The southern river terrapin, Batagur affinis is one of the world's 25 most endangered freshwater turtle species. The major portion of the global population is currently found in peninsular Malaysia, with the only remnant Indochinese population in southern Cambodia. For more than a decade, wild nests in this remnant Cambodian population have been fenced and hatchlings reared in captivity. Here we amplified 10 microsatellite markers from all 136 captive individuals, obtained 2,658 presumably unlinked and neutral single nucleotide polymorphisms from 72 samples with ddRAD-seq, and amplified 784 bp of mtDNA from 50 samples. Our results reveal that the last Indochinese population comprised only four kinship groups as of 2012, with all offspring sired from <10 individuals in the wild. We demonstrate an obvious decrease in genetic contributions of breeders in the wild from 2006-2012 and identify high-value breeders instrumental for ex-situ management of the contemporary genetic stock of the species.
The tropical climate in China exists in southeastern Xizang (Tibet), southwestern to southeastern Yunnan, southwestern Guangxi, southern Guangdon, southern Taiwan, and Hainan, and these southern Chinese areas contain tropical floras. I checked and synonymized native seed plants from these tropical areas in China and recognized 12,844 species of seed plants included in 2,181 genera and 227 families. In the tropical flora of southern China, the families are mainly distributed in tropical areas and extend into temperate zones and contribute to the majority of the taxa present. The genera with tropical distributions also make up the most of the total flora. In terms of geographical elements, the genera with tropical Asian distribution constitute the highest proportion, which implies tropical Asian or Indo-Malaysia affinity. Floristic composition and geographical elements are conspicuous from region to region due to different geological history and ecological environments, although floristic similarities from these regions are more than 90% and 64% at the family and generic levels, respectively, but lower than 50% at specific level. These differences in the regional floras could be influenced by historical events associated with the uplift of the Himalayas, such as the southeastward extrusion of the Indochina geoblock, clockwise rotation and southeastward movement of Lanping-Simao geoblock, and southeastward movement of Hainan Island. The similarity coefficients between the flora of southern China and those of Indochina countries are more than 96% and 80% at family and generic levels, indicating their close floristic affinity and inclusion in the same biogeographically floristic unit.
Cenococcum geophilum (C. geophilum) is a widely distributed ectomycorrhizal fungus that plays a crucial role in forest ecosystems worldwide. However, the specific ecological factors influencing its global distribution and how climate change will affect its range are still relatively unknown. In this study, we used the MaxEnt model optimized with the kuenm package to simulate changes in the distribution pattern of C. geophilum from the Last Glacial Maximum to the future based on 164 global distribution records and 17 environmental variables and investigated the key environmental factors influencing its distribution. We employed the optimal parameter combination of RM = 4 and FC = QPH, resulting in a highly accurate predictive model. Our study clearly shows that the mean temperature of the coldest quarter and annual precipitation are the key environmental factors influencing the suitable habitats of C. geophilum. Currently, appropriate habitats of C. geophilum are mainly distributed in eastern Asia, west-central Europe, the western seaboard and eastern regions of North America, and southeastern Australia, covering a total area of approximately 36,578,300 km2 globally. During the Last Glacial Maximum and the mid-Holocene, C. geophilum had a much smaller distribution area, being mainly concentrated in the Qinling-Huaihe Line region of China and eastern Peninsular Malaysia. As global warming continues, the future suitable habitat for C. geophilum is projected to shift northward, leading to an expected expansion of the suitable area from 9.21% to 21.02%. This study provides a theoretical foundation for global conservation efforts and biogeographic understanding of C. geophilum, offering new insights into its distribution patterns and evolutionary trends.
