A total of 30 specimens belonging to five species, namely; Cryptozona siamensis, Sarika resplendens and Sarika sp. from the family Ariophantidae as well as Quantula striata and Quantula sp. from the family Dyakiidae were collected from the Langkawi Island in Northern Peninsular Malaysia. All specimens were identified through comparisons of shell morphology and amplification of a 500 bp segment of the 16S rRNA mtDNA gene. To assess phylogenetic insights, the sequences were aligned using ClustalW and phylogenetic trees were constructed. The analyses showed two major lineages in both Maximum Parsimony and Neighbour Joining phylogenetic trees. Each putative taxonomic group formed a monophyletic cluster. Our study revealed low species and intraspecies genetic diversities based on the 16S rRNA gene sequences. Thus, this study has provided an insight of land snail diversity in populations of an island highly influenced by anthropogenic activities through complementary use of shell morphological and molecular data.
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.
A range of small- to moderate-scale studies of patterns in bacterial biodiversity have been conducted in Antarctica over the last two decades, most suggesting strong correlations between the described bacterial communities and elements of local environmental heterogeneity. However, very few of these studies have advanced interpretations in terms of spatially associated patterns, despite increasing evidence of patterns in bacterial biogeography globally. This is likely to be a consequence of restricted sampling coverage, with most studies to date focusing only on a few localities within a specific Antarctic region. Clearly, there is now a need for synthesis over a much larger spatial to consolidate the available data. In this study, we collated Antarctic bacterial culture identities based on the 16S rRNA gene information available in the literature and the GenBank database (n > 2,000 sequences). In contrast to some recent evidence for a distinct Antarctic microbiome, our phylogenetic comparisons show that a majority (~75 %) of Antarctic bacterial isolates were highly similar (≥99 % sequence similarity) to those retrieved from tropical and temperate regions, suggesting widespread distribution of eurythermal mesophiles in Antarctic environments. However, across different Antarctic regions, the dominant bacterial genera exhibit some spatially distinct diversity patterns analogous to those recently proposed for Antarctic terrestrial macroorganisms. Taken together, our results highlight the threat of cross-regional homogenisation in Antarctic biodiversity, and the imperative to include microbiota within the framework of biosecurity measures for Antarctica.
The blood clam, Tegillarca granosa, is widely cultivated in China. We isolated 6 microsatellite loci from T. granosa and used them to investigate genetic diversity and population structure of 5 widely distributed populations of blood clam collected from eastern and southeastern China. The allele number per locus varied from 4 to 9, and the polymorphism information content value was 0.301 to 0.830. The mean observed and expected heterozygosities varied from 0.304 to 0.460 and 0.556 to 0.621, respectively; the population from Yueqing had the smallest observed heterozygosity. In the neighbor-joining tree, Shandong, Fenghua and Yueqing populations clustered together, and there was geographic divergence between Shandong and Guangxi populations. Some microsatellite loci that were isolated from these mainland China samples were not found in blood clams collected from Malaysia.
A sum of 48 accessions of physic nut, Jatropha curcas L. were analyzed to determine the genetic diversity and association between geographical origin using RAPD-PCR markers. Eight primers generated a total of 92 fragments with an average of 11.5 amplicons per primer. Polymorphism percentages of J. curcas accessions for Selangor, Kelantan, and Terengganu states were 80.4, 50.0, and 58.7%, respectively, with an average of 63.04%. Jaccard's genetic similarity co-efficient indicated the high level of genetic variation among the accessions which ranged between 0.06 and 0.81. According to UPGMA dendrogram, 48 J. curcas accessions were grouped into four major clusters at coefficient level 0.3 and accessions from same and near states or regions were found to be grouped together according to their geographical origin. Coefficient of genetic differentiation (G(st)) value of J. curcas revealed that it is an outcrossing species.
