A new species of karst-adapted gekkonid lizard of the genus Cnemaspis Strauch is described from Gua Gunting and Gua Goyang in a karst region of Merapoh, Pahang, Peninsular Malaysia whose unique limestone formations are in immediate danger of being quarried. The new species differs from all other species of Cnemaspis based on its unique suite of morphological and color pattern characters. Its discovery underscores the unique biodiversity endemic to karst regions and adds to a growing list of karst-adapted reptiles from Peninsular Malaysia. We posit that new karst-adapted species endemic to limestone forests will continue to be discovered and these regions will harbor a significant percentage of Peninsular Malaysia's biodiversity and thusly should be conserved rather than quarried.
Fasciola gigantica and hybrid Fasciola flukes, responsible for the disease fasciolosis, are found in Southeast Asian countries. In the present study, we performed molecular species identification of Fasciola flukes distributed in Terengganu, Malaysia using multiplex PCR for phosphoenolpyruvate carboxykinase (pepck) and PCR-restriction fragment length polymorphism (RFLP) for DNA polymerase delta (pold). Simultaneously, phylogenetic analysis based on mitochondrial NADH dehydrogenase subunit 1 (nad1) was performed for the first time on Malaysian Fasciola flukes to infer the dispersal direction among neighboring countries. A total of 40 flukes used in this study were identified as F. gigantica. Eight nad1 haplotypes were identified in the F. gigantica population of Terengganu. Median-joining network analysis revealed that the Malaysian population was related to those obtained from bordering countries such as Thailand and Indonesia. However, genetic differentiation was detected using population genetics analyses. Nevertheless, the nucleotide diversity (π) value suggested that F. gigantica with the predominant haplotypes was introduced into Malaysia from Thailand and Indonesia. The dispersal direction suggested by population genetics in the present study may not be fully reliable since Fasciola flukes were collected from a single location in one state of Malaysia. Further studies analyzing more samples from many locations are required to validate the dispersal direction proposed herein.
A multilocus sequence analysis using mitochondria-encoded cytochrome c oxidase subunit I (COI), cytochrome B (CytB), NADH dehydrogenase subunit 5 (ND5); nuclear encoded 18S ribosomal RNA (18S) and 28S ribosomal RNA (28S) genes was performed to determine the levels of genetic variation between the closely related species Haematobia irritans Linnaeus and Haematobia exigua de Meijere. Among these five genes, ND5 and CytB genes were found to be more variable and informative in resolving the interspecific relationships of both species. In contrast, the COI gene was more valuable in inferring the intraspecific relationships. The ribosomal 18S and 28S sequences of H. irritans and H. exigua were highly conserved with limited intra- and inter-specific variation. Molecular evidence presented in this study demonstrated that both flies are genetically distinct and could be differentiated based on sequence analysis of mitochondrial genes.
Sundaland has a dynamic geographic history because its landmasses were periodically interconnected when sea levels fell during glacial periods. Superimposed on this geographic dynamism were environmental changes related to climatic oscillations. To investigate how tropical taxa responded to such changes, we studied the divergence and demographic history of two co-distributed rainforest passerine species, Arachnothera longirostra and Malacocincla malaccensis. We sampled birds primarily from Borneo and the Malay Peninsula, which straddle the now-submerged Sunda shelf, and analysed multilocus DNA data with a variety of coalescent and gene genealogy methods. Cross-shelf divergence in both species occurred well before the last glacial maximum, i.e., before the most recent land connection. However, post-divergence gene flow occurred, and it was more pronounced in A. longirostra (a highly vagile nectarivore/insectivore) than in M. malaccensis (an understory insectivore). Despite current habitat continuity on Borneo, the population of M. malaccensis in northeastern Borneo is substantially divergent from that on the rest of the island. The NE population experienced dramatic demographic fluctuations, probably because of competition with the other population, which expanded from western Borneo after the mid-Pleistocene. In contrast, the Borneo population of A. longirostra has little structure and appears to have experienced demographic expansion 16 kya, long after it had diverged from the Malay Peninsula population (630-690 kya). Malay Peninsula populations of both species have remained relatively stable. Overall, the most recent glacial period was not the chief determinant of the evolutionary dynamics of our study species, and in this respect, they are different from temperate species.
