: ConsÈquences nomenclaturales. Monitore Zoologico Italiano, Supplemento, 15(1), pp.225–284. doi: 10.1080/03749444.1981.10736637.

Eprilurahman, R. et al., 2021. A tiny new species of Microhyla Tschudi, 1838 (Amphibia: Anura: Microhylidae) from Belitung Island and Southeastern Sumatra, Indonesia. Zootaxa, 5027(4), pp.451–488. doi: 10.11646/zootaxa.5027.4.1.

Fouquet, A. et al., 2007. Underestimation of species richness in neotropical frogs revealed by mtDNA analyses. PLoS ONE, 2(10), e1109. doi: 10.1371/journal.pone.0001109.

Frost, D.R. et al., 2006. The amphibian tree of life. Bulletin of the American Museum of Natural History, (297), pp.1–291. doi: 10.1206/0003-0090(2006)297[0001:TATOL]2.0.CO;2.

Gabriel, K.R., 1971. The biplot graphic display of matrices with application to principal component analysis. Biometrika, 58(3), pp.453–467. doi: 10.1093/biomet/58.3.453.

Gadow, H., 1901. Amphibia and reptiles, London: Macmillan and Co. Ltd.

Hall, R., 2013. The palaeogeography of Sundaland and Wallacea since the Late Jurassic. In Journal of Limnology, 72(s2), e1. doi: 10.4081/jlimnol.2013.s2.e1.

Hamidy, A. et al., 2011. Morphological and genetic discordance in two species of Bornean Leptobrachium (Amphibia, Anura, Megophryidae). Molecular Phylogenetics and Evolution, 61(3), pp.904–913. doi: 10.1016/j.ympev.2011.08.020.

Hamidy, A. et al., 2012. Detection of cryptic taxa in Leptobrachium nigrops (Amphibia, Anura, Megophryidae), with description of two new species. Zootaxa, (3398), pp.22–39. doi: 10.11646/zootaxa.3398.1.2.

Hamidy, A. & Kurniati, H., 2015. A new species of tree frog genus Rhacophorus from Sumatra, Indonesia (Amphibia, Anura). Zootaxa, 3947(1), pp.49–66. doi: 10.11646/zootaxa.3947.1.3.

Hanifa, B.F. et al., 2023. New data on and the easternmost record of the Javan endemic Pearly Tree Frog, Nyctixalus margaritifer Boulenger, 1882 (Anura, Rhacophoridae). Check List, 19(6), pp.971–982. doi: 10.15560/19.6.971.

Hebert, P.D.N. et al., 2003. Biological identifications through DNA barcodes. Proceedings of the Royal Society B: Biological Sciences, 270(1512), pp.313-321. doi: 10.1098/rspb.2002.2218.

Hedges, S.B., 1994. Molecular evidence for the origin of birds. Proceedings of the National Academy of Sciences of the United States of America, 91(7), pp.2621–2624. doi: 10.1073/pnas.91.7.2621.

Huelsenbeck, J.P. & Hillis, D.M., 1993. Success of phylogenetic methods in the four taxon case. Systematic Biology, 42(3), pp.247–264. doi: 10.1093/sysbio/42.3.247.

Huelsenbeck, J.P. & Ronquist, F., 2001. MRBAYES: Bayesian inference of phylogenetic trees. Bioinformatics, 17(8), pp.754–755. doi: 10.1093/bioinformatics/17.8.754.

Inger, R.F., 1966. The systematics and zoogeography of the amphibia of Borneo, Chicago: Field Museum of Natural History.

Inger, R.F., 2005. The frog fauna of the Indo-Malayan region as it applies to the Wallace’s Line. Wallace in Sarawak– 150 Years Later. An International Conference on Biogeography and Biodiversity, (15), pp.82–90.

Inger, R.F. & Voris, H.K., 2001. The biogeographical relations of the frogs and snakes of Sundaland. Journal of Biogeography, 28(7), pp.863–891. doi: 10.1046/j.1365-2699.2001.00580.x.

Kaiser, H.F., 1960. The Application of Electronic Computers to Factor Analysis. Educational and Psychological Measurement, 20(1), pp.141–151. doi: 10.1177/001316446002000116.

Kambayashi, C. et al., 2022. Geography-Dependent Horizontal Gene Transfer from Vertebrate Predators to Their Prey. Molecular Biology and Evolution, 39(4), msac052. doi: 10.1093/molbev/msac052.

Katoh, K. et al., 2002. MAFFT: A novel method for rapid multiple sequence alignment based on fast Fourier transform. Nucleic Acids Research, 30(14), pp.3059–3066. doi: 10.1093/nar/gkf436.