All honey bee species (Apis spp) share the same sex determination mechanism using the complementary sex determination (csd) gene. Only individuals heterogeneous at the csd allele develop into females, and the homozygous develop into diploid males, which do not survive. The honeybees are therefore under selection pressure to generate new csd alleles. Previous studies have shown that the csd gene is under balancing selection. We hypothesize that due to the long separation from the mainland of Hainan Island, China, that the giant honey bees (Apis dorsata) should show a founder effect for the csd gene, with many different alleles clustered together, and these would be absent on the mainland.
The genus Curcuma is a member of the ginger family (Zingiberaceae) that has recently become popular for use as flowering pot plants, both indoors and as patio and landscape plants. We used PCR-based molecular markers (ISSRs) to assess genetic variation and relationships between five varieties of curcuma (Curcuma alismatifolia) cultivated in Malaysia. Sixteen ISSR primers generated 139 amplified fragments, of which 77% had high polymorphism among these varieties. These markers were used to estimate genetic similarity among the varieties using Jaccard's similarity coefficient. The similarity matrix was used to construct a dendrogram, and a principal component plot was developed to examine genetic relationships among varieties. Similarity coefficient values ranged from 0.40 to 0.58 (with a mean of 0.5) among the five varieties. The mean value of number of observed alleles, number of effective alleles, mean Nei's gene diversity, and Shannon's information index were 8.69, 1.48, 0.29, and 0.43, respectively.
The fruit fly Bactrocera caudata is a pest species of economic importance in Asia. Its larvae feed on the flowers of Cucurbitaceae such as Cucurbita moschata. To-date it is distinguished from related species based on morphological characters. Specimens of B. caudata from Peninsular Malaysia and Indonesia (Bali and Lombok) were analysed using the partial DNA sequences of cytochrome c oxidase subunit I (COI) and 16S rRNA genes. Both gene sequences revealed that B. caudata from Peninsular Malaysia was distinctly different from B. caudata of Bali and Lombok, without common haplotype between them. Phylogenetic analysis revealed two distinct clades, indicating distinct genetic lineage. The uncorrected 'p' distance for COI sequences between B. caudata of Malaysia-Thailand-China and B. caudata of Bali-Lombok was 5.65%, for 16S sequences from 2.76 to 2.99%, and for combined COI and 16S sequences 4.45 to 4.46%. The 'p' values are distinctly different from intraspecific 'p' distance (0-0.23%). Both the B. caudata lineages are distinctly separated from related species in the subgenus Zeugodacus - B. ascita, B. scutellata, B. ishigakiensis, B. diaphora, B. tau, B. cucurbitae, and B. depressa. Molecular phylogenetic analysis indicates that the B. caudata lineages are closely related to B. ascita sp. B, and form a clade with B. scutellata, B. ishigakiensis, B. diaphora and B. ascita sp. A. This study provides additional baseline for the phylogenetic relationships of Bactrocera fruit flies of the subgenus Zeugodacus. Both the COI and 16S genes could be useful markers for the molecular differentiation and phylogenetic analysis of tephritid fruit flies.
Mackerel (Scombridae; Rastrelliger) are small commercially important pelagic fish found in tropical regions. They serve as a cheap source of animal protein and are commonly used as live bait. By using a truss morphometrics protocol and RAPD analysis, we examined morphological and genetic variation among 77 individual mackerel that were caught using long lines and gillnets at 11 locations along the west coast of Peninsular Malaysia. Nineteen morphometric traits were evaluated and genetic information was estimated using five 10-base RAPD random primers. Total DNA was extracted from muscle tissue. Morphometric discriminant function analysis revealed that two morphologically distinct groups of Rastrelliger kanagurta and a single group of R. brachysoma can be found along the west coast of Peninsular Malaysia. We also found that the head-related characters and those from the anterior part of the body of Rastrelliger spp significantly contribute to stock assessment of this population. RAPD analysis showed a trend similar to that of the morphometric analysis, suggesting a genetic component to the observed phenotypic differentiation. These data will be useful for developing conservation strategies for these species.
