Plastid trnL-trnF and nuclear ribosomal ITS sequences were obtained from selected wild-type individuals of Polygonum minus Huds. in Peninsular Malaysia. The 380 bp trnL-trnF sequences of the Polygonum minus accessions were identical. Therefore, the trnL-trnF failed to distinguish between the Polygonum minus accessions. However, the divergence of ITS sequences (650 bp) among the Polygonum minus accessions was 1%, indicating that these accessions could be distinguished by the ITS sequences. A phylogenetic relationship based on the ITS sequences was inferred using neighbor-joining, maximum parsimony and Bayesian inference. All of the tree topologies indicated that Polygonum minus from Peninsular Malaysia is unique and different from the synonymous Persicaria minor (Huds.) Opiz and Polygonum kawagoeanum Makino.
Infection with Chloromyxum careni Mutschmann, 1999 was found in the Asian horned frog Megophrys nasuta from Malaysia and Indonesia. Kidney was the only organ infected. Coelozoic plasmodia up to 300 μm were localized in Bowman's space, embracing the glomerulus from all sides, or rarely in lumina of renal tubules. Plasmodia are polysporic, containing disporic pansporoblasts. Myxospores observed by light microscopy are colorless, variable in shape and size, measuring 6.0-8.5 × 5.0-6.5 μm, composed of two symmetrical valves joined by a meridian suture, containing four pyriform polar capsules 3.0-4.0 × 2.5-3.0 μm and a single sporoplasm. Each valve possesses 14-24 (median 21) fine longitudinal ridges clearly visible only in scanning electron microscopy. Rarely, atypical spores with a markedly pointed posterior pole and only 6-10 surface ridges are present in plasmodia together with typical spores. Both small subunit (SSU) and large subunit (LSU) rRNA gene sequences possess extremely long GU-rich inserts. In all SSU and LSU rDNA-based phylogenetic analyses, C. careni clustered as a distinct basal branch to the Myxobolus+Myxidium lieberkuehni clade, out of the marine Chloromyxum clade containing Chloromyxum leydigi, the type species of the genus. These morphological and phylogenetic data suggest erection of a new genus for the C. careni lineage, but we conservatively treat it as a Chloromyxum sensu lato until more information is available.
A diesel-degrading bacterium from Antarctica has been isolated. The isolate was tentatively identified as Pseudomonas sp. strain DRYJ3 based on partial 16S rDNA molecular phylogeny and Biolog GN microplate panels and Microlog database. Growth on diesel was supported optimally by ammonium sulphate, nitrate and nitrite. The bacterium grew optimally in between 10 and 15 degrees C, pH 7.0 and 3.5% (v/v) diesel. The biodegradation of diesel oil by the strain increased in efficiency from the second to the sixth day of incubation from 1.4 to 18.8% before levelling off on the eighth day n-alkane oxidizing and aldehyde reductase activities were detected in the crude enzyme preparation suggesting the existence of terminal n-alkane oxidizing activity in this bacterium.
Anopheles sundaicus s.l. is a malaria vector in coastal areas of Southeast Asia. Previous studies showed at least four distinct species within the complex. The present study investigated the phylogeography and the status of A. sundaicus s.l. populations from Cambodia, Thailand, Malaysia and Indonesia with regard to A. sundaicus s.s. from Sarawak, Malaysian Borneo and A. epiroticus in Vietnam and Thailand. Three lineages recovered by analyses of Cyt-b and COI (mtDNA) confirmed the presence of A. sundaicus s.s. in Malaysian Borneo, the distribution of A. epiroticus from southern Vietnam to peninsular Malaysia, and recognised a distinct form in Indonesia that is named A. sundaicus E. The phylogenetic and demographic analyses suggest that the three species were separated during the Early Pleistocene (1.8-0.78 Myr) and experienced bottlenecks followed by a genetic expansion in more recent times. Based on the results and knowledge of the biogeography of the area, we hypothesise that the combination of cyclical island and refugium creation was the cause of lineage isolation and bottleneck events during the Pleistocene.
Coptotermes gestroi, the Asian subterranean termite (AST), is an economically important structural and agricultural pest that has become established in many areas of the world. For the first time, phylogeography was used to illuminate the origins of new found C. gestroi in the US Commonwealth of Puerto Rico; Ohio, USA; Florida, USA; and Brisbane, Australia. Phylogenetic relationships of C. gestroi collected in indigenous locations within Malaysia, Thailand, and Singapore as well as from the four areas of introduction were investigated using three genes (16S rRNA, COII, and ITS) under three optimality criteria encompassing phenetic and cladistic assumptions (maximum parsimony, maximum likelihood, and neighbor-joining). All three genes showed consistent support for a close genetic relationship between C. gestroi samples from Singapore and Ohio, whereas termite samples from Australia, Puerto Rico, and Key West, FL were more closely related to those from Malaysia. Shipping records further substantiated that Singapore and Malaysia were the likely origin of the Ohio and Australia C. gestroi, respectively. These data provide support for using phylogeography to understand the dispersal history of exotic termites. Serendipitously, we also gained insights into concerted evolution in an ITS cluster from rhinotermitid species in two genera.
