The first complete mitochondrial genome of the critically endangered Malaysian giant turtle, Orlitia borneensis (Testudines: Geoemydidae).

We present here the complete mitochondrial sequence of the critically endangered Malaysian giant turtle, Orlitia borneensis. The assembled mitochondrial genome includes 13 protein-coding genes (PCGs), 22 transfer RNA (tRNA) genes, two ribosomal RNA genes (rRNAs), and one control region. This mitochondrial genome has been archived in the NCBI GenBank with accession number OQ808845. The Batagur control region is relatively smaller than O. borneensis and closer to Aldabrachelys gigantea, which suggests potentially that O. borneensis has undergone an expansion in the control region.

Evaluation of Fecundity, Fertilization, Hatching, and Gonadosomatic Index of Exotic Clarias gariepinus (Burchell, 1822) and Native Clarias macromystax (Gunther, 1864) under Semi-Arid Conditions of Nigeria.

The study evaluates the hybridizations between two African catfish, C. gariepinus and C. macromystax, using artificial reproduction. Fecundity and gonadosomatic index were assessed, and growth performance at different developmental stages was evaluated. The results indicated that both species possess a similar gonadosomatic index. The parent C. gariepinus possesses significantly (p < 0.05) higher male reproductive quality than C. macromystax. Fecundity was significantly higher (p < 0.05) in the female parent of C. macromystax and lower in C. gariepinus. The highest fertilization rates and hatching rates were achieved in the hybrid cross of ♀C. macromystax × â™‚C. gariepinus (♀Cm × â™‚Cg) which also recorded the lowest deformed larva rate. Survival rates at the larval stage were more than 80%, with the highest record of survival in the hybrid ♀Cm × â™‚Cg. However, survival rates in the fry stage were lower than 70%. The hybrid cross (C. macromystax × C. gariepinus) outperformed the parental cross of C. macromystax but was not significantly similar to the parental cross of C. gariepinus. The study revealed the potential of the hybrid ♀C. macromystax × â™‚C. gariepinus, and hybridizations of ♀C. macromystax × â™‚C. gariepinus (♀Cm × â™‚Cg) are indeed possible and proved to have a better growth and survival rate under semi-arid conditions, which would contribute significantly to the improvement of C. macromystax production in captivity.

Global Terrapin Character-Based DNA Barcodes: Assessment of the Mitochondrial COI Gene and Conservation Status Revealed a Putative Cryptic Species.

Technological and analytical advances to study evolutionary biology, ecology, and conservation of the Southern River Terrapin (Batagur affinis ssp.) are realised through molecular approaches, including DNA barcoding. We evaluated the use of COI DNA barcodes in Malaysia's Southern River Terrapin population to better understand the species' genetic divergence and other genetic characteristics. We evaluated 26 sequences, including four from field specimens of Southern River Terrapins obtained in Bota Kanan, Perak, Malaysia, and Kuala Berang, Terengganu, Malaysia, as well as 22 sequences from global terrapins previously included in the Barcode of Life Database (BOLD) Systems and GenBank. The species are divided into three families: eight Geoemydidae species (18%), three Emydidae species (6%), and one Pelomedusidae species (2%). The IUCN Red List assigned the 12 species of terrapins sampled for this study to the classifications of critically endangered (CR) for 25% of the samples and endangered (EN) for 8% of the samples. With new haplotypes from the world's terrapins, 16 haplotypes were found. The intraspecific distance values between the COI gene sequences were calculated using the K2P model, which indicated a potential cryptic species between the Northern River Terrapin (Batagur baska) and Southern River Terrapin (Batagur affinis affinis). The Bayesian analysis of the phylogenetic tree also showed both species in the same lineage. The BLASTn search resulted in 100% of the same species of B. affinis as B. baska. The Jalview alignment visualised almost identical sequences between both species. The Southern River Terrapin (B. affinis affinis) from the west coast of Peninsular Malaysia was found to share the same haplotype (Hap_1) as the Northern River Terrapin from India. However, B. affinis edwardmolli from the east coast of Peninsular Malaysia formed Hap_16. The COI analysis found new haplotypes and showed that DNA barcodes are an excellent way to measure the diversity of a population.