Although the crab-eating frog Fejervarya cancrivora is one of the most widely distributed species in Asian region, taxonomic relationships among different populations remain unclarified. In this study, we attempted to elucidate the taxonomic status of F. cancrivora from Indonesian and other Asian populations. Five populations of F. cancrivora from Selangor (Malaysia), Cianjur (Java, Indonesia), Trat (Thailand), Khulna (Bangladesh), and Makassar (Sulawesi, Indonesia) were morphologically observed and subjected to crossing experiments. Principal component and clustering analyses revealed that these five populations could be organized into three groups corresponding to three observed morphological types: a Selangor and Cianjur group (large-type), a Trat and Khulna group (mangrove-type), and a Makassar group (Sulawesi-type). The limited crossing experiments revealed that hybrids between Selangor females and Cianjur and Trat males developed normally, whereas hybrids between Selangor females and Khulna males showed incomplete gametic isolation. Histological observations of the testes of mature males revealed the presence of pycnotic nuclei in the hybrids between Selangor females and Khulna males in addition to normal bundles of spermatozoa. In contrast, no pycnotic nuclei were observed in the Selangor controls. Although meiotic metaphases in the controls were normal, those in hybrids showed several abnormalities, such as the appearance of univalents and an increase in rod-shaped bivalents. Based on our findings from the morphological observations and crossing experiments, we conclude that each of three identified types represents a distinct species. We propose that the large-type is F. cancrivora, the mangrove-type is F. moodiei, and the Sulawesi-type represents an undescribed species.
Isozyme and protein electrophoresis data from mycelial extracts of 27 isolates of Trichoderma harzianum, 10 isolates of T. aureoviride and 10 isolates of T. longibrachiatum from Southern Peninsular Malaysia were investigated. The eight enzyme and a single protein pattern systems were analyzed. Three isozyme and total protein patterns were shown to be useful for the detection of three Trichoderma species. The isozyme and protein data were analyzed using the Nei and Li Dice similarity coefficient for pairwise comparison between individual isolates, species isolate group, and for generating a distance matrix. The UPGMA cluster analysis showed a higher degree of relationship between T. harzianum and T. aureoviride than to T. longibrachiatum. These results suggested that the T. harzianum isolates had high levels of genetic variation compared to the other isolates of Trichoderma species.
Mitochondrial DNA cytochrome c oxidase II (COII) gene sequences of Malaysian Cercopithecidae were examined to ascertain their phylogenetic relationships. Colobinae were represented by the genera Presbytis, Trachypithecus and Nasalis, while the genus Macaca represented Cercopithecinae. DNA amplification and sequencing of the COII gene was performed on 16 samples. Symphalangus syndactylus (Hylobatidae) was used as the outgroup. Data were analyzed using both character (maximum parsimony) and distance (neighbor-joining) methods. Tree topologies indicated that Colobinae and Cercopithecinae have their own distinct monophyletic clade. This result was well supported by bootstrap values and genetic distances derived from the Kimura-2-parameter algorithm. Separation of Macaca nemestrina from M. fascicularis was also well supported by bootstrap values. In addition, tree topologies indicate a good resolution of the Colobinae phylogenetic relationships at the intergeneric level, but with low bootstrap support. The position of Nasalis remained problematic in both trees. Overall, COII is a good gene candidate for portraying the phylogenetic relationships of Malaysian primates at the inter- and intra-subfamily levels.
Insect larvae and adult insects found on human corpses provide important clues for the estimation of the postmortem interval (PMI). Among all necrophagous insects, flesh flies (Diptera: Sarcophagidae) are considered as carrion flies of forensic importance. DNA variations of 17 Malaysian, two Indonesian and one Japanese flesh fly species are analysed using the mitochondrial COI and COII. These two DNA regions were useful for identifying most species experimented. However, characterisation of the species was not sufficiently made in the case of Sarcophaga javanica. Seventeen Malaysian species of forensic importance were successfully clustered into distinct clades and grouped into the six species groups: peregrina, albiceps, dux, pattoni, princeps and ruficornis. These groups correspond with generic or subgeneric taxa of the subfamily Sarcophaginae: Boettcherisca, Parasarcophaga, Liosarcophaga, Sarcorohdendorfia-Lioproctia, Harpagophalla-Seniorwhitea and Liopygia. The genetic variations found in COI and COII can be applied not only to identify the species of forensic importance, but also to understand the taxonomic positions, generic or subgeneric status, of the sarcophagine species.