Symbiosis is increasingly recognized as being an important component in marine systems, and many such relationships are initiated when free-swimming larvae of one partner settle and become sedentary on a host partner. Therefore, several crucial questions emerge such as the larva's mechanism of locating a host, selection of substratum and finally settlement on the surface of its future partner. Here, we investigated these mechanisms by studying how larvae of the fire coral-associated barnacle Wanella milleporae move, settle and establish symbiosis with their host, Millepora tenera. Cyprids of W. milleporae possess a pair of specialized antennules with bell-shaped attachment discs that enable them to explore and settle superficially on the hostile surface of the fire coral. Intriguingly, the stinging polyps of the fire coral remain in their respective pores when the cyprids explore the fire coral surface. Even when cyprids come into contact with the nematocysts on the extended stinging polyps during the exploratory phase, no immobilization effects against the cyprids were observed. The exploratory phase of Wanella cyprids can be divided into a sequence of wide searching (large step length and high walking speed), close searching (small step length and low speed) and inspection behavior, eventually resulting in permanent settlement and metamorphosis. After settlement, xenogeneic interactions occur between the fire coral and the newly metamorphosed juvenile barnacle. This involved tissue necrosis and regeneration in the fire coral host, leading to a callus ring structure around the juvenile barnacle, enhancing survival rate after settlement. The complex exploratory and settlement patterns and interactions documented here represent a breakthrough in coral reef symbiosis studies to show how invertebrates start symbiosis with fire corals.
Mangosteen (Garcinia mangostana var. mangostana) is a popular tropical fruit, yet many aspects of its biology and evolutionary history are little known. Its origin remains contentious, although recent findings suggest G. mangostana L. var. malaccensis (Hook. f.) Nazre (synonym: G. malaccensis Hook. f.) as the sole progenitor. We review hypotheses on the origin of mangosteen and clarify points that have been affected by errors of fact and interpretation. The narrow focus and lack of detail in published results make their interpretation difficult. When possible, we support our interpretations with field observations and examination of herbarium specimens. We outline the main biological traits (e.g., dioecy, facultative apomixis, and polyploidy) of mangosteen and its wild relatives to infer traits that might have evolved during domestication of mangosteen. We find no clear indication that apomixis and polyploidy evolved during domestication. Polyploidy is known in the wild relatives, but apomixis has not yet been demonstrated. Also, we propose a testable new evolutionary-ecological framework that we call "Forest-Dusun Interface" to infer processes in the origin of mangosteen. Dusun (Malay) refers to subsistence orchards in this context. Lastly, we propose future studies to address identified knowledge gaps.
Monoculture farming is pervasive in industrial oil palm agriculture, including those RSPO plantations certified as sustainably managed. This farming practice does not promote the maintenance of farmland biodiversity. However, little scientific attention has been given to polyculture farming in oil palm production landscapes. Polyculture farming is likely to increase the floristic diversity and stand structural complexity that underpins biodiversity. Mist nets were used to sample birds at 120 smallholdings in Peninsular Malaysia. At each site, 12 vegetation structure characteristics were measured. We compared bird species richness, abundance, and composition between monoculture and polyculture smallholdings and used predictive models to examine the effects of habitat quality on avian biodiversity. Bird species richness was significantly greater in polyculture than that of monoculture smallholdings. The number of fallen and standing, dead oil palms were also important positive predictors of species richness. Bird abundance was also strongly increased by standing and dead oil palms and decreased with oil palm stand height. Our results indicate that polyculture farming can improve bird species richness in oil palm production landscapes. In addition, key habitat variables that are closely associated with farming practices, such as the removal of dead trees, should and can be managed by oil palm growers in order to promote biodiversity. To increase the sustainability of oil palm agriculture, it is imperative that stakeholders modify the way oil palms are currently planted and managed. Our findings can guide policy makers and certification bodies to promote oil palm production landscapes that will function more sustainably and increase existing biodiversity of oil palm landscapes.