Characterization of the complete mitochondrial genome of the critically endangered Southern River Terrapin, Batagur affinis affinis (Reptilia: Geoemydidae).

In this study, we report the nearly complete mitochondrial sequence of Batagur affinis affinis. The assembled mitogenome consists of 13 PCGs, 22 tRNA genes, two rRNAs and one near-complete D-loop region. Of the annotated genes, the ND6 subunit gene and eight tRNA genes were encoded on the L-strand, while the remaining genes were dispersed on the H-strand. Except for CO1, which has a GTG start codon, all protein-coding genes begin with ATG. The mitogenome has been deposited in NCBI GenBank under the accession number OQ409915. Phylogenetic tree analysis based on publicly available mitogenomes indicate the sister grouping of B. affinis affinis with B. kachuga.

Conservation Genetics of the Critically Endangered Southern River Terrapin (Batagur affinis) in Malaysia: Genetic Diversity and Novel Subspecies Distribution Ranges.

A population genetics study was carried out on the Southern River terrapin (Batagur affinis) from four places in Peninsular Malaysia: Pasir Gajah, Kemaman (KE), Terengganu; Bukit Pinang (BP), Kedah; Bota Kanan (BK), Perak; and Bukit Paloh, Kuala Berang (KB), Terengganu. The goal of this study is to identify genetic differences in two subspecies of B. affinis in Malaysia. No previous reports were available on the genetic diversity, phylogenetic relationships and matrilineal hereditary structure of these terrapin populations in Malaysia. The sequencing identified 46 single nucleotide polymorphisms that defined six mitochondrial haplotypes in the Southern River terrapins. Tajima's D test and Fu's Fs neutrality tests were performed to evaluate the signatures of recent historical demographic events. Based on the tests, the B. affinis edwardmolli was newly subspecies identified in the west coast-northern region of Kedah state. In addition, the B. affinis edwardmolli in Bukit Paloh, Kuala Berang (KB), Terengganu (Population 4), was shown to have a single maternal lineage compared to other populations. Low genetic diversity, but significant genetic differences, were detected among the studied Southern River terrapin populations.

The complete mitochondrial genomes of Pangasius nasutus and P. conchophilus (Siluriformes: Pangasiidae).

The catfish, Pangasius nasutus and P. conchophilus, are often misidentified between each other due to their similar morphology. Thus, the current study was conducted to differentiate them based on a molecular approach. The complete mitochondrial genomes of P. nasutus and P. conchophilus obtained from the Pahang River (Peninsular Malaysia) were sequenced, assembled, and annotated using next-generation sequencing (NGS). A 16,465 bp and 16,470 bp length mitogenome sequence of P. nasutus and P. conchophilus, respectively, was generated, each containing 13 protein genes, 22 tRNAs, and two rRNAs, typical of most vertebrates. This is the first report of the complete mitochondrial genome sequences of P. nasutus and P. conchophilus. These data are a valuable genetic resource for future studies of these two commercially important species.

Turtles in Malaysia: A Review of Conservation Status and a Call for Research.

Approximately 356 species of turtles inhabit saltwater and freshwater habitats globally, except in Antarctica. Twenty-four species of turtles have been reported in Malaysia, four of which are sea turtles. The state of Terengganu harbored the highest number of turtles, with 17 different reported species. Based on the IUCN Red List, 29% of turtle species in Malaysia are critically endangered. In comparison, another 25% are classified as endangered. Likewise, CITES reported that 67% of Malaysia's turtles are threatened, while 25% are classified as critically endangered. This review discusses the checklists, molecular genetics work, conservation status, recent trends, and recommendations for future research. Factors contributing to their population declines and current endangered status are also discussed.

Faecal DNA metabarcoding reveals novel bacterial community patterns of critically endangered Southern River Terrapin, Batagur affinis.