Little is known about the classification and phylogenetic relationships of the leaf monkeys (Presbytis). We analyzed mitochondrial DNA sequences of cytochrome b (Cyt b) and 12S rRNA to determine the phylogenetic relationships of the genus Presbytis. Gene fragments of 388 and 371 bp of Cyt b and 12S rRNA, respectively, were sequenced from samples of Presbytis melalophos (subspecies femoralis, siamensis, robinsoni, and chrysomelas), P. rubicunda and P. hosei. The genus Trachypithecus (Cercopithecidae) was used as an outgroup. The Cyt b NJ and MP phylogeny trees showed P. m. chrysomelas to be the most primitive, followed by P. hosei, whereas 12S rRNA tree topology only indicated that these two species have close relationships with the other members of the genus. In our analysis, chrysomelas, previously classified as a subspecies of P. melalophos, was not included in either the P. m. femoralis clade or the P. m. siamensis clade. Whether or not there should be a separation at the species level remains to be clarified. The tree topologies also showed that P. m. siamensis is paraphyletic with P. m. robinsoni, and P. m. femoralis with P. rubicunda, in two different clades. Cyt b and 12S rRNA are good gene candidates for the study of phylogenetic relationships at the species level. However, the systematic relationships of some subspecies in this genus remain unclear.
Erwinia mallotivora was isolated from papaya infected with dieback disease showing the typical symptoms of greasy, water-soaked lesions and spots on leaves. Phylogenetic analysis of 16S rRNA gene sequences showed that the strain belonged to the genus Erwinia and was united in a monophyletic group with E. mallotivora DSM 4565 (AJ233414). Earlier studies had indicated that the causal agent for this disease was E. papayae. However, our current studies, through Koch's postulate, have confirmed that papaya dieback disease is caused by E. mallotivora. To our knowledge, this is the first new discovery of E. mallotivora as a causal agent of papaya dieback disease in Peninsular Malaysia. Previous reports have suggested that E. mallotivora causes leaf spot in Mallotus japonicus. However, this research confirms it also to be pathogenic to Carica papaya.
In this work we report on the isolation of a local molybdenum-reducing bacterium. The bacterium reduced molybdate or Mo(6+) to molybdenum blue (oxidation states between 5+ to 6+). Electron donors that supported cellular growth were sucrose, maltose, mannitol, fructose, glucose and starch (in decreasing order) with sucrose supporting formation of the highest amount of molybdenum blue at 10 g/l after 24 hours of static incubation. The optimum molybdate and phosphate concentrations that supported molybdate reduction were 20 and 5 mM, respectively. Molybdate reduction was optimal at 37 degrees C. The molybdenum blue produced from cellular reduction exhibited a unique absorption spectrum with a maximum peak at 865 nm and a shoulder at 700 nm. The isolate was tentatively identified as S. marcescens strain Dr.Y9 based on carbon utilization profiles using Biolog GN plates and partial 16S rDNA molecular phylogeny. No inhibition of molybdenum-reducing activity was seen using electron transport system (ETS) inhibitors such as antimycin A, 1HQNO (Hydroxyquinoline-N-Oxide), sodium azide and cyanide suggesting that the ETS of this bacterium is not the site of molybdate reduction.