Hybridization is very common in flowering plants and it plays a significant role in plant evolution and adaptation. Melastoma L. (Melastomataceae) comprises about 80-90 species in tropical Asia and Oceania, among which 41 species occur in Borneo. Natural hybridization is frequently reported in Melastoma in China, but so far there have been no confirmed cases of hybridization in Southeast Asia (including Borneo), where most species occur. Here, we identified a case of natural hybridization between Melastoma malabathricum L. and Melastoma beccarianum Cogn. in Sarawak, Malaysia, by using sequence data of three nuclear genes and one chloroplast intergenic spacer. Melastoma malabathricum is the most widespread species of this genus, occurring in almost the whole range of this genus, while M. beccarianum is a local species endemic to northern Borneo. Our results showed that natural hybridization and introgression occur between M. malabathricum and M. beccarianum, and the introgression was asymmetrical, mainly from M. malabathricum to M. beccarianum. As adaptive traits can be transferred by introgression, our study suggests that natural hybridization should be a significant mechanism for the evolution and adaptation of Melastoma in Southeast Asia. However, introgression from the common species M. malabathricum to the relatively rare species M. beccarianum may cause the decline of M. beccarianum, incurring conservation concern. With a large number of species of Melastoma and almost year-around flowering in Southeast Asia, more cases of natural hybridization are expected to be found and identified in near future.
Beta diversity - the variation in species composition among spatially discrete communities - and sampling grain - the size of samples being compared - may alter our perspectives of diversity within and between landscapes before and after agricultural conversion. Such assumptions are usually based on point comparisons, which do not accurately capture actual differences in total diversity. Beta diversity is often not rigorously examined. We investigated the beta diversity of ground-foraging ant communities in fragmented oil palm and forest landscapes in Sabah, Malaysia, using diversity metrics transformed from Hill number equivalents to remove dependences on alpha diversity. We compared the beta diversities of oil palm and forest, across three hierarchically nested sampling grains. We found that oil palm and forest communities had a greater percentage of total shared species when larger samples were compared. Across all grains and disregarding relative abundances, there was higher beta diversity of all species among forest communities. However, there were higher beta diversities of common and very abundant (dominant) species in oil palm as compared to forests. Differences in beta diversities between oil palm and forest were greatest at the largest sampling grain. Larger sampling grains in oil palm may generate bigger species pools, increasing the probability of shared species with forest samples. Greater beta diversity of all species in forest may be attributed to rare species. Oil palm communities may be more heterogeneous in common and dominant species because of variable community assembly events. Rare and also common species are better captured at larger grains, boosting differences in beta diversity between larger samples of forest and oil palm communities. Although agricultural landscapes support a lower total diversity than natural forests, diversity especially of abundant species is still important for maintaining ecosystem stability. Diversity in agricultural landscapes may be greater than expected when beta diversity is accounted for at large spatial scales.
Marine fungal biodiversity remains vastly understudied, and even less is known of their biogeography and the processes responsible for driving these distributions in marine environments. We investigated the fungal communities associated with the seagrass Enhalus acoroides collected from Singapore and Peninsular Malaysia to test the hypothesis that fungal communities are homogeneous throughout the study area. Seagrass samples were separated into different structures (leaves, roots, and rhizomes), and a sediment sample was collected next to each plant. Amplicon sequencing of the fungal internal transcribed spacer 1 and subsequent analysis revealed significant differences in fungal communities collected from different locations and different structures. We show a significant pattern of distance decay, with samples collected close to each other having more similar fungal communities in comparison with those that are more distant, indicating dispersal limitations and/or differences in habitat type are contributing to the observed biogeographic patterns. These results add to our understanding of the seagrass ecosystem in an understudied region of the world that is also the global epicenter of seagrass diversity. This work has implications for seagrass management and conservation initiatives, and we recommend that fungal community composition be a consideration for any seagrass transplant or restoration programme.
Dual effects of spatial distance and environment shape archipelagic floras. In Malesia, there are multiple environmental stressors associated with increasing uplands, drought, and metal-rich ultramafic soils. Here, we examine the contrasting impacts of multifactorial environmental stress and spatial distance upon Lamiaceae species distributions. We used a phylogenetic generalized mixed effects model of species occurrence across Malesia's taxonomic database working group areas from Peninsular Malaysia to New Guinea. Predictor variables were environmental stress, spatial distance between areas and two trait principal component axes responsible for increasing fruit and leaf size and a negative correlation between flower size and plant height. We found that Lamiaceae species with smaller fruits and leaves are more likely to tolerate environmental stress and become widely distributed across megadiverse Malesian islands. How global species distribution and diversification are shaped by multifactorial environmental stress requires further examination.