Disjunctive distributions across paleotropical regions in the Indian Ocean Basin (IOB) often invoke dispersal/vicariance debates. Exacum (Gentianaceae, tribe Exaceae) species are spread around the IOB, in Africa, Madagascar, Socotra, the Arabian peninsula, Sri Lanka, India, the Himalayas, mainland Southeast Asia including southern China and Malaysia, and northern Australia. The distribution of this genus was suggested to be a typical example of vicariance resulting from the breakup of the Gondwanan supercontinent. The molecular phylogeny of Exacum is in principle congruent with morphological conclusions and shows a pattern that resembles a vicariance scenario with rapid divergence among lineages, but our molecular dating analysis demonstrates that the radiation is too recent to be associated with the Gondwanan continental breakup. We used our dating analysis to test the results of DIVA and found that the program predicted impossible vicariance events. Ancestral area reconstruction suggests that Exacum originated in Madagascar, and divergence dating suggests its origin was not before the Eocene. The Madagascan progenitor, the most recent common ancestor of Exacum, colonized Sri Lanka and southern India via long-distance dispersals. This colonizer underwent an extensive range expansion and spread to Socotra-Arabia, northern India, and mainland Southeast Asia in the northern IOB when it was warm and humid in these regions. This widespread common ancestor retreated subsequently from most parts of these regions and survived in isolation in Socotra-Arabia, southern India-Sri Lanka, and perhaps mainland Southeast Asia, possibly as a consequence of drastic climatic changes, particularly the spreading drought during the Neogene. Secondary diversification from these surviving centers and Madagascar resulted in the extant main lineages of the genus. The vicariance-like pattern shown by the phylogeny appears to have resulted from long-distance dispersals followed by extensive range expansion and subsequent fragmentation. The extant African species E. oldenlandioides is confirmed to be recently dispersed from Madagascar.
Sarcocystis nesbitti, using snakes as the definitive host, is a causative agent of acute human muscular sarcocystosis in Malaysia. Therefore, it is important to explore the distribution and prevalence of S. nesbitti in snakes. Nevertheless, epizootiological information of S. nesbitti in snakes remains insufficient because few surveys have assessed Sarcocystis infection in snakes in endemic countries. In Japan, snakes are popular exotic pet animals that are imported from overseas, but the degree of Sarcocystis infection in them remains unclear. The possibility exists that muscular sarcocystosis by S. nesbitti occurs in contact with captive snakes in non-endemic countries. For a total of 125 snake faecal samples from 67 snake species collected at animal hospitals, pet shops and a zoo, this study investigated the presence of Sarcocystis using polymerase chain reaction (PCR) for the 18S ribosomal RNA gene (18S rDNA). Four (3.2%) faecal samples were positive by PCR. Phylogenetic analysis of the 18S rDNA sequences obtained from four amplification products revealed one isolate from a beauty snake (Elaphe taeniura), Sarcocystis zuoi, which uses rat snakes as the definitive host. The isolate from a Macklot's python (Liasis mackloti) was closely related with unidentified Sarcocystis sp. from reticulated pythons in Malaysia. The remaining two isolates from tree boas (Corallus spp.) were closely related with Sarcocystis lacertae, Sarcocystis gallotiae and unidentified Sarcocystis sp. from smooth snakes, Tenerife lizards and European shrews, respectively. This report is the first of a study examining the distribution of Sarcocystis species in captive snakes in Japan.
Amplified fragment length polymorphism (AFLP) is a recently developed, PCR-based high resolution fingerprinting method that is able to generate complex banding patterns which can be used to delineate intraspecific genetic relationships among bacteria. In the present study, AFLP was evaluated for its usefulness in the molecular typing of Salmonella typhi in comparison to ribotyping and pulsed-field gel electrophoresis (PFGE). Six S. typhi isolates from diverse geographic areas (Malaysia, Indonesia, India, Chile, Papua New Guinea and Switzerland) gave unique, heterogeneous profiles when typed by AFLP, a result which was consistent with ribotyping and PFGE analysis. In a further study of selected S. typhi isolates from Papua New Guinea which caused fatal and non-fatal disease previously shown to be clonally related by PFGE, AFLP discriminated between these isolates but did not indicate a linkage between genotype with virulence. We conclude that AFLP (discriminatory index=0.88) has a higher discriminatory power for strain differentiation among S. typhi than ribotyping (DI=0.63) and PFGE (DI=0.74).
