Malayan box turtle (Cuora amboinensis) has been a wildlife-protected vulnerable turtle species in Malaysia since 2005. However, because of its purported usage in traditional medicine, tonic foods and feeds, clandestine black market trade is rampant. Several polymerase chain reaction (PCR) assays for the taxonomic detection and classification of turtle species have been proposed. These assays are based on long-length target amplicons which are assumed to break down under compromised states and, hence, might not be suitable for the forensic tracing and tracking of turtle trafficking. For the first time this paper develops a very short-amplicon-length PCR assay (120 bp) for the detection of Malayan box turtle meat in raw, processed and mixed matrices, and experimental evidence is produced that such an assay is not only more stable and reliable but also more sensitive than those previously published. We checked the assay specificity against 20 different species and no cross-species detection was observed. The possibility of any false-negative detection was eliminated by a universal endogenous control for eukaryotes. The assay detection limit was 0.0001 ng of box turtle DNA from pure meat and 0.01% turtle meat in binary and ternary admixtures and commercial meatballs. Superior target stability and sensitivity under extreme treatments of boiling, autoclaving and microwave cooking suggested that this newly developed assay would be suitable for any forensic and/or archaeological identification of Malayan box turtle species, even in severely degraded specimens. Further, in silico studies indicated that the assay has the potential to be used as a universal probe for the detection of nine Cuora species, all of which are critically endangered.
Presbytis chrysomelas chrysomelas endemic only in Sarawak and Kalimantan was categorized by IUCN as a critically endangered primate that require special attention from research and conservation perspectives. A qualitative study on ranging patterns of P. c. chrysomelas was conducted in the Samunsam Wildlife Sanctuary, Sarawak. The study was conducted over a period of 13 months from December 2004 to December 2005 with 213 days of observation. Behavioural observation covered 17 groups with special emphasis on two main groups and 1 subadult group. Scanning and focal sampling were employed as the observation methods. Results indicated that P. c. chrysomelas had vertical, straight horizontal, and cross-horizontal types of movement patterns. P. c. chrysomelas was recorded to have a short movement distance (31.8-54.3 m). Distribution, abundance types, and food resources might be the factors that shaped the patterns of movement and distance in P. c. chrysomelas.
Next-Gen sequencing was used to recover the complete mitochondrial genome of Cherax tenuimanus. The mitogenome consists of 15,797 base pairs (68.14% A + T content) containing 13 protein-coding genes, two ribosomal subunit genes, 22 transfer RNAs, and a 779 bp non-coding AT-rich region. Mitogenomes have now been recovered for all six species of Cherax native to Western Australia.
Over half of globally threatened animal species have experienced rapid geographic range loss. Identifying the parts of species' distributions most vulnerable to local extinction would benefit conservation planning. However, previous studies give little consensus on whether ranges decline to the core or edge. We built on previous work by using empirical data to examine the position of recent local extinctions within species' geographic ranges, address range position as a continuum, and explore the influence of environmental factors. We aggregated point-locality data for 125 Galliform species from across the Palearctic and Indo-Malaya into equal-area half-degree grid cells and used a multispecies dynamic Bayesian occupancy model to estimate rates of local extinctions. Our model provides a novel approach to identify loss of populations from within species ranges. We investigated the relationship between extinction rates and distance from range edge by examining whether patterns were consistent across biogeographic realm and different categories of land use. In the Palearctic, local extinctions occurred closer to the range edge than range core in both unconverted and human-dominated landscapes. In Indo-Malaya, no pattern was found for unconverted landscapes, but in human-dominated landscapes extinctions tended to occur closer to the core than the edge. Our results suggest that local and regional factors override general spatial patterns of recent local extinction within species' ranges and highlight the difficulty of predicting the parts of a species' distribution most vulnerable to threat.
A total of 1951 species of freshwater and marine fishes belonging to 704 genera and 186 families are recorded in Malaysia. Almost half (48%) are currently threatened to some degree, while nearly one third (27%) mostly from the marine and coral habitats require urgent scientific studies to evaluate their status. Freshwater habitats encompass the highest percentage of threatened fish species (87%) followed by estuarine habitats (66%). Of the 32 species of highly threatened (HT) species, 16 are freshwater and 16 are largely marine-euryhaline species. Fish extinctions in Malaysia are confined to two freshwater species, but both freshwater and marine species are being increasingly threatened by largely habitat loss or modification (76%), overfishing (27%) and by-catch (23%). The most important threat to freshwater fishes is habitat modification and overfishing, while 35 species are threatened due to their endemism. Brackish-water, euryhaline and marine fishes are threatened mainly by overfishing, by-catch and habitat modification. Sedimentation (pollution) additionally threatens coral-reef fishes. The study provides recommendations to governments, fish managers, scientists and stakeholders to address the increasing and unabated extinction risks faced by the Malaysian fish fauna.
