We report here the complete mitochondrial (mt) genomes of six individuals of Cheilinus undulatus (Napoleon Wrasse), an endangered marine fish species. The six mt DNA sequences had an average size of 17,000 kb and encoded 22 tRNA, two sRNA, 13 highly conserved protein coding genes and a control region. The polymorphic variation (control region) in these six individuals suggests their potential use as a specific marker for phylogeographic conservation. Moreover, the sequence polymorphism within the control region (D-loop) suggests that this locus can be applied for phylogenetic studies.
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.
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.
The Tapirus indicus, also known as Malayan tapir, has been listed as a rapidly declining animal species in the past decades, along with being declared and categorized as an endangered species by the International Union for Conservation of Nature (IUCN) 2016. This tapir species is geographically distributed across several countries in Southeast Asia such as Peninsular Malaysia, Indonesia (Sumatra), South Thailand, and Myanmar. Amongst these countries, the Peninsula Malaysia forest is recorded to contain the highest number of Malayan tapir population. Unfortunately, in the past decades, the population of Malayan tapirs has declined swiftly due to serious deforestation, habitat fragmentation, and heavy vehicle accidents during road crossings at forest routes. Concerned by this predicament, the Department of Wildlife and National Parks (DWNP) Peninsular Malaysia collaborated with a few local universities to conduct various studies aimed at increasing the population number of tapirs in Malaysia. Several studies were conducted with the aim of enhancing the well-being of tapirs in captivity. Veterinarians face problems when it comes to selecting healthy and suitable tapirs for breeding programs at conservation centers. Conventional molecular methods using high-throughput sequencing provides a solution in determining the health condition of Malayan tapirs using the Next-Generation Sequencing (NGS) technology. Unaware by most, gut microbiome plays an important role in determining the health condition of an organism by various aspects: (1) digestion control; (2) benefiting the immune system; and (3) playing a role as a "second brain." Commensal gut bacterial communities (microbiomes) are predicted to influence organism health and disease. Imbalance of unhealthy and healthy microbes in the gut may contribute to weight gain, high blood sugar, high cholesterol, and other disorders. In infancy, neonatal gut microbiomes are colonized with maternal and environmental flora, and mature toward a stable composition in two to three years. Interactions between the microorganism communities and the host allow for the establishment of microbiological roles. Identifying the core microbiome(s) are essential in the prediction of diseases and changes in environmental behavior of microorganisms. The dataset of 16S rRNA amplicon sequencing of Malayan tapir was deposited in the MG-RAST portal. Parameters such as quality control, taxonomic prediction (unknown and predicted), diversity (rarefaction), and diversity (alpha) were analyzed using sequencing approaches (Amplicon sequencing). Comparisons of parameters, according to the type of sequencing, showed significant differences, except for the prediction variable. In the Amplicon sequencing datasets, the parameters Rarefaction and Unknown had the highest correlation, while Alpha and Predicted had the lowest. Firmicutes, Bacteroidetes, Proteobacteria, Bacilli, and Bacteroidia were the most representative genera in Malayan tapir amplicon sequences, which indicated that most of the tapirs were healthy. However, continuous assessment to maintain the well-being of tapir for long term is still required. This chapter focuses on the introduction of 16S rRNA amplicon metagenomics in analyzing Malayan tapir gut microbiome dataset.
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.
New records of the Japanese seahorse Hippocampus mohnikei from Cambodia, Malaysia, Thailand and Vietnam, along with recently published studies from India and Singapore, have greatly expanded the known range of H. mohnikei within Southeast Asia. These new records reveal novel habitat preferences and threats to H. mohnikei in the region. Although the global conservation status of H. mohnikei is classified as Data Deficient according to the IUCN Red List of Threatened Species, new sightings indicate that this species is found in similar habitats and faces similar threats as other Hippocampus species that are considered Vulnerable.
Demand for pangolin scales in East Asia has increased dramatically in the past two decades, raising concern to the pangolin survival and bringing them to the brink of local extinction. Enumerating the number of individuals from the seized pangolin scales primarily goes undocumented, mostly due to the unavailability of the appropriate methods. In this study, we developed a Pangolin Indexing System, a multi-locus STR panel of eight dinucleotide microsatellites that showed promising results in individualization and assignment of scales into Chinese and Indian pangolins. The combined power of exclusion was 0.83 and 0.99 for Chinese and Indian pangolin. The select panel of eight polymorphic STRs exhibited the cumulative probability of identity 3.7 × 10-9 for Indian pangolin and 3.6 × 10-7 for Chinese pangolin and identified 51 unique genotypes from the 74 scales selected from the four pangolin seizures. The study demonstrated the first report of cross-species validation of STRs developed from Malayan pangolin to Indian pangolin and showed the potential application of Pangolin Indexing System in screening of large seizures through DNA profiling from the scales of Indian and Chinese pangolin.