Kimura, M., 1980. A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences. Journal of Molecular Evolution, 16(2), pp.111–120. doi: 10.1007/BF01731581.

Kumar, S. et al., 2018. MEGA X: Molecular evolutionary genetics analysis across computing platforms. Molecular Biology and Evolution, 35(6), 1547. doi: 10.1093/molbev/msy096.

Kurniawan, N. et al., 2023. A New Species of Theloderma Tschudi, 1838 (Amphibia: Rhacoporidae) from Central Java Allied to T. horridum (Boulenger, 1903). Asian Herpetological Research, 14(1), pp.1–23. doi: 10.16373/j.cnki.ahr.220033.

Leaché, A.D. & Reeder, T.W., 2002. Molecular systematics of the Eastern Fence Lizard (Sceloporus undulatus): A comparison of parsimony, likelihood, and Bayesian approaches. Systematic Biology, 51(1), pp.44–68. doi: 10.1080/106351502753475871.

Liem, S.S., 1970. The morphology, systematics, and evolution of the Old World treefrogs (Rhacophoridae and Hyperoliidae), Chicago, USA: Field Museum of Natural History.

Marx, K.W., 1975. A substitute name, Edwardtayloria for a genus of tree frogs from southeast Asia (Anura: Rhacophoridae). Scientific Publications of the Science Museum of Minnesota, (2), pp.1–3.

Matsui, M., 1984. Morphometric variation analyses and revision of the Japanese toads (genus Bufo, Bufonidae). Contributions from the Biological Laboratory, Kyoto University, 26, pp.209–428.

Matsui, M., 2015. A new species of Limnonectes from the border of East Kalimantan and Sarawak, Borneo Island (Anura, Dicroglossidae). Current Herpetology, 34(2), pp.120–127. doi: 10.5358/hsj.34.120.

Matsui, M. et al., 2010. Phylogenetic relationships of megophryid frogs of the genus Leptobrachium (Amphibia, Anura) as revealed by mtDNA gene sequences. Molecular Phylogenetics and Evolution, 56(1), pp.259–272. doi: 10.1016/j.ympev.2010.03.014.

Matsui, M., Hamidy, A. & Eto, K., 2013. Description of a new species of Microhyla from Bali, Indonesia (Amphibia, Anura). Zootaxa, 3670(4), pp.579–590. doi: 10.11646/zootaxa.3670.4.9.

Meegaskumbura, M. et al., 2011. Taruga (Anura: Rhacophoridae), a new genus of foam-nesting tree frogs endemic to Sri Lanka. Ceylon Journal of Science (Biological Sciences), 39(2), pp.75–94. doi: 10.4038/cjsbs.v39i2.2995.

Minh, B.Q., Nguyen, M.A.T. & Von Haeseler, A., 2013. Ultrafast approximation for phylogenetic bootstrap. Molecular Biology and Evolution, 30(5), pp.1188–1195. doi: 10.1093/molbev/mst024.

Munir, M. et al., 2018. A New Megophrys Kuhl and Van Hasselt (Amphibia: Megophryidae) from southwestern Sumatra, Indonesia. Zootaxa, 4442(3), pp.389–412. doi: 10.11646/zootaxa.4442.3.3.

Nguyen, T.T., Matsui, M. & Eto, K., 2015. Mitochondrial phylogeny of an Asian tree frog genus Theloderma (Anura: Rhacophoridae). Molecular Phylogenetics and Evolution, 85, pp.59–67. doi: 10.1016/j.ympev.2015.02.003.

O’Connell, K.A. et al., 2016. Testing the inland seas hypothesis using an endemic Sumatran frog radiation, USA.

Palacký, J., 1898. Die Verbreitung der Batrachier auf der Erde. Verhandlungen der Kaiserlich-Königlichen Zoologisch-Botanischen Gesellschaft in Wien, 48, pp.374–382.

Peters, W.C.H., 1871. Monatsberichte der Königlichen Preussische Akademie des Wissenschaften zu Berlin, Berlin: Königliche Akademie der Wissenschaften.

Posada, D., 2008. jModelTest: Phylogenetic model averaging. Molecular Biology and Evolution, 25(7), pp.1253–1256. doi: 10.1093/molbev/msn083.

Poyarkov, N.A. et al., 2015. Sorting out moss frogs: MtDNA data on taxonomic diversity and phylogenetic relationships of the indochinese species of the genus Theloderma (Anura, Rhacophoridae). Russian Journal of Herpetology, 22(4), pp.241–280.