Genetic variation among Malaysian isolates of Salmonella typhi was determined by analysis of ribosomal RNA gene restriction patterns. Of the 20 isolates analyzed, eight different pattern combinations were detected. The amount of variation observed was also dependent upon the restriction endonuclease used; PstI produced more different patterns than did SmaI. The results suggested that disease activity was due to a number of different clones circulating simultaneously rather than a single strain. Further implications of the data are discussed.
For elucidation of the taxonomic status of the Japanese Fasciola species, whole mitochondrial DNA of Fasciola hepatica from Australia, F. gigantica from Malaysia, and Fasciola sp. from Japan was digested with three four-base-cutting endonucleases: HinfI, MspI, and RsaI. The resulting digestion patterns showed that for each enzyme there were some bands specific for each geographical isolate and that the Japanese Fasciola sp. shared more bands with F. gigantica than with F. hepatica. Nucleotide sequences of two regions, the second internal transcribed spacer (ITS2) of the nuclear ribosomal RNA cluster and mitochondrial cytochrome c oxidase subunit I (COI), were also compared among them. The ITS2 sequence was highly conserved among the three isolates. F. gigantica and the Japanese Fasciola sp. were identical, but they differed from the Australian F. hepatica at six sites, one of which was a deletion. The COI sequence was less conserved but implied a similar relationship between the isolates. There seems no reason to regard the Japanese Fasciola sp. as anything other than a strain of F. gigantica.
Forty-nine isolates of Burkholderia pseudomallei from sporadic cases of melioidosis in Malaysia over the past 18 years were examined by BamHI ribotyping and pulsed-field gel electrophoresis (PFGE) of XbaI digests of total deoxyribonucleic acid (DNA). Twenty-four patients had septicaemic melioidosis with a mortality of 70%; mortality in the non-septicaemic disease was 16%. Five ribotype patterns were identified, 2 of which accounted for 90% of all isolates. PFGE revealed a number of different strains within these ribotypes, but some pairs of isolates from unrelated cases gave closely similar DNA profiles. These results are in agreement with Australian studies which showed a high prevalence of a few ribotypes of B. pseudomallei which are further divisible by genotyping, in areas where melioidosis is endemic.
The taxonomic status of two actinomycetes isolated from the wall of a hypogean Roman catacomb was established based on a polyphasic investigation. The organisms were found to have chemical and morphological markers typical of members of the genus Amycolatopsis. They also shared a range of chemical, molecular and phenotypic markers which served to separate them from representatives of recognized Amycolatopsis species. The new isolates formed a branch in the Amycolatopsis 16S rRNA gene sequence tree with Amycolatopsis minnesotensis NRRL B-24435(T), but this association was not supported by a particularly high bootstrap value or by the product of the maximum-parsimony tree-making algorithm. The organisms were distinguished readily from closely related Amycolatopsis species based on a combination of phenotypic properties and from all Amycolatopsis strains by their characteristic menaquinone profiles, in which tetra-hydrogenated menaquinones with 11 isoprene units predominated. The combined genotypic and phenotypic data indicate that the isolates merit recognition as representing a novel species of the genus Amycolatopsis. The name proposed for this novel species is Amycolatopsis nigrescens sp. nov., with type strain CSC17Ta-90(T) (=HKI 0330(T)=DSM 44992(T)=NRRL B-24473(T)).
This study focused on the isolation and characterization of high cadmium-resistant bacterial strains, possible exploitation of its cadmium-accumulation and cadmium-induced proteins. Cadmium-resistant bacterial strains designated as RZ1 and RZ2 were isolated from industrial wastewater of Penang, Malaysia. These isolates were identified as Enterobacter mori and Enterobacter sp. WS12 on the basis of phenotypic, biochemical and 16S rDNA sequence based molecular phylogenetic characteristics. Both isolates were Gram negative, cocci, and growing well in Lauria-Bertani broth medium at 35 °C temperature and pH 7.0. Results also indicated that Enterobacter mori and Enterobacter sp. WS12are capable to remove 87.75 and 85.11% of the cadmium from 100 µg ml(-1) concentration, respectively. This study indicates that these strains can be useful as an inexpensive and efficient bioremediation technology to remove and recover the cadmium from wastewater.