Southern River Terrapin, Batagur affinis, is a freshwater turtle listed as critically endangered on the IUCN Red List since 2000. Many studies suggest that faecal DNA metabarcoding can shield light on the host-associated microbial communities that play important roles in host health. Thus, this study aimed to characterise and compare the faecal bacterial community between captive and wild B. affinis using metabarcoding approaches. A total of seven faeces samples were collected from captive (N = 5) and wild (N = 2) adult B. affinis aseptically, crossing the East and West coast of peninsular Malaysia. The DNA was extracted from the faeces samples, and the 16S rRNA gene (V3-V4 region) was amplified using polymerase chain reaction (PCR). The amplicon was further analysed using SILVA and DADA2 pipelines. In total, 297 bacterial communities taxonomic profile (phylum to genus) were determined. Three phyla were found in high abundance in all faeces samples, namely Firmicutes (38.69%), Bacteroidetes (24.52%), and Fusobacteria (6.95%). Proteobacteria were detected in all faeces samples (39.63%), except the wild sample, KBW3. Under genus level, Cetobacteriumwas found as the most abundant genus (67.79%), followed by Bacteroides (24.56%) and Parabacteroides (21.78%). The uncultured genus had the highest abundance (88.51%) even though not detected in the BK31 and KBW2 samples. The potential probiotic genera (75.00%) were discovered to be more dominant in B. affinis faeces samples. Results demonstrated that the captive B. affinis faeces samples have a greater bacterial variety and richness than wild B. affinis faeces samples. This study has established a starting point for future investigation of the gut microbiota of B. affinis.

Characterization of the first mitogenomes of the smallest fish in the world, Paedocypris progenetica, from peat swamp of Peninsular Malaysia, Selangor, and Perak.

The two complete mitochondrial genomes (mitogenomes) of Paedocypris progenetica, the smallest fish in the world which belonged to the Cyprinidae family, were sequenced and assembled. The circular DNA molecules of mitogenomes P1-P. progenetica and S3-P. progenetica were 16,827 and 16,616 bp in length, respectively, and encoded 13 protein-coding genes, 22 transfer RNA genes, two ribosomal RNA genes, and one control region. The gene arrangements of P. progenetica were identical to those of other Paedocypris species. BLAST and phylogenetic analyses revealed variations in the mitogenome sequences of two Paedocypris species from Perak and Selangor. The circular DNA molecule of P. progenetica yield a standard vertebrate gene arrangement and an overall nucleotide composition of A 33.0%, T 27.2%, C 23.5%, and G 15.5%. The overall AT content of this species was consistent with that of other species in other genera. The negative GC-skew and positive ATskew of the control region in P. progenetica indicated rich genetic variability and AT nucleotide bias, respectively. The results of this study provide genomic variation information and enhance the understanding of the mitogenome of P. progenetica. They could later deliver highly valuable new insight into data for phylogenetic analysis and population genetics.

Mitochondrial Markers Identify a Genetic Boundary of the Green Tiger Prawn (Penaeus semisulcatus) in the Indo-Pacific Ocean.

A population genetics study of the commercially important Green Tiger Prawn (Penaeus semisulcatus) was conducted in the Indo-Pacific Ocean with a focus on the Indo-Malay Archipelago waters of the South China Sea (SCS), Sulu Sea (SLS), Celebes Sea (CLS) and the Strait of Malacca (SOM), the latter being the main waterway that connects the Indian Ocean with the Pacific Ocean. A 548-base-pair region of mitochondrial COI and 571 base pairs of the control region (CR) were analysed in 284 specimens from 15 locations. Genetic divergences (Tamura 3-parameter) for COI ranged from 0.1% to 7.2% and CR 2.3% to 21.7%, with Bagan Pasir (BGP) in central SOM being the most genetically different from other populations (COI: 3.3-4.2%; CR: 7.1-16.5%). All populations were differentiated into two lineages with a genetic break in the vicinity of BGP; Lineage I comprised populations south of this site (SCS, SLS, CLS and part of SOM) and Lineage II comprised populations north of BGP (part of the SOM). Specifically, most individuals of Bagan Pasir (BGP) and another site just south of it, Batu Pahat (BPT), clustered in Lineage I, while all SOM populations to the north of these sites clustered in Lineage II. The BGP population is believed to be a mixed gene pool between the two lineages. The results could be attributed to the fluctuations of Pleistocene sea levels and a possible influence of the One Fathom Bank in SOM. High genetic diversity was recorded, π (Lineage I: COI: 3.4%; CR: 7.4%) (Lineage II: COI: 3.8%; CR: 12.6%) and, h (Lineage I: COI: 0.81; CR: 1.0) (Lineage II: COI: 0.57; CR: 0.99). Demographic statistics revealed that both lineages underwent a sudden expansion and consequent stabilisation in genetic variability. The findings of this study have wide implications for fisheries in the Indo-Pacific. The increased sampling effort within a narrower geographical scale by the current study permitted a precise locality of the genetic break for this species within the Indo-Pacific Ocean to be identified. The substantial genetic diversity within both lineages should be considered in fishery management and aquaculture development programs of this species in this region.

