Information on the diversity and distribution of harmful microalgae in the Gulf of Thailand is very limited and mainly based on microscopic observations. Here, we collected 44 water samples from the Gulf of Thailand and its adjacent water (Perhentian Island, Malaysia) for comparison in 2018. DNA metabarcoding was performed targeting the partial large subunit ribosomal RNA gene (LSU rDNA D1-D3) and the internal transcribed spacers (ITS1 and ITS2). A total of 50 dinoflagellate genera (made up of 72 species) were identified based on the LSU rDNA dataset, while the results of ITS1 and ITS2 datasets revealed 33 and 32 dinoflagellate genera comprising 69 and 64 species, respectively. Five potentially toxic Pseudo-nitzschia (Bacillariophyceae) species were detected, with four as newly recorded species in the water (Pseudo-nitzschia americana/brasilliana, Pseudo-nitzschia simulans/delicatissima, P. galaxiae and P. multistriata). The highest relative abundances of P. galaxiae and P. multistriata were found in Trat Bay and Chumphon (accounting for 0.20% and 0.06% of total ASVs abundance, respectively). Three paralytic shellfish toxin producing dinoflagellate species were detected: Alexandrium tamiyavanichii, Alexandrium fragae, and Gymnodinium catenatum. The highest abundance of A. tamiyavanichii was found in the surface sample of Chumphon (CHO7 station), accounting for 1.95% of total ASVs abundance. Two azaspiracid producing dinoflagellate species, Azadinium poporum ribotype B, Azadinium spinosum ribotype A, and a pinnatoxin producing dinoflagellate species Vulcanodinium rugosum, with two ribotypes B and C, were revealed from the datasets although with very low abundances. Six fish killing dinoflagellate species, including Margalefidinium polykrikoides group IV, Margalefidinium fulvescens, Karenia mikimotoi, Karenia selliformis ribotype B, Karlodinium australe, and Karlodinium digitatum were detected and all representing new records in this area. The findings of numerous harmful microalgal species in the Gulf of Thailand highlight the potential risk of human intoxication and fish killing events.
Members of the genus Aglaia have been reported to contain bioactive phytochemicals. The genus, belonging to the Meliaceae family, is represented by at least 120 known species of woody trees or shrubs in the tropical rain forest. As some of these species are very similar in their morphology, taxonomic identification can be difficult. A reliable and definitive molecular method which can identify Aglaia to the level of the species will hence be useful in comparing the content of specific bioactive compounds between the species of this genus. Here, we report the analysis of DNA sequences in the internal transcribed spacer (ITS) of the nuclear ribosomal DNA and the observation of a unique nucleotide signature in the ITS that can be used for the identification of Aglaia stellatopilosa. The nucleotide signature consists of nine bases over the length of the ITS sequence (654 bp). This uniqueness was validated in 37 samples identified as Aglaia stellatopilosa by an expert taxonomist, whereas the nucleotide signature was lacking in a selection of other Aglaia species and non-Aglaia genera. This finding suggests that molecular typing could be utilized in the identification of Aglaia stellatopilosa.
A study on the morphology and phylogeny of 18 strains of Pseudo-nitzschia established from the Strait of Malacca, Peninsular Malaysia, was undertaken. Morphological data combined with molecular evidence show that they constitute three new species, for which the names, P. batesiana sp. nov., P. lundholmiae sp. nov., and P. fukuyoi sp. nov., are proposed. The three new species closely resemble species in the P. pseudodelicatissima complex sensu lato. Morphologically, P. batesiana differs from other species in the complex by having a smaller part of cell overlapping in the chain, whereas P. lundholmiae differs by having fewer poroid sectors and P. fukuyoi by having a distinct type of poroid sectors. Nucleotide sequences of the LSU rDNA (D1-D3) of the three new species reveal significant nucleotide sequence divergence (0.1%-9.3%) from each other and from other species in the P. pseudodelicatissima complex s.l. The three species are phylogenetically closely related to species in the P. pseudodelicatissima complex, with P. batesiana appearing as a sister taxon to P. circumpora, P. caciantha, and P. subpacifica; whereas P. lundholmiae and P. fukuyoi are more closely related to P. pseudodelicatissima and P. cuspidata. The three species show 2-3 compensatory base changes (CBCs) in their ITS2 transcripts when compared to the closely related species. The ITS2 with its structural information has proven its robustness in constructing a better resolved phylogenetic framework for Pseudo-nitzschia.