A century ago, tigers (Panthera tigris Linnaeus, 1758) were so common in parts of Southeast Asia as to be considered pests, and governments sponsored their killing. Habitat loss and fragmentation, market-driven poaching and loss of prey have since led to the disappearance of Indochinese tigers from most their former range. Despite 15 years of dedicated tiger conservation funding, national estimates of Indochinese tiger subpopulations can at best only be roughly approximated. The future for the subspecies appears grim unless very focused efforts can be applied to stabilize and recover subpopulations. On a regional scale, the 2 proposed subspecies Panthera tigris corbetti and P. tigris jacksoni are effectively managed as separate conservation units. Evaluating where to place conservation efforts should consider the vulnerability (likelihood of extinction) and irreplaceability (likelihood that an area contributes uniquely to regional conservation) of tiger subpopulations. Only 1 site in Thailand supporting <200 individuals (Huai Kha Khaeng-Thung Yai) is considered low vulnerability, and is irreplaceable. Five sites in Lao, Thailand and Peninsular Malaysia are medium vulnerability and irreplaceable. Priorities at these 6 sites are to double tiger numbers within 10 years through protection and monitoring. Seven sites in Lao, Thailand and Myanmar are high vulnerability and irreplaceable, and might be recovered if government commitment to tigers, staff capacity and legal frameworks for tiger protection are established. Tigers are extremely vulnerable or even extinct in Cambodia's Eastern Plains and the site is irreplaceable for tigers because it represents the only large (>10,000 km(2) ) block of dry forest habitat available in the region. A reintroduction program is the only option to recover tigers there.
The endangered Asian tapir (Tapirus indicus) is threatened by large-scale habitat loss, forest fragmentation and increased hunting pressure. Conservation planning for this species, however, is hampered by a severe paucity of information on its ecology and population status. We present the first Asian tapir population density estimate from a camera trapping study targeting tigers in a selectively logged forest within Peninsular Malaysia using a spatially explicit capture-recapture maximum likelihood based framework. With a trap effort of 2496 nights, 17 individuals were identified corresponding to a density (standard error) estimate of 9.49 (2.55) adult tapirs/100 km(2) . Although our results include several caveats, we believe that our density estimate still serves as an important baseline to facilitate the monitoring of tapir population trends in Peninsular Malaysia. Our study also highlights the potential of extracting vital ecological and population information for other cryptic individually identifiable animals from tiger-centric studies, especially with the use of a spatially explicit capture-recapture maximum likelihood based framework.
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.
With only 5% of the world's wild tigers (Panthera tigris Linnaeus, 1758) remaining since the last century, conservationists urgently need to know whether or not the management strategies currently being employed are effectively protecting these tigers. This knowledge is contingent on the ability to reliably monitor tiger populations, or subsets, over space and time. In the this paper, we focus on the 2 seminal methodologies (camera trap and occupancy surveys) that have enabled the monitoring of tiger populations with greater confidence. Specifically, we: (i) describe their statistical theory and application in the field; (ii) discuss issues associated with their survey designs and state variable modeling; and, (iii) discuss their future directions. These methods have had an unprecedented influence on increasing statistical rigor within tiger surveys and, also, surveys of other carnivore species. Nevertheless, only 2 published camera trap studies have gone beyond single baseline assessments and actually monitored population trends. For low density tiger populations (e.g. <1 adult tiger/100 km(2)) obtaining sufficient precision for state variable estimates from camera trapping remains a challenge because of insufficient detection probabilities and/or sample sizes. Occupancy surveys have overcome this problem by redefining the sampling unit (e.g. grid cells and not individual tigers). Current research is focusing on developing spatially explicit capture-mark-recapture models and estimating abundance indices from landscape-scale occupancy surveys, as well as the use of genetic information for identifying and monitoring tigers. The widespread application of these monitoring methods in the field now enables complementary studies on the impact of the different threats to tiger populations and their response to varying management intervention.
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.