New Insights on the Evolution of the Sweet Taste Receptor of Primates Adapted to Harsh Environments.

Taste perception is an essential function that provides valuable dietary and sensory information, which is crucial for the survival of animals. Studies into the evolution of the sweet taste receptor gene (TAS1R2) are scarce, especially for Bornean endemic primates such as Nasalis larvatus (proboscis monkey), Pongo pygmaeus (Bornean orangutan), and Hylobates muelleri (Muller's Bornean gibbon). Primates are the perfect taxa to study as they are diverse dietary feeders, comprising specialist folivores, frugivores, gummivores, herbivores, and omnivores. We constructed phylogenetic trees of the TAS1R2 gene for 20 species of anthropoid primates using four different methods (neighbor-joining, maximum parsimony, maximum-likelihood, and Bayesian) and also established the time divergence of the phylogeny. The phylogeny successfully separated the primates into their taxonomic groups as well as by their dietary preferences. Of note, the reviewed time of divergence estimation for the primate speciation pattern in this study was more recent than the previously published estimates. It is believed that this difference may be due to environmental changes, such as food scarcity and climate change, during the late Miocene epoch, which forced primates to change their dietary preferences. These findings provide a starting point for further investigation.

Population Structure of the Blue Swimmer Crab Portunus pelagicus in Coastal Areas of Malaysia Inferred from Microsatellites.

Chuan Jian Chai, Yuzine Bin Esa, Muhammad Fadhil Syukri Ismail, and Mohd. Salleh Kamarudin (2017) Portunus pelagicus, distributed throughout the Indo-West Paci c region, is one of the large and edible species of blue swimmer crabs. Increasing demand for the frozen and canned crabmeat industry worldwide has now relied mainly on P. pelagicus which in turn generates splendid income for the fisherman communities. In the present study, the population genetic structure of P. pelagicus was examined using six pairs of microsatellite loci. A total of 87 crab samples were collected from five different coastal areas of Malaysia. Genomic DNA was extracted from each sample for polymerase chain reaction (PCR) amplification and fragment analysis. Four out of six microsatellite primers revealed polymorphic loci in P. pelagicus sampled. The number of alleles per locus in P. pelagicus ranged from 14 to 34. Microsatellites analyses indicated low levels of genetic differentiation among the P. pelagicus populations. The average observed heterozygosity (HO = 0.48) obtained was lower than the standard heterozygosity found in most marine populations (H O = 0.79). The high F IS values (mean F IS = 0.4756) and low F ST values (mean F ST = 0.0413) also suggested the existence of inbreeding among different populations of P. pelagicus. In conclusion, this study was able to shed light on the population structure of P. pelagicus in coastal areas of Malaysia.

Phylogenetics and population genetics of Plotosus canius (Siluriformes: Plotosidae) from Malaysian coastal waters.