Any forms of valorization of microorganisms would require accurate identity recognition to ensure repeatability, reproducibility and quality assurance. This study aimed to evaluate the effectiveness of different primers for identifying cultured eukaryotic microalgae using a simple 18S rDNA approach. A total of 34 isolated microalgae and one culture collection were utilized in the search for an effective molecular identification method for microalgae. Ammonium formate was applied to marine microalgae prior to DNA extraction. The microalgal DNA was extracted using a commercial kit and subjected directly to PCR amplification using four different published 18S rDNA primers. The DNA sequences were analysed using Basic Local Alignment Search Tool (BLAST) and phylogenetic trees to determine the microalgae identity. The identity was further validated with conventional morphological taxonomic identification, and the relationship of microalgal morphology and genetic materials was also determined. The microalgal DNA was successfully amplified, including marine species without prior cleaning. In addition, the ss5 + ss3 primer pair was found to be an ideal primer set among the tested primers for identifying microalgae. Overall, molecular identification showed relative matching with morphological identification (82.86%). This study is important because it serves as a platform to develop a standardized eukaryotic microalgae identification method. In addition, this method could help to ease the eukaryotic microalgae identification process and enrich the current reference databases such as GenBank.
Forty-eight isolates of Pseudo-nitzschia species were established from the Miri coast of Sarawak (Malaysian Borneo) and underwent TEM observation and molecular characterization. Ten species were found: P. abrensis, P. batesiana, P. fukuyoi, P. kodamae, P. lundholmiae, P. multistriata, P. pungens, P. subfraudulenta, as well as two additional new morphotypes, herein designated as P. bipertita sp. nov. and P. limii sp. nov. This is the first report of P. abrensis, P. batesiana, P. kodamae, P. fukuyoi, and P. lundholmiae in coastal waters of Malaysian Borneo. Pseudo-nitzschia bipertita differs from its congeners by the number of sectors that divide the poroids, densities of band striae, and its cingular band structure. Pseudo-nitzschia limii, a pseudo-cryptic species in the P. pseudodelicatissima complex sensu lato, is distinct by having wider proximal and distal mantles, a higher number of striae, and greater poroid height in the striae of the valvocopula. The species were further supported by the phylogenetic reconstructions of the nuclear-encoded large subunit ribosomal gene and the second internal transcribed spacer. Phylogenetically, P. bipertita clustered with its sister taxa (P. subpacifica + P. heimii); P. limii appears as a sister taxon to P. kodamae and P. hasleana in the ITS2 tree. Pairwise comparison of ITS2 transcripts with its closest relatives revealed the presence of both hemi- and compensatory base changes. Toxicity analysis showed detectable levels of domoic acid in P. abrensis, P. batesiana, P. lundholmiae, and P. subfraudulenta, but both new species tested below the detection limit.
Three species of Opisthomonorcheides Parukhin, 1966 are reported for the first time from Indonesian waters: O. pampi (Wang, 1982) Liu, Peng, Gao, Fu, Wu, Lu, Gao & Xiao, 2010 and O. ovacutus (Mamaev, 1970) Machida, 2011 from Parastromateus niger (Bloch), and O. decapteri Parukhin, 1966 from Atule mate (Cuvier). Both O. pampi and O. ovacutus can now be considered widespread in the Indo-Pacific region, with earlier records of these species being from Fujian Province, China and Penang, Malaysia, respectively. We redescribe O. decapteri from one of its original hosts, Atule mate, off New Caledonia, and report this species from Jakarta Bay, Indonesia, extending its range throughout the Indian Ocean into the south-western Pacific. All three species possess a genital atrium that is long, sometimes very long, and a genital pore that is located in the forebody. This validates the interpretation that the original description was erroneous in reporting the genital pore in the hindbody, well posterior to the ventral sucker. These observations verify the synonymy of Retractomonorchis Madhavi, 1977 with Opisthomonorcheides. A major discrepancy between the species of Opisthomonorcheides is that some are described with the uterus entering the terminal organ laterally and some with it entering terminally; this feature needs further analysis. Based on the length of the genital atrium and the posterior extent of the vitellarium, the 27 species of Opisthomonorcheides considered valid can be divided into four groups. Among the 53 host records analysed, the families Carangidae (53% of records), Stromateidae (17%) and Serranidae (5.7%) are the most common; the reports are overwhelmingly from members of the Perciformes (91%), with further records in the Clupeiformes (5.7%), Gadiformes (1.9%) and Pleuronectiformes (1.9%). Two fish genera (Parastromateus Bleeker and Pampus Bonaparte) dominate the recorded hosts, with the black pomfret Parastromateus niger harbouring six species, the silver pomfret Pampus argenteus (Euphrasen) harbouring six, and the Chinese silver pomfret P. chinensis (Euphrasen) two. A host-parasite checklist is presented. We discuss the host-specificity of members of the genus, questioning some records such as that of O. decapteri in a deep-sea macrourid. We also comment on the morphological similarity, but phylogenetic distance, between the various Pomfret species, advancing the possibility that a series of host misidentifications has occurred. Sequences of the ITS2 rDNA gene generated for O. pampi and O. ovacutus are briefly discussed and molecular data are lodged in the GenBank database.
