The application of high-throughput sequencing (HTS) for metabarcoding of mixed samples offers new opportunities in conservation biology. Recently, the successful detection of prey DNA from the guts of leeches has raised the possibility that these, and other blood-feeding invertebrates, might serve as useful samplers of mammals. Yet little is known about whether sympatric leech species differ in their feeding preferences, and whether this has a bearing on their relative suitability for monitoring local mammalian diversity. To address these questions, we collected spatially matched samples of two congeneric leech species Haemadipsa picta and Haemadipsa sumatrana from lowland rainforest in Borneo. For each species, we pooled ~500 leeches into batches of 10 individuals, performed PCR to target a section of the mammalian 16S rRNA locus and undertook sequencing of amplicon libraries using an Illumina MiSeq. In total, we identified sequences from 14 mammalian genera, spanning nine families and five orders. We found greater numbers of detections, and higher diversity of OTUs, in H. picta compared with H. sumatrana, with rodents only present in the former leech species. However, comparison of samples from across the landscape revealed no significant difference in mammal community composition between the leech species. We therefore suggest that H. picta is the more suitable iDNA sampler in this degraded Bornean forest. We conclude that the choice of invertebrate sampler can influence the detectability of different mammal groups and that this should be accounted for when designing iDNA studies.
Tropical forests are being rapidly altered by logging and cleared for agriculture. Understanding the effects of these land use changes on soil bacteria, which constitute a large proportion of total biodiversity and perform important ecosystem functions, is a major conservation frontier. Here we studied the effects of logging history and forest conversion to oil palm plantations in Sabah, Borneo, on the soil bacterial community. We used paired-end Illumina sequencing of the 16S rRNA gene, V3 region, to compare the bacterial communities in primary, once-logged, and twice-logged forest and land converted to oil palm plantations. Bacteria were grouped into operational taxonomic units (OTUs) at the 97% similarity level, and OTU richness and local-scale α-diversity showed no difference between the various forest types and oil palm plantations. Focusing on the turnover of bacteria across space, true β-diversity was higher in oil palm plantation soil than in forest soil, whereas community dissimilarity-based metrics of β-diversity were only marginally different between habitats, suggesting that at large scales, oil palm plantation soil could have higher overall γ-diversity than forest soil, driven by a slightly more heterogeneous community across space. Clearance of primary and logged forest for oil palm plantations did, however, significantly impact the composition of soil bacterial communities, reflecting in part the loss of some forest bacteria, whereas primary and logged forests did not differ in composition. Overall, our results suggest that the soil bacteria of tropical forest are to some extent resilient or resistant to logging but that the impacts of forest conversion to oil palm plantations are more severe.
A species of Simulium in the Simulium melanopus species-group of the subgenus Simulium (formerly misidentified as S. laterale Edwards from Sabah and Sarawak, Malaysia) is suspected to have dimorphic male scutal color patterns linked with different numbers of upper-eye facets. This study aimed to confirm whether or not these two forms of adult males represent a single species.
Alexandrium ostenfeldii (Paulsen) Balech and Tangen and A. peruvianum (Balech and B.R. Mendiola) Balech and Tangen are morphologically closely related dinoflagellates known to produce potent neurotoxins. Together with Gonyaulax dimorpha Biecheler, they constitute the A. ostenfeldii species complex. Due to the subtle differences in the morphological characters used to differentiate these species, unambiguous species identification has proven problematic. To better understand the species boundaries within the A. ostenfeldii complex we compared rDNA data, morphometric characters and toxin profiles of multiple cultured isolates from different geographic regions. Phylogenetic analysis of rDNA sequences from cultures characterized as A. ostenfeldii or A. peruvianum formed a monophyletic clade consisting of six distinct groups. Each group examined contained strains morphologically identified as either A. ostenfeldii or A. peruvianum. Though key morphological characters were generally found to be highly variable and not consistently distributed, selected plate features and toxin profiles differed significantly among phylogenetic clusters. Additional sequence analyses revealed a lack of compensatory base changes in ITS2 rRNA structure, low to intermediate ITS/5.8S uncorrected genetic distances, and evidence of reticulation. Together these data (criteria currently used for species delineation in dinoflagellates) imply that the A. ostenfeldii complex should be regarded a single genetically structured species until more material and alternative criteria for species delimitation are available. Consequently, we propose that A. peruvianum is a heterotypic synonym of A. ostenfeldii and this taxon name should be discontinued.