Poyarkov, N.A. et al., 2018. A new species of the genus Theloderma Tschudi, 1838 (Amphibia: Anura: Rhacophoridae) from Tay Nguyen Plateau, central Vietnam. Zoological research, 39(3), pp.158–184. doi: 10.24272/j.issn.2095-8137.2018.018.

Priambodo, B. et al., 2021. Rediscovery of pearly tree frog, nyctixalus margaritifer boulenger, 1882 (Amphibia: Rhacophoridae) from mt. wilis after 135 years. Turkish Journal of Zoology, 45(4), pp.329–334. doi: 10.3906/zoo-2104-22.

R Core Team, 2019. R: A language and environment for statistical computing. R Foundation for Statistical Computing.

Rambaut, A., 2016, ‘FigTree – Tree Figure Drawing Tool, version 1.4.3’, in Molecular evolution, phylogenetics and epidemiology, viewed from http://tree.bio.ed.ac.uk/software/.

Riyanto, A. & Kurniati, H., 2014. Three new species of Chiromantis Peters 1854 (Anura: Rhacophoridae) from Indonesia. Russian Journal of Herpetology, 21(1), pp.65–73. doi: 10.30906/1026-2296-2014-21-1-65-73

Ronquist, F. et al., 2012. Mrbayes 3.2: Efficient bayesian phylogenetic inference and model choice across a large model space. Systematic Biology, 61(3), pp.539–542. doi: 10.1093/sysbio/sys029.

Roux, J., 1905. La famille des Ranidae. Notes sur les genres gui la composent, suivie dun tableau de détermination. Zoologischer Anzeiger, 28, pp.777–785.

Sambrook, J., Fritsch, E.F. & Maniatis, T., 1989. Molecular cloning: A laboratory manual (2nd edition), USA: Cold Spring Harbor Laboratory Press

Segura-Delorme, M. et al., 2005. Nouveautés taxinomiques. Une nouvelle classification générique et subgénérique de la tribu des Philautini (Amphibia, Anura, Ranidae, Rhacophorinae). Bulletin de la Société Linnéenne de Lyon, 74(5), pp.165–171.

Segura-Delorme, M. & Dubois, A., 2004. Phylogénie des ranidae rhacophorinae : confrontations des analyses moléculaires et morphologiques, et étude de caractères. Paris: National Museum of Natural History.

Sivongxay, N. et al., 2016. A new small-sized Theloderma (Anura: Rhacophoridae) from Laos. Zootaxa, 4147(4), pp.433–442. doi: 10.11646/zootaxa.4147.4.5.

Smith, M.A., 1931. The herpetology of Mt. Kinabalu, North Borneo, 13,455 ft. The Bulletin of The Raffles Museum, pp.8–32.

Taylor, E.H., 1920. Philippine Amphibia. The Philippine journal of science, 16, pp.213–359. doi: 10.5962/bhl.part.4751.

Taylor, E.H., 1962. The Amphibian Fauna of Thailand. The University of Kansas science bulletin, 43(8), pp.265–599. doi: 10.5962/bhl.part.13347.

Trifinopoulos, J. et al., 2016. W-IQ-TREE: a fast online phylogenetic tool for maximum likelihood analysis. Nucleic Acids Research, 44(W1), pp.w232–w235. doi: 10.1093/NAR/GKW256.

van Kampen, P.N., 1923. The amphibia of the Indo-Australian archipelago, Leiden: E. J. Brill, Ltd.

van Kampen, P.N., 1907. Zoologische Ergebnisse einer reise in Niederländisch Ost-Indien, Leiden: E.J. Brill.

Vences, M., 1999. Phylogenetic Studies on Ranoid Frogs (Amphibia. Anura). With a discussion of the origin and evolution of the vertebrate clades of Madagascar. Rheinische Friedrich-Wilhelms-Universität Bonn.

Vences, M. et al., 2012. DNA barcoding amphibians and reptiles. Methods in Molecular Biology, 858, pp.79–107. doi: 10.1007/978-1-61779-591-6_5.

Voris, H.K., 2000. Maps of Pleistocene sea levels in Southeast Asia: Shorelines, river systems and time durations. Journal of Biogeography, 27(5), pp.1153–1167. doi: 10.1046/j.1365-2699.2000.00489.x.

Wilkinson, J.A., Drewes, R.C. & Tatum, O.L., 2002. A molecular phylogenetic analysis of the family Rhacophoridae with an emphasis on the Asian and African genera. Molecular Phylogenetics and Evolution, 24(2), pp.265–273. doi: 10.1016/S1055-7903(02)00212-9.