Two female and one male adult hookworms were recovered from a female patient in Thailand. Based on gross and microscopic morphology, the three hookworms are members of Necator americanus. Phylogenetic reconstruction based on partial NADH dehydrogenase subunit 1 (nad1) mitochondrial gene sequences shows that these hookworms belong to the same genetic lineage as N. americanus adult worm from Zhejiang, China. The male and female hookworms were genetically distinct, belonging to two different nad1-haplotypes. This is the first report targeting the nad1 gene on the identification and genetic characterization of the human hookworms originated from infected patient. The nad1 gene marker is useful for species and higher taxa differentiation of hookworms.
The demersal brown banded bamboo shark Chiloscyllium punctatum is a major component of sharks landed in Malaysia. However, little is known about their population structure and the effect of high fishing pressure on these weak swimming sharks. Both mitochondrial DNA control region (1072 bp) and NADH dehydrogenase subunit 2 (1044 bp) were used to elucidate the genetic structure and connectivity of C. punctatum among five major areas within the Sundaland region. Our findings revealed (i) strong genetic structure with little present day mixing between the major areas, (ii) high intra-population genetic diversity with unique haplotypes, (iii) significant correlation between genetic differentiation and geographical distance coupled with detectable presence of fine scale geographical barriers (i.e. the South China Sea), (iv) historical directional gene flow from the east coast of Peninsular Malaysia towards the west coast and Borneo, and (v) no detectable genetic differentiation along the coastline of east Peninsular Malaysia. Genetic patterns inferred from the mitochondrial DNA loci were consistent with the strong coastal shelf association in this species, the presence of contemporary barriers shaped by benthic features, and limited current-driven egg dispersal. Fine scale population structure of C. punctatum highlights the need to improve genetic understanding for fishery management and conservation of other small-sized sharks.
Mitochondrial Subunit ND1 (mtND1) gene is involved in the first step of the electron transport chain of oxidative phosphorylation (OXPHOS). Alteration of the electron transport components by mutations in mtDNA may compromise the normal electron flow. This could lead to an increase of bifurcation and generation of superoxidase radicals and increase oxidative stress in various types of cancer cells. Genomic DNA was extracted from thirty matched primary colorectal tumour tissues and matching non-tumour tissues. Blood samples were obtained from twenty-five normal people. The mtNDI coding region was amplified by step-down PCR. The purified products were then subjected to direct sequencing and subsequently, the DNA sequences obtained were compared with the revised Cambridge Reference Sequence (rCRS) and MITOMAP. From the analysis, the mtND1 gene showed 11 (45.8%) different mutations and also 13 (54.2%) polymorphisms. The heteroplasmic mutation A4123A/G (I273I/V) might have a pathogenic significance as it fulfills various pathogenic criteria. Three mutations, T3394C (Y30H), A3434G (Y43C) and C3497T (A64V) which occur in a highly conserved region were likely to alter the structure and function of the ND1 protein. We suggest that these mutations, and in combination with the polymorphic variance in mtDNA, may cause slight changes that generate subtly higher levels of toxic reactive oxygen species (ROS).
The male of Phlebotomus (Larroussius) betisi is described from Malayan caves. Several males have been caught in association with P. betisi females. Males and females have been associated by ecology, biogeography, morphology and molecular biology (homology of the ND4 mtDNA sequences).