Pangolins, unique mammals with scales over most of their body, no teeth, poor vision, and an acute olfactory system, comprise the only placental order (Pholidota) without a whole-genome map. To investigate pangolin biology and evolution, we developed genome assemblies of the Malayan (Manis javanica) and Chinese (M. pentadactyla) pangolins. Strikingly, we found that interferon epsilon (IFNE), exclusively expressed in epithelial cells and important in skin and mucosal immunity, is pseudogenized in all African and Asian pangolin species that we examined, perhaps impacting resistance to infection. We propose that scale development was an innovation that provided protection against injuries or stress and reduced pangolin vulnerability to infection. Further evidence of specialized adaptations was evident from positively selected genes involving immunity-related pathways, inflammation, energy storage and metabolism, muscular and nervous systems, and scale/hair development. Olfactory receptor gene families are significantly expanded in pangolins, reflecting their well-developed olfaction system. This study provides insights into mammalian adaptation and functional diversification, new research tools and questions, and perhaps a new natural IFNE-deficient animal model for studying mammalian immunity.
In 2008, the IUCN threat status of the Asian tapir (Tapirus indicus) was reclassified from 'vulnerable' to 'endangered'. The latest distribution map from the IUCN Red List suggests that the tapirs' native range is becoming increasingly fragmented in Peninsular Malaysia, but distribution data collected by local researchers suggest a more extensive geographical range. Here, we compile a database of 1261 tapir occurrence records within Peninsular Malaysia, and demonstrate that this species, indeed, has a much broader geographical range than the IUCN range map suggests. However, extreme spatial and temporal bias in these records limits their utility for conservation planning. Therefore, we used maximum entropy (MaxEnt) modeling to elucidate the potential extent of the Asian tapir's occurrence in Peninsular Malaysia while accounting for bias in existing distribution data. Our MaxEnt model predicted that the Asian tapir has a wider geographic range than our fine-scale data and the IUCN range map both suggest. Approximately 37% of Peninsular Malaysia contains potentially suitable tapir habitats. Our results justify a revision to the Asian tapir's extent of occurrence in the IUCN Red List. Furthermore, our modeling demonstrated that selectively logged forests encompass 45% of potentially suitable tapir habitats, underscoring the importance of these habitats for the conservation of this species in Peninsular Malaysia.
Habitat destruction and overhunting are two major drivers of mammal population declines and extinctions in tropical forests. The construction of roads can be a catalyst for these two threats. In Southeast Asia, the impacts of roads on mammals have not been well-documented at a regional scale. Before evidence-based conservation strategies can be developed to minimize the threat of roads to endangered mammals within this region, we first need to locate where and how roads are contributing to the conversion of their habitats and illegal hunting in each country. We interviewed 36 experts involved in mammal research from seven Southeast Asian countries to identify roads that are contributing the most, in their opinion, to habitat conversion and illegal hunting. Our experts highlighted 16 existing and eight planned roads - these potentially threaten 21% of the 117 endangered terrestrial mammals in those countries. Apart from gathering qualitative evidence from the literature to assess their claims, we demonstrate how species-distribution models, satellite imagery and animal-sign surveys can be used to provide quantitative evidence of roads causing impacts by (1) cutting through habitats where endangered mammals are likely to occur, (2) intensifying forest conversion, and (3) contributing to illegal hunting and wildlife trade. To our knowledge, ours is the first study to identify specific roads threatening endangered mammals in Southeast Asia. Further through highlighting the impacts of roads, we propose 10 measures to limit road impacts in the region.
