Molecular Ecology of the Sarawak Microhyla (Amphibian: Anura: Microhylidae)

Nooraina Atira, Binti Alaudin (2019) Molecular Ecology of the Sarawak Microhyla (Amphibian: Anura: Microhylidae). Masters thesis, Universiti Malaysia Sarawak (UNIMAS).

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Abstract

Genus Microhyla in Sarawak that populate the primary lowland forest are intolerant to habitat destruction. Habitat disturbance that cause a drop in the number of frog species. The forest had been open for logging, agriculture, converted to the oil palm plantation and human settlements. The species listed under genus Microhyla was a Microhyla berdmorei, Microhyla borneensis, Microhyla malang, Microhyla perpava and Microhyla petrigena. The species of the Sarawak Microhyla have a complex taxonomy and causes the misidentified among researchers. Not only that, because of their small body size, they always not be found, and their habitat have been disturbed. The aims of this study are i) to infer the phylogenetic relationships among genus Microhyla in Sarawak based on the mitochondrial DNA, ii) to define the habitat preferences of Microhyla and iii) to infer the genetic variation among populations M. borneensis using the Cytochrome oxidase I (COI). Total of 26 individuals of Microhyla was collected through visual encounter surveys from nine study sites. The tissues were extracted from thigh muscles and proceed to polymerase chain reaction using 16 small ribosomal RNA (16SrRNA) and Cytochrome b (Cyt b). Then, the phylogram was constructed using PAUP. From the results, 26 samples and only 18 individuals was succesfully amplified for 16SrRNA for Cyt b respectively. The phylogram produced for both genes showed that, M. malang and M. borneensis was paraphylectic while M. petrigena and M. perpava was monophylectic with high bootstrap support. There were two clades that can be recovered, based on their morphological traits; namely, Clade A (M. malang, M. borneensis and M. nepenthicola), and Clade B (M. petrigena and M. perpava). Clade A shows a species complex comprising three species as their morphologically mimics each other. The ecology data for each individual was recorded during the sampling time. Then, to test the species association with habitat preferences, all the data was analyzed by using Multivariate Statistical Package (MVSP), t-test and Principle Component Analysis (PCA). The result of habitat and microhabitat character showed that the variables used could discriminate the frog species. Out of 63 variables, only 14 variables have been utilised by Sarawak Microhyla with the dominant resource namely, heath forest, distant from a water body, resting on seedlings and herbaceous plant and surface of the leaf. M. nepenthicola and M. borneensis found on perching on a leaf or on the forest floor for their substrates, M. malang and M. perpava were at the permanent pond, M. petrigena was found inhabiting the edge MDF. Next, the population genetics of the M. borneensis was inferred using 465 base pairs (bp) of the Cytochrome oxidase I (COI) gene. The findings showed that the M. borneensis populations were separated into two haplogroups, namely Haplogroup 1 (containing KNP and MWC) and Haplogroup 2 (BNP, STB, DKNP and GNP). The occurrence of two haplogroups ware address to unclear status of M. borneensis. Low genetic divergence detected between the haplogroup supports their taxonomic status as they cause from the same species. The KNP population was said to undergo bottleneck effect while the other population do not undergo bottleneck effect. As a conclusion, molecular studies based on 16SrRNA, and Cyt b and COI is a good marker to infer the genetic variation of Sarawak Microhyla and showed M. borneensis is in panmixia. Further studies are required based on larger populations’ size, more individual per population and more variable markers to validate the findings. Keywords: Microhyla, habitat, COI, genetic, Cyt b

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