Three new species and one new variety of bioluminescent Mycena collected from Peninsular Malaysia are described herein. All new species belong to Mycena sect. Calodontes in what is known as the Mycena pura complex. Comprehensive descriptions, photographs, illustrations and comparisons with phenetically similar species are provided. Molecular sequences data from the nuclear internal transcribed spacers (ITS-1 and ITS-2, including the 5.8S rRNA) were used to infer relationships within sect. Calodontes. Axenic cultures were obtained to provide data on culture morphology. This is the first published photographic documentation of bioluminescent basidiomes of members of Mycena sect. Calodontes. Also, this addition brings the total known bioluminescent fungi to 77 species.
Genetic hybridization, sequence and karyotypic analyses of natural Saccharomyces yeasts isolated in different regions of Taiwan revealed three biological species: Saccharomyces arboricola, Saccharomyces cerevisiae and Saccharomyces kudriavzevii. Intraspecies variability of the D1/D2 and ITS1 rDNA sequences was detected among S. cerevisiae and S. kudriavzevii isolates. According to molecular and genetic analyses, the cosmopolitan species S. cerevisiae and S. kudriavzevii contain local divergent populations in Taiwan, Malaysia and Japan. Six of the seven known Saccharomyces species are documented in East Asia: S. arboricola, S. bayanus, S. cerevisiae, S. kudriavzevii, S. mikatae, and S. paradoxus.
The biodiversity and the killer activity of yeasts isolated from various types of fermented food in Malaysia were investigated in this study. Of 252 yeasts isolated from 48 fermented food samples in this study, 19 yeast species were identified based on sequence analysis of the ITS1-5.8S-ITS2 partial fragments of the yeasts. A total of 29 (11.5%) of the yeast isolates demonstrated killer activity to at least one Candida species tested in this study; including 22 isolates of Trichosporon asahii, 4 isolates of Pichia anomala, and one isolate each of Pichia norvegensis, Pichia fermentans and Issatchenkia orientalis, respectively. The presence of killer yeasts reflects antagonism that occurs during microbial interaction in the fermented food, whereby certain yeasts produce killer toxins and possibly other toxic substances in competition for limited nutrients and space. The anti-Candida activity demonstrated by killer yeasts in this study should be further explored for development of alternative therapy against candidiasis.
This study describes the killer phenotypes of tropical environmental yeasts and the inhibition effects of the culture filtrates on the biofilm of Candida albicans. A total of 26 (10.5%) of 258 yeast isolates obtained from an environmental sampling study demonstrated killer activity to Candida species. The killer yeasts were identified as species belonging to the genus Aureobasidium, Pseudozyma, Ustilago and Candida based on sequence analysis of the ITS1-5.8S-ITS2 region of the yeasts. Pseudozyma showed the broadest killing effects against sensitive strains of Candida. New species of Ustilago and Pseudozyma demonstrating killer phenotypes were identified in this study. Interestingly, more than 50% reduction in the metabolic activity of Candida albicans biofilm was noted after exposure to the culture filtrates of the nine killer yeasts. Purification and characterization of toxin and metabolites are essential for understanding the yeast killing effects.
Molecular techniques are invaluable for investigation on the biodiversity of Anopheles mosquitoes. This study aimed at investigating the spatial-genetic variations among Anopheles mosquitoes from different areas of Peninsular Malaysia, as well as deciphering evolutionary relationships of the local Anopheles mosquitoes with the mosquitoes from neighbouring countries using the anopheline ITS2 rDNA gene.