Freshwater mussels of the family Unionidae exhibit a particular form of mitochondria inheritance called double uniparental inheritance (DUI), in which the mitochondria are inherited by both male and female parents. The (M)ale and (F)emale mitogenomes are highly divergent within species. In the present study, we determine and describe the complete M and F mitogenomes of the Endangered freshwater mussel Potomida littoralis (Cuvier, 1798). The complete M and F mitogenomes sequences are 16 451 bp and 15 787 bp in length, respectively. Both F and M have the same gene content: 13 protein-coding genes (PCGs), 22 transfer RNA (trn) and 2 ribosomal RNA (rrn) genes. Bayesian analyses based on the concatenated nucleotide sequences of 12 PCGs and 2 rrn genes of both genomes, including mitogenome sequences available from related species, were performed. Male and Female lineages are monophyletic within the family, but reveal distinct phylogenetic relationships.
A new species of Bent-toed Gecko, Cyrtodactylus gunungsenyumensis sp. nov. of the sworderi complex, is described from Hutan Lipur Gunung Senyum, Pahang, Peninsular Malaysia and is differentiated from all other species in the sworderi complex by having a unique combination of characters including a maximum SVL of 74.7 mm; low, rounded, weakly keeled, body tubercles; 34-40 paravertebral tubercles; weak ventrolateral body fold lacking tubercles; 38-41 ventral scales; an abrupt transition between the posterior and ventral femoral scales; 20-23 subdigital lamellae on the fourth toe; enlarged femoral scales; no femoral or precloacal pores; no precloacal groove; wide caudal bands; and an evenly banded dorsal pattern. Cyrtodactylus gunungsenyumensis sp. nov. is a scansorial, karst forest-adapted specialist endemic to the karst ecosystem surrounding Gunung Senyum and occurs on the vertical walls of the limestone towers as well as the branches, trunks, and leaves of the vegetation in the associated karst forest. Cyrtodactylus gunungsenyumensis sp. nov. is the seventh species of karst forest-adapted Cyrtodactylus and the sixteenth endemic species of karst ecosystem reptile discovered in Peninsular Malaysia in the last seven years from only 12 different karst forests. This is a clear indication that many species remain to be discovered in the approximately 558 isolated karst ecosystems in Peninsular Malaysia not yet surveyed. These data continue to underscore the importance of karst ecosystems as reservoirs of biodiversity and microendemism and that they constitute an important component of Peninsular Malaysia's natural heritage and should be protected from the quarrying interests of foreign industrial companies.
With less than 3200 wild tigers in 2010, the heads of 13 tiger-range countries committed to doubling the global population of wild tigers by 2022. This goal represents the highest level of ambition and commitment required to turn the tide for tigers in the wild. Yet, ensuring efficient and targeted implementation of conservation actions alongside systematic monitoring of progress towards this goal requires that we set site-specific recovery targets and timelines that are ecologically realistic. In this study, we assess the recovery potential of 18 sites identified under WWF's Tigers Alive Initiative. We delineated recovery systems comprising a source, recovery site, and support region, which need to be managed synergistically to meet these targets. By using the best available data on tiger and prey numbers, and adapting existing species recovery frameworks, we show that these sites, which currently support 165 (118-277) tigers, have the potential to harbour 585 (454-739) individuals. This would constitute a 15% increase in the global population and represent over a three-fold increase within these specific sites, on an average. However, it may not be realistic to achieve this target by 2022, since tiger recovery in 15 of these 18 sites is contingent on the initial recovery of prey populations, which is a slow process. We conclude that while sustained conservation efforts can yield significant recoveries, it is critical that we commit our resources to achieving the biologically realistic targets for these sites even if the timelines are extended.
Climatic differences across a taxon's range may be associated with specific bioenergetic demands and may result in genetics-based metabolic adaptation, particularly in aquatic ectothermic organisms that rely on heat exchange with the environment to regulate key physiological processes. Extending down the east coast of Australia, the Great Dividing Range (GDR) has a strong influence on climate and the evolutionary history of freshwater fish species. Despite the GDR acting as a strong contemporary barrier to fish movement, many species, and species with shared ancestries, are found on both sides of the GDR, indicative of historical dispersal events. We sequenced complete mitogenomes from the four extant species of the freshwater cod genus Maccullochella, two of which occur on the semi-arid, inland side of the GDR, and two on the mesic coastal side. We constructed a dated phylogeny and explored the relative influences of purifying and positive selection in the evolution of mitogenome divergence among species. Results supported mid- to late-Pleistocene divergence of Maccullochella across the GDR (220-710 thousand years ago), bringing forward previously reported dates. Against a background of pervasive purifying selection, we detected potentially functionally relevant fixed amino acid differences across the GDR. Although many amino acid differences between inland and coastal species may have become fixed under relaxed purifying selection in coastal environments rather than positive selection, there was evidence of episodic positive selection acting on specific codons in the Mary River coastal lineage, which has consistently experienced the warmest and least extreme climate in the genus.