Urban expansion has caused major deforestation and forest fragmentation in the tropics. The impacts of habitat fragmentation on biodiversity are understudied in urban forest patches, especially in the tropics and little is known on the conservation value of the patches for maintaining mammalian biodiversity. In this study, camera trapping was used to determine the species composition and species richness of medium- and large-sized mammals in three urban forest patches and a contiguous forest in Peninsular Malaysia. We identified the key vegetation attributes that predicted mammal species richness and occurrence of herbivores and omnivores in urban forest patches. A total number of 19 mammal species from 120 sampling points were recorded. Contiguous forest had the highest number of species compared to the urban forest patches. Sunda Pangolin and Asian Tapir were the only conservation priority species recorded in the urban forest patches and contiguous forest, respectively. Top predators such as Malayan Tiger and Melanistic Leopard were completely absent from the forest patches as well as the contiguous forest. This was reflected by the abundance of wild boars. We found that mammal species richness increased with the number of trees with DBH less than 5 cm, trees with DBH more than 50 cm, and dead standing trees. In the future, the remaining mammal species in the urban forest patches are expected to be locally extinct as connecting the urban forest patches may be infeasible due to land scarcity. Hence, to maintain the ecological integrity of urban forest patches, we recommend that stakeholders take intervention measures such as reintroduction of selected species and restocking of wild populations in the urban forest patches to regenerate the forest ecosystems.
For community ecologists, "neutral or not?" is a fundamental question, and thus, rejecting neutrality is an important first step before investigating the deterministic processes underlying community dynamics. Hubbell's neutral model is an important contribution to the exploration of community dynamics, both technically and philosophically. However, the neutrality tests for this model are limited by a lack of statistical power, partly because the zero-sum assumption of the model is unrealistic. In this study, we developed a neutrality test for local communities that implements non-zero-sum community dynamics and determines the number of new species (N sp) between observations. For the non-zero-sum neutrality test, the model distributed the expected N sp, as calculated by extensive simulations, which allowed us to investigate the neutrality of the observed community by comparing the observed N sp with distributions of the expected N sp derived from the simulations. For this comparison, we developed a new "non-zero-sum N sp test," which we validated by running multiple neutral simulations using different parameter settings. We found that the non-zero-sum N sp test rejected neutrality at a near-significance level, which justified the validity of our approach. For an empirical test, the non-zero-sum N sp test was applied to real tropical tree communities in Panama and Malaysia. The non-zero-sum N sp test rejected neutrality in both communities when the observation interval was long and N sp was large. Hence, the non-zero-sum N sp test is an effective way to examine neutrality and has reasonable statistical power to reject the neutral model, especially when the observed N sp is large. This unique and simple approach is statistically powerful, even though it only employs two temporal sequences of community data. Thus, this test can be easily applied to existing datasets. In addition, application of the test will provide significant benefits for detecting changing biodiversity under climate change and anthropogenic disturbance.
A large body of evidence indicates that evolutionary innovations of novel organs have facilitated the subsequent diversification of species. Investigation of the evolutionary history of such organs should provide important clues for understanding the basis for species diversification. An Asian natricine snake, Rhabdophis tigrinus, possesses a series of unusual organs, called nuchal glands, which contain cardiotonic steroid toxins known as bufadienolides. Rhabdophis tigrinus sequesters bufadienolides from its toad prey and stores them in the nuchal glands as a defensive mechanism. Among more than 3,500 species of snakes, only 17 Asian natricine species are known to possess nuchal glands or their homologues. These 17 species belong to three nominal genera, Balanophis, Macropisthodon, and Rhabdophis. In Macropisthodon and Rhabdophis, however, species without nuchal glands also exist. To infer the evolutionary history of the nuchal glands, we investigated the molecular phylogenetic relationships among Asian natricine species with and without nuchal glands, based on variations in partial sequences of Mt-CYB, Cmos, and RAG1 (total 2,767 bp). Results show that all species with nuchal glands belong to a single clade (NGC). Therefore, we infer that the common ancestor of this clade possessed nuchal glands with no independent origins of the glands within the members. Our results also imply that some species have secondarily lost the glands. Given the estimated divergence time of related species, the ancestor of the nuchal gland clade emerged 19.18 mya. Our study shows that nuchal glands are fruitful subjects for exploring the evolution of novel organs. In addition, our analysis indicates that reevaluation of the taxonomic status of the genera Balanophis and Macropisthodon is required. We propose to assign all species belonging to the NGC to the genus Rhabdophis, pending further study.