The chloroplastic trnL intron and the nuclear internal transcribed spacer (ITS) region were sequenced for 11 Nepenthes species recorded in Peninsular Malaysia to examine their phylogenetic relationship and to evaluate the usage of trnL intron and ITS sequences for phylogenetic reconstruction of this genus.
The genus Gymnodinium includes many morphologically similar species, but molecular phylogenies show that it is polyphyletic. Eight strains of Gymnodinium impudicum, Gymnodinium dorsalisulcum and a novel Gymnodinium-like species from Chinese and Malaysian waters and the Mediterranean Sea were established. All of these strains were examined with light microscopy, scanning electron microscopy and transmission electron microscopy. SSU, LSU and internal transcribed spacers rDNA sequences were obtained. A new genus, Wangodinium, was erected to incorporate strains with a loop-shaped apical structure complex (ASC) comprising two rows of amphiesmal vesicles, here referred to as a new type of ASC. The chloroplasts of Wangodinium sinense are enveloped by two membranes. Pigment analysis shows that peridinin is the main accessory pigment in W. sinense. Wangodinium differs from other genera mainly in its unique ASC, and additionally differs from Gymnodinium in the absence of nuclear chambers, and from Lepidodinium in the absence of Chl b and nuclear chambers. New morphological information was provided for G. dorsalisulcum and G. impudicum, e.g., a short sulcal intrusion in G. dorsalisulcum; nuclear chambers in G. impudicum and G. dorsalisulcum; and a chloroplast enveloped by two membranes in G. impudicum. Molecular phylogeny was inferred using maximum likelihood and Bayesian inference with independent SSU and LSU rDNA sequences. Our results support the classification of Wangodinium within the Gymnodiniales sensu stricto clade and it is close to Lepidodinium. Our results also support the close relationship among G. dorsalisulcum, G. impudicum, and Barrufeta. Further research is needed to assign these Gymnodinium species to Barrufeta or to erect new genera.
Haliotrema susanae sp. nov. is described from the gills of the pinecone soldierfish, Myripristis murdjan off Langkawi Island, Malaysia. This species is differentiated from other Haliotrema species especially those from holocentrids in having a male copulatory organ with bract-like extensions at the initial of the copulatory tube, grooved dorsal anchors and ventral anchors with longer shafts. The maximum likelihood (ML) analysis based on partial 28S rDNA sequences of H. susanae sp. nov. and 47 closely related monogeneans showed that H. susanae sp. nov. is recovered within a monophyletic clade consisting of only species from the genus Haliotrema. It is also observed that H. susanae sp. nov. forms a clade with H. cromileptis and H. epinepheli which coincides with a similar grouping by Young based on solely morphological characteristics. The morphological and molecular results validate the identity of H. susanae sp. nov. as belonging to the genus Haliotrema.
A new species of toxic benthic dinoflagellate is described based on laboratory cultures isolated from two locations from Brazil, Rio de Janeiro and Bahia. The morphology was studied with SEM and LM. Cells are elliptical in right thecal view and flat. They are 37-44μm long and 29-36μm wide. The right thecal plate has a V shaped indentation where six platelets can be identified. The thecal surface of both thecal plates is smooth and has round or kidney shaped and uniformly distributed pores except in the central area of the cell, and a line of marginal pores. Some cells present an elongated depression on the central area of the apical part of the right thecal plate. Prorocentrum caipirignum is similar to Prorocentrum lima in its morphology, but can be differentiated by the general cell shape, being elliptical while P. lima is ovoid. In the phylogenetic trees based on ITS and LSU rDNA sequences, the P. caipirignum clade appears close to the clades of P. lima and Prorocentrum hoffmannianum. The Brazilian strains of P. caipirignum formed a clade with strains from Cuba, Hainan Island and Malaysia and it is therefore likely that this new species has a broad tropical distribution. Prorocentrum caipirignum is a toxic species that produces okadaic acid and the fast acting toxin prorocentrolide.