Identification of three Asian otter species (Aonyx cinereus, Lutra sumatrana, and Lutrogale perspicillata) using a novel noninvasive PCR-RFLP analysis.

Four species of otters occur in tropical Asia, and all face multiple threats to their survival. Studies of distribution and population trends of these otter species in Asia, where they occur sympatrically, are complicated by their elusive nature and difficulties with reliable identification of species in field surveys. In Malaysia, only three species, the smooth-coated otter, Asian small-clawed otter, and hairy-nosed otter have been reliably reported as residents. We designed a replicable and cost-efficient PCR-RFLP protocol to identify these three species. Using published reference sequences of mitochondrial regions, we designed and tested three PCR-RFLP protocols on DNA extracted from reference samples and 33 spraints of wild otters collected along the North Central Selangor Coast of Malaysia. We amplified and sequenced two fragments (450 and 200 bp) of the mt D-loop region and a 300-bp fragment of the mt ND4 gene using primer sets TanaD, TanaD-Mod, and OTR-ND4, respectively. Amplification products were digested with restriction enzymes to generate species-specific RFLP profiles. We analyzed the costs of all three protocols and compared these with the costs of sequencing for species identification. Amplification success was highest for the smallest PCR product, with the TanaD-Mod primer amplifying DNA from all 33 spraints. TanaD and OTR-ND4 primers amplified DNA from 60.6% and 63.6% spraints, respectively. PCR products of TanaD-Mod provided the expected species-specific RFLP profile for 32 (97%) of the spraints. PCR products of OTR-ND4 provided the expected RFLP profile for all 21 samples that amplified, but TanaD produced spurious bands and inconsistent RFLP profiles. The OTR-ND4 primer-enzyme protocol was the least expensive (437 USD) for processing 100 samples, followed by TanaD-Mod (455 USD). We suggest the use of both OTR-ND4 and TanaD-Mod protocols that show potential for highly efficient and reliable species identification from noninvasive genetic sampling of three Asian otter species. We expect our novel noninvasive PCR-RFLP analysis methods to facilitate population monitoring, ecological and behavioral studies on otters in tropical and subtropical Asia.

Mitochondrial DNA Profiling Reveals Two Lineages of Sun Bears in East and West Malaysia.

Sun bear populations are fragmented and at risk from habitat loss and exploitation for body parts. These threats are made worse by significant gaps in knowledge of sun bear population genetic diversity, population connectivity, and taxonomically significant management units. Using a complete sun bear mitochondrial genome, we developed a set of mitochondrial markers to assess haplotype variation and the evolutionary history of sun bears from Peninsular (West) Malaysia and Sabah (East Malaysia). Genetic samples from 28 sun bears from Peninsular Malaysia, 36 from Sabah, and 18 from Thailand were amplified with primers targeting a 1800 bp region of the mitochondrial genome including the complete mitochondrial control region and adjacent genes. Sequences were analyzed using phylogenetic methods. We identified 51 mitochondrial haplotypes among 82 sun bears. Phylogenetic and network analyses provided strong support for a deep split between Malaysian sun bears and sun bears in East Thailand and Yunnan province in China. The Malaysian lineage was further subdivided into two clades: Peninsular Malaysian and Malaysian Borneo (Sabah). Sun bears from Thailand occurred in both Sabah and Peninsular Malaysian clades. Our study supports recent findings that sun bears from Sundaland form a distinct clade from those in China and Indochina with Thailand possessing lineages from the three clades. Importantly, we demonstrate a more recent and clear genetic delineation between sun bears from the Malay Peninsula and Sabah indicating historical barriers to gene flow within the Sundaic region.

The efficiency of universal mitochondrial DNA barcodes for species discrimination of Pomacea canaliculata and Pomacea maculata.

Invasive apple snails, Pomacea canaliculata and P. maculata, have a widespread distribution globally and are regarded as devastating pests of agricultural wetlands. The two species are morphologically similar, which hinders species identification via morphological approaches and species-specific management efforts. Advances in molecular genetics may contribute effective diagnostic tools to potentially resolve morphological ambiguity. DNA barcoding has revolutionized the field of taxonomy by providing an alternative, simple approach for species discrimination, where short sections of DNA, the cytochrome c oxidase subunit I (COI) gene in particular, are used as 'barcodes' to delineate species boundaries. In our study, we aimed to assess the effectiveness of two mitochondrial markers, the COI and 16S ribosomal deoxyribonucleic acid (16S rDNA) markers for DNA barcoding of P. canaliculata and P. maculata. The COI and 16S rDNA sequences of 40 Pomacea specimens collected from six localities in Peninsular Malaysia were analyzed to assess their barcoding performance using phylogenetic methods and distance-based assessments. The results confirmed both markers were suitable for barcoding P. canaliculata and P. maculata. The phylogenies of the COI and 16S rDNA markers demonstrated species-specific monophyly and were largely congruent with the exception of one individual. The COI marker exhibited a larger barcoding gap (6.06-6.58%) than the 16S rDNA marker (1.54%); however, the magnitude of barcoding gap generated within the barcoding region of the 16S rDNA marker (12-fold) was bigger than the COI counterpart (approximately 9-fold). Both markers were generally successful in identifying P. canaliculata and P. maculata in the similarity-based DNA identifications. The COI + 16S rDNA concatenated dataset successfully recovered monophylies of P. canaliculata and P. maculata but concatenation did not improve individual datasets in distance-based analyses. Overall, although both markers were successful for the identification of apple snails, the COI molecular marker is a better barcoding marker and could be utilized in various population genetic studies of P. canaliculata and P. maculata.