To elucidate genetic divergence and evolutionary relationship in Fejervarya cancrivora from Indonesia and other Asian countries, allozyme and molecular analyses were carried out using 131 frogs collected from 24 populations in Indonesia, Thailand, Bangladesh, Malaysia, and the Philippines. In the allozymic survey, seventeen enzymatic loci were examined for 92 frogs from eight representative localities. The results showed that F. cancrivora is subdivided into two main groups, the mangrove type and the large- plus Pelabuhan ratu types. The average Nel's genetic distance between the two groups was 0.535. Molecular phylogenetic trees based on nucleotide sequences of the 16S rRNA and Cyt b genes and constructed with the ML, MP, NJ, and BI methods also showed that the individuals of F. cancrivora analyzed comprised two clades, the mangrove type and the large plus Pelabuhan ratu / Sulawesi types, the latter further split into two subclades, the large type and the Pelabuhan ratu / Sulawesi type. The geographical distribution of individuals of the three F. cancrivora types was examined. Ten Individuals from Bangladesh, Thailand, and the Philippines represented the mangrove type; 34 Individuals from Malaysia and Indonesia represented the large type; and 11 individuals from Indonesia represented the Pelabuhan ratu / Sulawesi type. Average sequence divergences among the three types were 5.78-10.22% for the 16S and 12.88-16.38% for Cyt b. Our results suggest that each of the three types can be regarded as a distinct species.
Chikungunya fever swept across many South and South-east Asian countries, following extensive outbreaks in the Indian Ocean Islands in 2005. However, molecular epidemiological data to explain the recent spread and evolution of Chikungunya virus (CHIKV) in the Asian region are still limited. This study describes the genetic Characteristics and evolutionary relationships of CHIKV strains that emerged in Sri Lanka and Singapore during 2006-2008. The viruses isolated in Singapore also included those imported from the Maldives (n=1), India (n=2) and Malaysia (n=31). All analysed strains belonged to the East, Central and South African (ECSA) lineage and were evolutionarily more related to Indian than to Indian Ocean Islands strains. Unique genetic characteristics revealed five genetically distinct subpopulations of CHIKV in Sri Lanka and Singapore, which were likely to have emerged through multiple, independent introductions. The evolutionary network based on E1 gene sequences indicated the acquisition of an alanine to valine 226 substitution (E1-A226V) by virus strains of the Indian sublineage as a key evolutionary event that contributed to the transmission and spatial distribution of CHIKV in the region. The E1-A226V substitution was found in 95.7 % (133/139) of analysed isolates in 2008, highlighting the widespread establishment of mutated CHIKV strains in Sri Lanka, Singapore and Malaysia. As the E1-A226V substitution is known to enhance the transmissibility of CHIKV by Aedes albopictus mosquitoes, this observation has important implications for the design of vector control strategies to fight the virus in regions at risk of chikungunya fever.
Unusual tumour-like pathologies caused by mysterious cells termed 'X-cells' have been reported from numerous fish groups worldwide. After nearly 100 years of research, the tumour-like growths have recently been shown to be caused by a protozoan parasite. In the present study, histopathology and small subunit ribosomal DNA (SSU rDNA) sequences are used to assess whether the X-cell parasite infecting Atlantic dab Limanda limanda L. is distinct from the X-cell parasite infecting Japanese flounder and goby, and to determine their systematic position within the protists. SSU rDNA from Scottish dab was 89.3% and 86.7% similar to Japanese X-cell sequences from flounder and goby respectively, indicating that the parasite infecting dab in the Atlantic is distinct from the Pacific species. Histological studies revealed significant gill pathology and demonstrated the precise location of the parasites within the gill tissues using specific in situ hybridization probes. Phylogenetic analyses showed that the X-cell parasites from Scotland and Japan form a monophyletic group within the Myzozoa, and are basal alveolates. However, ultrastructure of X-cells from dab fails to confirm this systematic placement.
Hydnangium echinulatum, described originally from a single specimen collected in Malaysia, has been recollected, and based on morphological and molecular characters is recognized as representing a new gasteroid genus of boletes with affinities to the Boletineae, herein named Durianella. Diagnostic features include an epigeous, ovoid, pyramidal-warted, durian fruit-like basidiome with gelatinized glebal locules and a columella that turns indigo blue upon exposure, and subglobose basidiospores with long, curved, thin-walled and collapsible spines. A redescription, phylogenetic analysis and comparison with allied taxa are presented.