Plotosus canius (Hamilton, 1822) is a significant marine species in Malaysia from nutritional and commercial perspectives. Despite numerous fundamental research on biological characteristics of P. canius, there are various concerns on the level of population differentiation, genomic structure, and the level of genetic variability among their populations due to deficiency of genetic-based studies. Deficiency on basic contexts such as stock identification, phylogenetic relationship and population genetic structure would negatively impact their sustainable conservation. Hence, this study was conducted to characterize the genetic structure of P. canius for the first time through the application of mitochondrial Cytochrome Oxidase I (COI) gene, cross amplification of Tandanus tandanus microsatellites, and a total of 117 collected specimens across five selected populations of Malaysia. The experimental results of the mitochondrial analysis revealed that the haplotype diversity and nucleotide diversity varied from 0.395-0.771 and 0.033-0.65 respectively. Moreover, the statistical analysis of microsatellites addressed a considerable heterozygote insufficiency in all populations, with average observed heterozygosity (Ho ) value of 0.2168, which was lower than the standard heterozygosity in marine populations (Ho = 0.79). This alongside the high Fis values estimation, high pairwise differentiation among populations and low within population variations are supposed to be associated with small sample size, and inbreeding system. Besides, the significant finding of this study was the sharing of common haplotype KR086940, which reflects a historical genetic connectivity between Peninsular Malaysia and Borneo populations due to the geological history of Southeast Asia during Pleistocene era. Demographic analyses showed that all populations were in an equilibrium state with no significant evidence of population expansion. To put it briefly, the current study has managed to provide an initial genomic database toward understanding of the genetic characterization, phylogenetic, molecular diversification and population structure in P. canius, and should be necessary highlighted for appropriate management and conservation of species. Further studies must be carried out involving more geographical and sampling sites, larger population size per site, and utilization of species specific microsatellites loci.

DNA barcoding and phylogenetic analysis of Malaysian groupers (Subfamily: Epinephelinae) using mitochondrial Cytochrome c oxidase I (COI) gene.

The present study was carried out to examine the species identification and phylogenetic relationships of groupers in Malaysia using mitochondrial Cytochrome c Oxidase I (COI) gene, commonly known as barcoding gene. A total of 63 individuals comprising 10 species from three genera were collected from the coastal areas of Johor, Kelantan, Pahang, Perak, Selangor and Terengganu. All the individuals were morphologically identified and molecular works involved polymerase chain reaction (PCR) and sequencing of COI barcoding fragment (655 base pairs). Results from the BLAST search showed that 55 sequences could be assigned to 10 grouper species with high percentage identity index (≥95% to 100%), while eight grouper individuals showed discrepancies in their taxonomic identification based on the morphology and the COI barcoding results. The histogram of distances showed that there was a clear-cut barcode gap present in the sequences indicating a clear separation between intraspecific and interspecific distances. The pairwise genetic distances showed lowest pairwise distance between P. leopardus and P. maculatus (4.4%), while the highest pairwise distance was between E. bleekeri and P. maculatus (23.5%), supporting their morphological and habitat similarities and differences. Phylogenetic analysis (Neighbor-Joining) showed the presence of two major clades (1) genus Epinephelus vs (2) genus Plectropomus and Cephalopholis). In conclusion, the present study has managed to show the accuracy of DNA barcoding method for species identification, and utilization of COI gene for phylogenetic study among groupers. ?

Comparative growth and survival of diploid and triploid Mozambique tilapia (Oreochromism ossambicus) reared in indoor tanks.

In the present study, the effect of cold shock on fertilized eggs of Oreochromis mossambicus was during triploid production. Two males (150 ? 224 g) and six gravid females (150 g to 264 g) were collected. The females were injected with ovulin at a dosage rate of 0.5ml kg(-1), the eggs were stripped into a dry bowl, fertilised with milt, divided into two groups (A and B) and incubated. After three minutes, group A was cold shocked at 50C for a period for 5 min, while group B was incubated normally. Tilapia larvae hatchlings were triplicated and stocked at a density of 200 larvae / liter in the aquaria for four weeks. Results showed that triploid fish had a lower hatchability (58%) and survival rate (47%) than diploid fish (of 85 and 70%, respectively), but a higher specific growth rate of 1.097 as compared to diploid at 0.931. While the improved growth rates have great commercial applications. Further research should be conducted to improve the lower hatchability and survivability of triploid tilapia. ?