The opportunistic pathogen Exophiala dermatitidis has been frequently isolated from tropical regions of the world. However, there is no report of environmental isolation of Exophiala spp. from Malaysia. The information regarding the ecology of this microbe is important for a better understanding of the opportunism. This study aims to conduct a survey of natural distribution of Exophiala spp. in Malaysia. Forty-seven strains of Exophiala-like was isolated by using selective media. These isolates from the fields were molecularly identified based on the ITS region. The biochemical activity of these microbes was tested by conducting various tests, i.e. DNase test, proteinase activity, and urea hydrolysis. Overall, 22 strains of E. dermatitidis were successfully obtained and identified from burnt tree bark, oil dripped soil sample, hot spring biofilm, railway track stones, tar road contaminated with petrol hydrocarbon, drain and deep mud of Sungai Pinang besides the new discovery from pigeon droppings. A single strain of E. heteromorpha was identified from tar road contaminated with petrol hydrocarbon. Genotypes of the isolated E. dermatitidis were identified by the neighbor-joining tree and grouped into Genotype A, A2 and B. The existence of new Genotype A4 was confirmed by a similar cladogram position in both neighbor-joining and maximum likelihood tree. The survival of E. dermatitidis in the hydrocarbon contaminated environment was studied by supplying engine oil and observing the growth pattern. The results of this study suggest that the opportunistic Exophiala spp. was isolated from nutrient limited and harsh conditions in the natural environment.
The genus Halophila shows the highest species diversity within the seagrass genera. Southeast Asian countries where several boundary lines exist were considered as the origin of seagrasses. We hypothesize that the boundary lines, such as Wallace's and Lydekker's Lines, may act as marine geographic barriers to the population structure of Halophila major. Seagrass samples were collected at three islands in Vietnamese waters and analyzed by the molecular maker ITS. These sequences were compared with published ITS sequences from seagrasses collected in the whole region of interest. In this study, we reveal the haplotype and nucleotide diversity, linking population genetics, phylogeography, phylogenetics and estimation of relative divergence times of H. major and other members of the Halophila genus. The morphological characters show variation. The results of the ITS marker analysis reveal smaller groups of H. major from Myanmar, Shoalwater Bay (Australia) and Okinawa (Japan) with high supporting values. The remaining groups including Sri Lanka, Viet Nam, the Philippines, Thailand, Malaysia, Indonesia, Two Peoples Bay (Australia) and Tokushima (Japan) showed low supporting values. The Wallacea region shows the highest haplotype and also nucleotide diversity. Non-significant differences were found among regions, but significant differences were presented among populations. The relative divergence times between some members of section Halophila were estimated 2.15-6.64 Mya.
Amphidoma languida, a marine thecate dinoflagellate that produces the lipophilic toxin azaspiracids (AZAs), is primarily found in the Atlantic. Although this species has not been recorded in the Asian Pacific, environmental DNAs related to Am. languida have been widely detected in the region by metabarcoding analysis. Their morphology and AZA production remain unclear. In this study, the morphology, ultrastructure, phylogeny, and AZA production of nine Amphidoma strains isolated from Japan, Malaysia, and Philippines were investigated. Phylogenetic trees inferred from rDNAs (SSU, ITS, and LSU rDNA) showed monophyly of the nine Pacific strains and were sister to the Am. languida clade, including the toxigenic strains from the Atlantic. Cells were ellipsoid, 8.7-16.7 µm in length and 7.4-14.0 µm in width, with a conspicuous apical pore complex. A large nucleus in the hyposome, parietal chloroplast with a spherical pyrenoid in the episome, and refractile bodies were observed. Thecal tabulation was typical of Amphidoma, Po, cp, X, 6', 6'', 6C, 5S, 6''', 2''''. A ventral pore was located on the anterior of 1' plate, beside the suture to 6' plate. The presence of a ventral depression, on the anterior of anterior sulcal plate, was different from Am. languida. A large antapical pore, containing approximately 10 small pores, was observed. Cells were apparently smaller than Am. trioculata, a species possessing three pores (ventral pore, ventral depression, and antapical pore). TEM showed the presence of crystalline structures, resembling guanine crystals, and cytoplasmic invaginations into the pyrenoid matrix. Flagellar apparatus lacking the striated root connective is similar to peridinioids and related dinoflagellates. AZAs were not detected from the Pacific strains by LC-MS/MS. This non-toxigenic Amphidoma species, here we propose as Amphidoma fulgens sp. nov., is widely distributed in the Asian Pacific. Moreover, molecular comparison also suggested that most of the environmental DNA sequences previously reported as Am. languida or related sequences from the Asian Pacific were attributable to Am. fulgens.