Adaptive differences across species' ranges can have important implications for population persistence and conservation management decisions. Despite advances in genomic technologies, detecting adaptive variation in natural populations remains challenging. Key challenges in gene-environment association studies involve distinguishing the effects of drift from those of selection and identifying subtle signatures of polygenic adaptation. We used paired-end restriction site-associated DNA sequencing data (6,605 biallelic single nucleotide polymorphisms; SNPs) to examine population structure and test for signatures of adaptation across the geographic range of an iconic Australian endemic freshwater fish species, the Murray cod Maccullochella peelii. Two univariate gene-association methods identified 61 genomic regions associated with climate variation. We also tested for subtle signatures of polygenic adaptation using a multivariate method (redundancy analysis; RDA). The RDA analysis suggested that climate (temperature- and precipitation-related variables) and geography had similar magnitudes of effect in shaping the distribution of SNP genotypes across the sampled range of Murray cod. Although there was poor agreement among the candidate SNPs identified by the univariate methods, the top 5% of SNPs contributing to significant RDA axes included 67% of the SNPs identified by univariate methods. We discuss the potential implications of our findings for the management of Murray cod and other species generally, particularly in relation to informing conservation actions such as translocations to improve evolutionary resilience of natural populations. Our results highlight the value of using a combination of different approaches, including polygenic methods, when testing for signatures of adaptation in landscape genomic studies.
The Bukit Merah Orang Utan Island (OUI) Foundation has been conducting behavioral and veterinary research on orangutans as an attempt at ex situ conservation. Since 2010, the Primate Research Institute, Kyoto University has been collaborating with OUI to promote environmental enrichment and infant rearing by biological mothers in addition to the continuous efforts of refining the veterinary management of the endangered species. In 2011, three Bornean orangutans (Pongo pygmaeus pygmaeus) were released on an island, called BJ Island, adjacent to OUI. This island is approximately 5.6 ha in size, and 635 trees belonging to 102 plant species were identified prior to their release. Behavioral monitoring of the released individuals has been conducted to evaluate their behavioral adaptation to the new environment. Two of the three released orangutans were born in the wild, whereas the youngest individual was born on OUI and expected to learn forest survival strategies from the two older individuals. One of the orangutans was pregnant at the time of release and subsequently gave birth to two male infants on BJ Island. The behavioral monitoring indicated that these orangutans traveled more and spent more time on trees following their release onto BJ Island. However, resting was longer for two females both on OUI and BJ Island when compared to other populations. The orangutans consumed some natural food resources on BJ Island. The release into a more naturalistic environment may help the orangutans to develop more naturalistic behavioral patterns that resemble their wild counterparts.
The genetic diversity of the endangered crocodile Tomistoma schlegelii was characterized using the protein coding ND 6-tRNA(glu)-cyt b and the cytochrome b-control region (cyt b-CR) markers. Concatenate data revealed six haplotypes with an overall haplotype diversity of 0.769 ± 0.039; nucleotide diversity was 0.00535 ± 0.00172. A nearest-neighbor analysis showed that all individuals clustered with four geographic regions (Sumatra, Peninsular Malaysia, Sarawak, and East Kalimantan) and were genetically differentiated. With the exception of the individuals from haplotype H2, which occurred in both Peninsular Malaysia and Sarawak, all other haplotypes were geographically distinct. The H4 lineage, which was found to be the most divergent, clustered exclusively in the basal clade in all phylogenetic trees, and the haplotype network was unconnected at the 95% reconnection limit, suggesting further investigation to establish its possible status as a distinct evolutionary significant unit or a cryptic species.