Genetic structure and preliminary findings of cryptic diversity of the Malaysian Mahseer (Tor tambroides Valenciennes: Cyprinidae) inferred from mitochondrial DNA and microsatellite analyses.

This study examines the population genetic structure of Tor tambroides, an important freshwater fish species in Malaysia, using fifteen polymorphic microsatellite loci and sequencing of 464 base pairs of the mitochondrial cytochrome c oxidase I (COI) gene. A total of 152 mahseer samples were collected from eight populations throughout the Malaysia river system. Microsatellites results found high levels of intrapopulation variations, but mitochondrial COI results found high levels of interpopulations differentiation. The possible reasons for their discrepancies might be the varying influence of genetic drift on each marker or the small sample sizes used in most of the populations. The Kelantan population showed very low levels of genetic variations using both mitochondrial and microsatellite analyses. Phylogenetic analysis of the COI gene found a unique haplotype (ER8∗), possibly representing a cryptic lineage of T. douronensis, from the Endau-Rompin population. Nevertheless, the inclusion of nuclear microsatellite analyses could not fully resolve the genetic identity of haplotype ER8∗ in the present study. Overall, the findings showed a serious need for more comprehensive and larger scale samplings, especially in remote river systems, in combination with molecular analyses using multiple markers, in order to discover more cryptic lineages or undescribed "genetic species" of mahseer.

Phylogenetic analysis of hampala fishes (subfamily Cyprininae) in Malaysia inferred from partial mitochondrial cytochrome B DNA sequences.

This study examined 396 base pairs of the mitochondrial cytochrome b gene from 110 individuals belonging to the genus Hampala, a group of freshwater cyprinids that inhabit Southeast Asia. The samples were taken from various locations throughout Sarawak, Sabah, and peninsular Malaysia. The nucleotide sequences were subjected to phylogenetic analyses by using the neighbor-joining, maximum parsimony, and maximum likelihood methods. All three methods revealed the reciprocally monophyletic relationship of Hampala macrolepidota to the other Hampala forms, thus strongly supporting its status as a distinct species. Phylogenetic analysis also discovered the existence of two H. bimaculata lineages endemic to Borneo: (1) a newly identified species from the southern and central part of Sarawak assigned as H. bimaculata Type A and (2) the previously described H. bimaculata from northern Sarawak and the west coast of Sabah assigned as H. bimaculata Type B. However, the status of H. sabana and an intermediate form were not elucidated. The results suggest that the intermediate form from the Tawau population is actually a subpopulation of H. sabana, while the highly divergent intermediate form from Kalabakan could represent a cryptic species. The sharing of H. macrolepidota haplotypes in the southern peninsular Malaysia and southern and central Sarawak samples (Hm1 and Hm2) reflected the recent disconnection of the two regions, during the late Pleistocene. Overall, the partial sequence of the mitochondrial cytochrome b gene was useful for resolving the phylogenetic relationships among Hampala fishes in Malaysia.

Mammalian mitogenomic relationships and the root of the eutherian tree.

The strict orthology of mitochondrial (mt) coding sequences has promoted their use in phylogenetic analyses at different levels. Here we present the results of a mitogenomic study (i.e., analysis based on the set of protein-coding genes from complete mt genomes) of 60 mammalian species. This number includes 11 new mt genomes. The sampling comprises all but one of the traditional eutherian orders. The previously unrepresented order Dermoptera (flying lemurs) fell within Primates as the sister group of Anthropoidea, making Primates paraphyletic. This relationship was strongly supported. Lipotyphla ("insectivores") split into three distinct lineages: Erinaceomorpha, Tenrecomorpha, and Soricomorpha. Erinaceomorpha was the basal eutherian lineage. Sirenia (dugong) and Macroscelidea (elephant shrew) fell within the African clade. Pholidota (pangolin) joined the Cetferungulata as the sister group of Carnivora. The analyses identified monophyletic Pinnipedia with Otariidae (sea lions, fur seals) and Odobenidae (walruses) as sister groups to the exclusion of Phocidae (true seals).