Genetic variation in mitochondrial genes could underlie metabolic adaptations because mitochondrially encoded proteins are directly involved in a pathway supplying energy to metabolism. Macquarie perch from river basins exposed to different climates differ in size and growth rate, suggesting potential presence of adaptive metabolic differences. We used complete mitochondrial genome sequences to build a phylogeny, estimate lineage divergence times and identify signatures of purifying and positive selection acting on mitochondrial genes for 25 Macquarie perch from three basins: Murray-Darling Basin (MDB), Hawkesbury-Nepean Basin (HNB) and Shoalhaven Basin (SB). Phylogenetic analysis resolved basin-level clades, supporting incipient speciation previously inferred from differentiation in allozymes, microsatellites and mitochondrial control region. The estimated time of lineage divergence suggested an early- to mid-Pleistocene split between SB and the common ancestor of HNB+MDB, followed by mid-to-late Pleistocene splitting between HNB and MDB. These divergence estimates are more recent than previous ones. Our analyses suggested that evolutionary drivers differed between inland MDB and coastal HNB. In the cooler and more climatically variable MDB, mitogenomes evolved under strong purifying selection, whereas in the warmer and more climatically stable HNB, purifying selection was relaxed. Evidence for relaxed selection in the HNB includes elevated transfer RNA and 16S ribosomal RNA polymorphism, presence of potentially mildly deleterious mutations and a codon (ATP6113) displaying signatures of positive selection (ratio of nonsynonymous to synonymous substitution rates (dN/dS) >1, radical change of an amino-acid property and phylogenetic conservation across the Percichthyidae). In addition, the difference could be because of stronger genetic drift in the smaller and historically more subdivided HNB with low per-population effective population sizes.
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.
Haloxylon persicum is an endangered western Asiatic desert plant species, which survives under extreme environmental conditions. In this study, we focused on transcriptome analysis of H. persicum to understand the molecular mechanisms associated with drought tolerance. Two different periods of polyethylene glycol (PEG)-induced drought stress (48 h and 72 h) were imposed on H. persicum under in vitro conditions, which resulted in 18 million reads, subsequently assembled by de novo method with more than 8000 transcripts in each treatment. The N50 values were 1437, 1467, and 1524 for the control sample, 48 h samples, and 72 h samples, respectively. The gene ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG) pathway analysis resulted in enrichment of mitogen-activated protein kinase (MAPK) and plant hormone signal transduction pathways under PEG-induced drought conditions. The differential gene expression analysis (DGEs) revealed significant changes in the expression pattern between the control and the treated samples. The KEGG analysis resulted in mapping transcripts with 138 different pathways reported in plants. The differential expression of drought-responsive transcription factors depicts the possible signaling cascades involved in drought tolerance. The present study provides greater insight into the fundamental transcriptome reprogramming of desert plants under drought.
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.
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 proboscis monkey, Nasalis larvatus, is an endemic species to the island of Borneo. It is listed in the IUCN Red List as Endangered with a decreasing population trend. Nevertheless, biological information, especially on the genetic diversity of the species, is still incomplete. Its fragmented distribution poses difficulties in gathering genetic samples along with its widespread distribution across Borneo. This study aims to determine the genetic variation and structure of N. larvatus with an emphasis on Malaysian Borneo populations to elucidate its gene flow. The genetic variation and structure of N. larvatus were examined using 50 sequences of the 1,434-bp cytochrome oxidase subunit I (COI) gene region of mitochondrial DNA. The COI sequences revealed low genetic variation among N. larvatus populations in Malaysian Borneo. This low genetic variability could be the result of inbreeding pressure that may have occurred due to the absence of population expansion in this species over the last 30,000 years. This is supported in our analysis of molecular variance, which showed that groups of N. larvatus are significantly differentiated possibly due to natural geographic barriers. This study provides baseline information on the genetic diversity among proboscis monkey populations in Borneo for the future genetic assessment of the species.
Tor tor is an important game and food fish of India with a distribution throughout Asia from the trans-Himalayan region to the Mekong River basin to Malaysia, Pakistan, Bangladesh and Indonesia. A new cell line named TTCF was developed from the caudal fin of T. tor for the first time. The cell line was optimally maintained at 28°C in Leibovitz-15 (L-15) medium supplemented with 20% fetal bovine serum (FBS). The propagation of TTCF cells showed a high plating efficiency of 63.00%. The cytogenetic analysis revealed a diploid count of 100 chromosomes at passage 15, 30, 45 and 60 passages. The viability of the TTCF cell line was found to be 72% after 6 months of cryopreservation in liquid nitrogen (-196°C). The origin of the cell lines was confirmed by the amplification of 578- and 655-bp sequences of 16S rRNA and cytochrome oxidase subunit I (COI) genes of mitochondrial DNA (mtDNA) respectively. TTCF cells were successfully transfected with green fluorescent protein (GFP) reporter plasmids. Further, immunocytochemistry studies confirm its fibroblastic morphology of cells. Genotoxicity assessment of H₂O₂ in TTCF cell line revealed the utility of TTCF cell line as in vitro model for aquatic toxicological studies.