The sequences of the mitochondrial ND4 gene (1339 bp) and the ND4L gene (290 bp) were determined for all the 14 extant taxa of the Drosophila nasuta subgroup. The average A + T content of ND4 genes is 76.5% and that of ND4L genes is 83.5%. A total of 114 variable sites were scored. The ND4 gene sequence divergence ranged from 0 to 5.4% within the subgroup. The substitution rate of the ND4 gene is about 1.25% per million years. The base substitution of the genes is strongly transition biased. Neighbor-joining and parsimony were used to construct a phylogeny based on the resultant sequence data set. According to these trees, five distinct mtDNA clades can be identified. D. niveifrons represents the most diverged lineage. D. sulfurigaster bilimbata and D. kepulauana form two independent lineages. The other two clades are the kohkoa complex and the albomicans complex. The kohkoa complex consists of D. sulfurigaster sulfurigaster, D. pulaua, D. kohkoa, and Taxon-F. The albomicans complex can be divided into two groups: D. nasuta, D. sulfurigaster neonasuta, D. sulfurigaster albostrigata, and D. albomicans from Chiangmai form one group; and D. pallidifrons, Taxon-I, Taxon-J, and D. albomicans from China form the other group. High genetic differentiation was found among D. albomicans populations. Based on our phylogenetic results, we hypothesize that D. niveifrons diverged first from the D. nasuta subgroup in Papua New Guinea about 3.5 Mya. The ancestral population spread to the north and when it reached Borneo, it diversified sequentially into the kohkoa complex, D. s. bilimbata, and D. kepulauana. About 1 Mya, another radiation occurred when the ancestral populations reached the Indo-China Peninsula, forming the albomicans complex. Discrepancy between morphological groupings and phylogenetic results suggests that the male morphological traits may not be orthologous.
Food fraud is a global problem raising increased concerns during the past decades and food authenticity is now a burning issue. Beef, buffalo, chicken, duck, goat, sheep, and pork are heavily consumed meats bearing nutritional, economic and cultural/religious importance and are often found to be adulterated in raw and processed states. To authenticate these species, we developed and validated a highly specific multiplex (heptaplex) PCR assay targeting short length amplicons (73-263 bp) using seven pairs of species-specific primer sets targeting mitochondrial cytochrome b (cytb) and NADH dehydrogenase subunit 5 (ND5) genes. Specificity checking (in silico and in vitro) against 25 non-target species revealed no cross-species amplification. The developed multiplex assay was validated with various adulterated and heat-treated (boiled, microwaved and autoclaved) meatball products and were found to show high sensitivity and stability under all processing conditions. The assay was sensitive enough to detect 0.01-0.005 ng of DNA from raw meat and 0.5% (w/w) adulterated meat in mixed matrices. A market survey revealed mislabelling of 95% beef and 15% chicken products while pork products were found pure. Given some advantageous features including short sizes of amplicons, exceptional stability and superior sensitivity, the developed assay could be conveniently used for discriminatory detection of target species with a variety of raw meat as well as processed meat products undergoing extreme processing treatments.
To examine the phylogeographical pattern of Tetrancistrum nebulosi (Monogenea, Dactylogyridae) in the South China Sea, fragments of mitochondrial cytochrome c oxidase subunit I and NADH dehydrogenase subunit 2 genes were obtained for 220 individuals collected from 8 localities along the southeast coast of China and 1 locality in Terengganu, Malaysia. Based on these two genes, two and three distinct clades with geographic signals were revealed on the phylogenetic trees respectively. The divergence between these clades was estimated to occur in the late Pleistocene. Analysis of molecular variance and pairwise FSTsuggested a high rate of gene flow among individuals sampled from the Chinese coast, but with obvious genetic differentiation from the Malaysian population. Mismatch distribution and neutrality tests indicated that the T. nebulosi population experienced expansion in Pleistocene low sea level periods. Vicariance was considered to account for the genetic divergence between Chinese and Malaysian populations, while sea level fluctuations and mainland-island connections during glacial cycles were associated with the slight genetic divergence between the populations along the mainland coast of China and those off Sanya. On the contrary, oceanographic circulations and host migration could lead to genetic homogeneity of populations distributed along the mainland coast of China.
A combination of a modified Feret' (Silvae Genet. 1971, 20, 46-50) extraction buffer and two types of electrophoresis with acrylamide and starch gels were used to characterize allozymes in mature vegetative tissue of a commercially high value species of rattans (Calamus subinermis). From the analysis of allelic segregation from single maternal rattans and their offspring, genetic control of the 16 observed banding zones, which were consistently scorable, was assumed. Seventeen gene loci were identified. The percentage of polymorphic loci within Calamus subinermis was much higher (70.5%) than expected levels of genetic diversity for tropical woody and non-woody species. It is thought that the protocol described may be applied to the analysis of the genetic diversity of all the endangered Calamus species.