The mango (Mangifer indica L.) is an important species of the family Anacardiaceae and is one of the most important crops cultivated commercially in many parts of the world. Hence, a better understanding of the phylogeny in this species is crucial as it is the basis knowledge of improving its genetic resources which is beneficial for breeding programs. Phylogenetic relationships among 13 mango cultivars from Indonesia, Malaysia and Taiwan were carried out by comparing DNA sequence data sets derived from the Internal Transcribed Spacer (ITS) region pfnuclear ribosomal DNA (nrDNA). Analysis using parsimony method showed that the cultivars were classified into three major groups. The first group composed almost Malaysian cultivars although with low bootstrap value, the second group consisted of mainly Taiwan cultivars and the last group included mostly Indonesia one. The results indicated that some cultivars have a close relationships with each other even it is originated from different countries. With regards to the relationship among these cultivars, this gives better insight for generating new cultivar.
The benthic pennate diatom Nitzschia navis-varingica, known for producing domoic acid (DA) and its isomers, is widely distributed in the Western Pacific (WP) region. To investigate the genetic differentiation and gene flow patterns among the populations in the WP, the genetic diversity of 354 strains of N. navis-varingica was analysed using two nuclear-encoded rDNA loci: the large subunit rDNA (LSU rDNA) and the internal transcribed spacer 2 (ITS2). Frustule morphology of each strain was examined by TEM. The LSU rDNA phylogeny revealed a monophyletic lineage encompassing all strains, with sequence divergences of <0.9 %. Phylogenetic and population genetic analyses of ITS2 identified eight distinct clades (designated as Groups A to H) with moderate to high genetic heterogeneity (0.5-19.7 %). The low genetic differentiations between the geographically separated populations (pairwise FST of <0.03) suggested high gene flow and lack of spatial genetic structuring. Molecular clock analysis of the ITS2 phylogeny traced the evolutionary history of N. navis-varingica to the Eocene Epoch, and the split between clades likely occurred from the mid-Miocene to Pleistocene Epochs (10.8-1.2 Ma). The population dispersal in the WP were likely influenced by historical events like the Quarternary glacial cycles during the period, contributing to its homogenous distributions in the region.
Natural hybridization has been considered a source of taxonomic complexity in Cryptocoryne. A combined study of DNA sequencing data from the internal transcribed spacer (ITS) of nuclear ribosomal DNA and the trnK-matK region of chloroplast DNA was used to identify the parents of Cryptocoryne putative hybrids from Peninsular Malaysia. Based on the intermediate morphology and sympatric distribution area, the plants were tentatively identified as the hybrid Cryptocoryne ×purpurea nothovar. purpurea. The plants were pollen sterile and had long been considered as hybrids, supposedly between two related and co-existing species, C. cordata var. cordata and C. griffithii. The status of C. ×purpurea nothovar. purpurea was independently confirmed by the presence of an additive ITS sequence pattern from these two parental species in hybrid individuals. An analysis of the chloroplast trnK-matK sequences showed that the hybridization is bidirectional with the putative hybrids sharing identical sequences from C. cordata var. cordata and C. griffithii, indicating that both putative parental species had been the maternal parent in different accessions.
In 1994 Corner published five new species within the genus Psathyrella, all having been collected on the Malay Peninsula between 1929 and 1930. Three of these species belong to the genus Hebeloma and with their vinaceous colored lamellae and spore print, when fresh, they belong to H. sect. Porphyrospora. Of these three species, only one, P. flavidifolia, was validly published and thus we herewith recombine it as H. flavidifolium. The other two species, P. splendens and P. verrucispora, are synonyms of H. parvisporum and H. lactariolens, respectively. We also describe a new Malayan species, H. radicans, which also belongs to H. sect. Porphyrospora. These findings confirm the western Pacific Rim as a diversity hotspot for H. sect. Porphyrospora. The records described within this paper, represent the first recognition that the genus Hebeloma, and indeed that members of the ectomycorrhizal Hymenogastraceae, are present on the Malay Peninsula.