Surface-enhanced Raman scattering (SERS) based DNA biosensors have considered as excellent, fast and ultrasensitive sensing technique which relies on the fingerprinting ability to produce molecule specific distinct spectra. Unlike conventional fluorescence based strategies SERS provides narrow spectral bandwidths, fluorescence quenching and multiplexing ability, and fitting attribute with short length probe DNA sequences. Herein, we report a novel and PCR free SERS based DNA detection strategy involving dual platforms and short DNA probes for the detection of endangered species, Malayan box turtle (MBT) (Cuora amboinensis). In this biosensing feature, the detection is based on the covalent linking of the two platforms involving graphene oxide-gold nanoparticles (GO-AuNPs) functionalized with capture probe 1 and gold nanoparticles (AuNPs) modified with capture probe 2 and Raman dye (Cy3) via hybridization with the corresponding target sequences. Coupling of the two platforms generates locally enhanced electromagnetic field 'hot spot', formed at the junctions and interstitial crevices of the nanostructures and consequently provide significant amplification of the SERS signal. Therefore, employing the two SERS active substrates and short-length probe DNA sequences, we have managed to improve the sensitivity of the biosensors to achieve a lowest limit of detection (LOD) as low as 10 fM. Furthermore, the fabricated biosensor exhibited sensitivity even for single nucleotide base-mismatch in the target DNA as well as showed excellent performance to discriminate closely related six non-target DNA sequences. Although the developed SERS biosensor would be an attractive platform for the authentication of MBT from diverse samples including forensic and/or archaeological specimens, it could have universal application for detecting gene specific biomarkers for many diseases including cancer.
Confiscated slow lorises (Nycticebus spp.) at Bangpra Water-Bird Breeding Center (BWBC) in Thailand provided an opportunity to demonstrate the application of noninvasive genetic approaches for species identification when morphology of the animals was ambiguous. The slow lorises at BWBC had been assigned to either N. bengalensis or N. pygmaeus, based on body size. However, the morphology of N. bengalensis is highly variable and overlaps with that of N. coucang (sensu stricto). Phylogenetic analysis of cytochrome b and d-loop mitochondrial regions placed all confiscated N. pygmaeus with the published sequences of N. pygmaeus and distinguished them from other Nycticebus. All other confiscated individuals formed a monophyletic clade, most individuals grouping with published N. bengalensis sequences from wild populations in Vietnam and distinct from Peninsular Malaysian and Sumatran N. coucang, Javan N. javanicus and Bornean N. menagensis. Six individuals within the N. bengalensis clade formed a separate subgroup that did not group with any reference material as indicated by phylogenetic and haplotype network analyses. Whether these trafficked individuals are undiscovered wild populations will require further investigation. Additional genetic studies of wild slow loris populations in different regions are therefore urgently required for reference to aid the protection and conservation of these threatened species.
The identification of Aquilaria species from their resinous non-wood product, the agarwood, is challenging as conventional techniques alone are unable to ascertain the species origin. Aquilaria is a highly protected species due to the excessive exploitation of its precious agarwood. Here, we applied the DNA barcoding technique to generate barcode sequences for Aquilaria species and later applied the barcodes to identify the source species of agarwood found in the market. We developed a reference DNA barcode library using eight candidate barcode loci (matK, rbcL, rpoB, rpoC1, psbA-trnH, trnL-trnF, ITS, and ITS2) amplified from 24 leaf accessions of seven Aquilaria species obtained from living trees. Our results indicated that all single barcodes can be easily amplified and sequenced with the selected primers. The combination of trnL-trnF+ITS and trnL-trnF+ITS2 yielded the greatest species resolution using the least number of loci combination, while matK+trnL-trnF+ITS showed potential in detecting the geographical origins of Aquilaria species. We propose trnL-trnF+ITS2 as the best candidate barcode for Aquilaria as ITS2 has a shorter sequence length compared to ITS, which eases PCR amplification especially when using degraded DNA samples such as those extracted from processed agarwood products. A blind test conducted on eight agarwood samples in different forms using the proposed barcode combination proved successful in their identification up to the species level. Such potential of DNA barcoding in identifying the source species of agarwood will contribute to the international timber trade control, by providing an effective method for species identification and product authentication.
The Malayan tapir (Tapirus indicus) is the largest among the four tapir species and is listed as an endangered species. Ultrasound examination and description of the external anatomy of the female reproductive system of three adult females were performed, whereas the internal anatomy was investigated in necropsied samples of four adult females and one subadult female. Descriptions of the male external genitalia were conducted on one adult male. Gross examination revealed the presence of a bicornuate uterus. The uterine cervix is firm and muscular with projections towards its lumen, which is also evident on ultrasonography. The elongated and relatively small ovaries, which have a smooth surface, could not be imaged on ultrasonography, due to their anatomical position. The testes are located inside a slightly pendulous scrotum that is sparsely covered with soft, short hairs. The penis has one dorsal and two lateral penile projections just proximal to the glans penis.