Seed dispersal governs the distribution of plant propagules in the landscape and hence forms the template on which density-dependent processes act. Dispersal is therefore a vital component of many species coexistence and forest dynamics models and is of applied value in understanding forest regeneration. Research on the processes that facilitate forest regeneration and restoration is given further weight in the context of widespread loss and degradation of tropical forests, and provides impetus to improve estimates of seed dispersal for tropical forest trees. South-East Asian lowland rainforests, which have been subject to severe degradation, are dominated by trees of the Dipterocarpaceae family which constitute over 40% of forest biomass. Dipterocarp dispersal is generally considered to be poor given their large, gyration-dispersed fruits. However, there is wide variability in fruit size and morphology which we hypothesize mechanistically underpins dispersal potential through the lift provided to seeds mediated by the wings. We explored experimentally how the ratio of fruit wing area to mass ("inverse wing loading," IWL) explains variation in seed dispersal kernels among 13 dipterocarp species by releasing fruit from a canopy tower. Horizontal seed dispersal distances increased with IWL, especially at high wind speeds. Seed dispersal of all species was predominantly local, with 90% of seed dispersing <10 m, although maximum dispersal distances varied widely among species. We present a generic seed dispersal model for dipterocarps based on attributes of seed morphology and provide modeled seed dispersal kernels for all dipterocarp species with IWLs of 1-50, representing 75% of species in Borneo.
The Sunda pangolin (Manis javanica) is the most widely distributed Asian pangolin species, occurring across much of Southeast Asia and in southern China. It is classified as Critically Endangered and is one of the most trafficked mammals in the world, which not only negatively impacts wild Sunda pangolin populations but also poses a potential disease risk to other species, including humans and livestock. Here, we aimed to investigate the species' phylogeography across its distribution to improve our understanding of the species' evolutionary history, elucidate any taxonomic uncertainties and enhance the species' conservation genetic management and potential wildlife forensics applications. We sequenced mtDNA genomes from 23 wild Sunda pangolins of known provenance originating from Malaysia to fill sampling gaps in previous studies, particularly in Borneo. To conduct phylogenetic and population genetic analyses of Sunda pangolins across their range, we integrated these newly generated mitochondrial genomes with previously generated mtDNA and nuclear DNA data sets (RAD-seq SNP data). We identified an evolutionarily distinct mtDNA lineage in north Borneo, estimated to be ~1.6 million years divergent from lineages in west/south Borneo and the mainland, comparable to the divergence time from the Palawan pangolin. There appeared to be mitonuclear discordance, with no apparent genetic structure across Borneo based on analysis of nuclear SNPs. These findings are consistent with the 'out of Borneo hypothesis', whereby Sunda pangolins diversified in Borneo before subsequently migrating throughout Sundaland, and/or a secondary contact scenario between mainland and Borneo. We have elucidated possible taxonomic issues in the Sunda/Palawan pangolin complex and highlight the critical need for additional georeferenced samples to accurately apportion its range-wide genetic variation into appropriate taxonomic and conservation units. Additionally, these data have improved forensic identification testing involving these species and permit the implementation of geographic provenance testing in some scenarios.