Metabarcoding analysis is an effective technique for monitoring the domoic acid-producing Pseudo-nitzschia species in marine environments, uncovering high-levels of molecular diversity. However, such efforts may result in the overinterpretation of Pseudo-nitzschia species diversity, as molecular diversity not only encompasses interspecies and intraspecies diversities but also exhibits extensive intragenomic variations (IGVs). In this study, we analyzed the V4 region of the 18S rDNA of 30 strains of Pseudo-nitzschia multistriata collected from the coasts of China. The results showed that each P. multistriata strain harbored about a hundred of unique 18S rDNA V4 sequence varieties, of which each represented by a unique amplicon sequence variant (ASV). This study demonstrated the extensive degree of IGVs in P. multistriata strains, suggesting that IGVs may also present in other Pseudo-nitzschia species and other phytoplankton species. Understanding the scope and levels of IGVs is crucial for accurately interpreting the results of metabarcoding analysis.
We establish a new interstitial polyclad species, Theama japonica sp. nov., based on specimens collected from coarse-sandy habitats in three Japanese main islands (Hokkaido, Honshu, and Shikoku) along the coasts of the Pacific Ocean and the Sea of Japan. Theama japonica is characterized by i) two pairs of cerebral eyespots and four to six precerebral eyespots; ii) eosinophilic secretion glands distributed in the distal half of the inner ventral part of the prostatic vesicle; iii) a conical penis papilla, bent up dorsally, with a sclerotized inner wall; iv) the prostatic sheath with an inner angular fold on the dorso-distal side; and v) the external cilia longer dorsally than ventrally. Partial sequences of the cytochrome c oxidase subunit I (COI) gene from 20 specimens collected at eight localities along Japanese coasts represented 19 haplotypes. The uncorrected p-distances among these COI haplotypes fell within intraspecific variations observed in other polyclads. A network analysis based on these COI haplotypes suggested a geographically non-cohesive genetic structure of the species, possibly indicating the species' high dispersibility. Molecular phylogenetic analyses based on a concatenated dataset of 18S and 28S rDNA sequences showed T. japonica formed a clade with other Theama species. The resulting tree also indicates that our new species is more closely related to Theama sp. from Colombia than species from Panama and Croatia.
This study used morphological characterization and phylogenetic analysis of the internal transcribed spacer (ITS) region of nuclear ribosomal DNA to investigate the phylogeny of Passiflora species. The samples were collected from various regions of East Malaysia, and discriminant function analysis based on linear combinations of morphological variables was used to classify the Passiflora species. The biplots generated five distinct groups discriminated by morphological variables. The group consisted of cultivars of P. edulis with high levels of genetic similarity; in contrast, P. foetida was highly divergent from other species in the morphological biplots. The final dataset of aligned sequences from nine studied Passiflora accessions and 30 other individuals obtained from GenBank database (NCBI) yielded one most parsimonious tree with two strongly supported clades. Maximum parsimony (MP) tree showed the phylogenetic relationships within this subgenus Passiflora support the classification at the series level. The constructed phylogenic tree also confirmed the divergence of P. foetida from all other species and the closeness of wild and cultivated species. The phylogenetic relationships were consistent with results of morphological assessments. The results of this study indicate that ITS region analysis represents a useful tool for evaluating genetic diversity in Passiflora at the species level.
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.
The most economically important form of aquaculture is fish farming, which is an industry that accounts for an ever increasing share of world fishery production. Molecular markers can be used to enhance the productivity of the aquaculture and fish industries to meet the increasing demand. Molecular markers can be identified via a DNA test regardless of the developmental stage, age or environmental challenges experienced by the organism. The application of 16s and cytochrome b markers has enabled rapid progress in investigations of genetic variability and inbreeding, parentage assignments, species and strain identification and the construction of high resolution genetic linkage maps for aquaculture fisheries. In this review, the advantages of principles and potential power tools of 16s and cytochrome b markers are discussed. Main findings in term of trend, aspects and debates on the reviewed issue made from the model of aquatic species for the benefit of aquaculture genomics and aquaculture genetics research are discussed. The concepts in this review are illustrated with various research examples and results that relate theory to reality and provide a strong review of the current status of these biotechnology topics.