Intensive land expansion of commercial oil palm agricultural lands results in reducing the size of peat swamp forests, particularly in Southeast Asia. The effect of this land conversion on macrofungal biodiversity is, however, understudied. We quantified macrofungal biodiversity by identifying mushroom sporocarps throughout four different habitats; logged peat swamp forest, large-scale oil palm plantation, monoculture, and polyculture smallholdings. We recorded a total of 757 clusters of macrofungi belonging to 127 morphospecies and found that substrates for growing macrofungi were abundant in peat swamp forest; hence, morphospecies richness and macrofungal clusters were significantly greater in logged peat swamp forest than converted oil palm agriculture lands. Environmental factors that influence macrofungi in logged peat swamp forests such as air temperature, humidity, wind speed, soil pH, and soil moisture were different from those in oil palm plantations and smallholdings. We conclude that peat swamp forests are irreplaceable with respect to macrofungal biodiversity. They host much greater macrofungal biodiversity than any of the oil palm agricultural lands. It is imperative that further expansion of oil palm plantation into remaining peat swamp forests should be prohibited in palm oil producing countries. These results imply that macrofungal distribution reflects changes in microclimate between habitats and reduced macrofungal biodiversity may adversely affect decomposition in human-modified landscapes.
Invasive species can encounter environments different from their source populations, which may trigger rapid adaptive changes after introduction (niche shift hypothesis). To test this hypothesis, we investigated whether postintroduction evolution is correlated with contrasting environmental conditions between the European invasive and source ranges in the Asian tiger mosquito Aedes albopictus. The comparison of environmental niches occupied in European and source population ranges revealed more than 96% overlap between invasive and source niches, supporting niche conservatism. However, we found evidence for postintroduction genetic evolution by reanalyzing a published ddRADseq genomic dataset from 90 European invasive populations using genotype-environment association (GEA) methods and generalized dissimilarity modeling (GDM). Three loci, among which a putative heat-shock protein, exhibited significant allelic turnover along the gradient of winter precipitation that could be associated with ongoing range expansion. Wing morphometric traits weakly correlated with environmental gradients within Europe, but wing size differed between invasive and source populations located in different climatic areas. Niche similarities between source and invasive ranges might have facilitated the establishment of populations. Nonetheless, we found evidence for environmental-induced adaptive changes after introduction. The ability to rapidly evolve observed in invasive populations (genetic shift) together with a large proportion of unfilled potential suitable areas (80%) pave the way to further spread of Ae. albopictus in Europe.
The genetic benefits individuals receive from mate choice have been the focus of numerous studies, with several showing support for both intrinsic genetic benefits and compatibility effects on fertilization success and offspring viability. However, the robustness of these effects have rarely been tested across an ecologically relevant environmental gradient. In particular, sperm environment is a crucial factor determining fertilization success in many species, especially those with external fertilization. Here, we test the importance of sperm environment in mediating compatibility-based selection on fertilization using a factorial breeding design. We detected a significant intrinsic male effect on fertilization success at only one of four sperm concentrations. Compatibility effects were significant at the two highest sperm concentrations and, interestingly, the magnitude of the compatibility effect consistently increased with sperm concentration. This suggests that females are able to modify the probability of sperm-egg fusion as the amount of sperm available increases.
Climate change threatens biodiversity in a range of ways, including changing animal body sizes. Despite numerous examples of size declines related to increasing temperatures, patterns of size change are not universal, suggesting that one or more primary mechanisms impacting size change are unknown. Precipitation is likely to influence the size different from and in conjunction with changes in temperature, yet tests of the interaction between these variables are rare. In this study, we show that a crossover interaction between temperature and precipitation impacts the body size of frogs as the climate warms. Using more than 3000 museum frog specimens from Borneo and climate records spanning more than 100 years, we found that frogs are larger in wet conditions than in dry conditions at cool temperatures, suggesting that resource availability determines body size at colder temperature. Conversely, frogs are larger in dry conditions than in wet conditions at warm temperatures, resulting in a crossover to desiccation resistance as the main determinant of body size as climates warm. Our results demonstrate that global warming can alter the impact of precipitation on life-history traits. We suggest that increased attention be paid to such interactive effects of climate variables, to identify complex mechanisms driving climate-induced size changes.
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.