Coolia is a widespread and ecologically important genus of benthic marine dinoflagellates found in tropical regions. Historically, there has been taxonomic confusion about the taxonomy and toxicity of this group. The goal of this study was to resolve morphological questions concerning Coolia tropicalis and determine the taxonomic identity of the Australian Coolia isolate which has been reported to produce cooliatoxins. To accomplish this, the morphology of tropical strains from Belize (the type locality of C. tropicalis), Malaysia, Indonesia, and Australia were examined and compared to published reports. The morphological analysis showed that C. tropicalis differs from the original description in that it has a slightly larger size (35-47 μm long by 30-45 μm wide versus 23-40 μm long by 25-39 μm wide), and the shape of fourth apical plate, and the length of Po plate (7.4-12 μm versus 7 μm). Based on both morphology and phylogenetic analysis using LSU D1- D3 rDNA sequences, the clones of C. tropicalis from Malaysia, Indonesia, and Belize were found to form a monophyletic clade within the genus. The strain producing cooliatoxin was found to be C. tropicalis, not Coolia monotis as originally assumed. To explore the factors influencing the growth of Coolia species, the growth rates of C. tropicalis and Coolia malayensis were determined at different temperatures and salinities. Both species tolerated a wide range of temperatures, but cannot survive at temperatures <20°C or >35°C. C. monotis, the dominant species reported in the literature, probably does not produce toxins.
This study aimed to determine the distribution of Candida species in the oral cavity and differentiate the species based on PCR amplification, including HinfI and MspI digestion, in order to assess the effectiveness of using the rDNA region for species identification. Samples from saliva as well as palate, tongue and cheek mucosa surfaces were collected from 45 individuals, consisting of three groups: periodontal disease patients; denture-wearers; and the control group. The samples were serially diluted, spread on BHI and YPD agar plates and scored for colony-forming units (CFUs). Fifteen random candidal colonies were isolated and subjected to genomic DNA extraction, based on glass beads disruption. Four primers were used to amplify regions in the rDNA, and the ITSI-5.8S-ITSII PCR product was digested by HinfI and MspI restriction enzymes. The microbial loads on all sites of the denture-wearers were found to be significantly higher than control, while in the periodontal disease group only the microbial loads on the tongue were significantly higher than control. Meanwhile, there was no significant difference at other sites. The restriction fragment lengths of the clinical samples were compared to those of seven control species, allowing the differentiation of all seven species and the identification of 14 species from the clinical samples. The MspI restriction digest was not able to distinguish between C. albicans and C. dubliniensis, whereas the HinfI digest could not distinguish between C. tropicalis and C. parapsilosis. It was concluded that PCR-RFLP of the candidal rDNA region has potential for species identification. This study demonstrates the potential use of candidal rDNA as a means for identifying Candida species, based on genotype. The results also indicate the possibility of constructing genetic probes that target specific restriction fragments in the ITSI-5.8S-ITSII region, enabling swift and precise identification of Candida species.
This study compared morphological and molecular data for identification of Kaempferia species. Each species was deposited in Institute of Bioscience (IBS), Universiti Putra Malaysia (UPM) as voucher specimens and ITS sequences of each species deposited in NCBI (https://www.ncbi.nlm.nih.gov/) as GenBank accessions. DNA was extracted using a modified CTAB method and PCR amplification was completed using Internal Transcribed Spacer (ITS4 and ITS5) markers. PCR amplification of products were viewed under gel electrophoresis. Sequencing was performed and sequence characteristics of ITS rDNA in Kaempferia is shown. Qualitative and qualitative scoring of morphological characters and measuring techniques for Kaempferia species are included. In addition, a brief review of molecular markers used in phylogenetic studies of Zingiberaceae is included in this dataset.
Candida albicans is an important opportunistic fungal pathogen capable of causing fatal systemic infections in humans. Presently in Malaysia, there is little information available on the genetic diversity of this organism and trends in behavioural characteristics. In this project, three genotyping methods: 25S rDNA genotyping, Alternative Lengthening of Telomerase (ALT) sequence typing and Multi-Locus Sequence Typing (MLST) were applied to study the genetic diversity of strains from infected hospital in-patients and asymptomatic individuals in the community. The results showed that, with the 25S rDNA genotyping, as in other parts of the world, the most common genotype was type A which accounted for approximately 70% of the 111 isolates tested. Further typing with the ALT sequence showed type 3 to be the most common in the isolates tested. MLST analysis revealed many possibly novel sequence types, as well as a statistically significant association between pathogenicity and a group of closely related isolates, most of which were from hospital samples. Further work on genotypes associated with enhanced virulence will help to clarify the value of genotyping for clinical and epidemiological investigations.
Aging is a complex biological process that occurs in all living organisms. Aging is initiated by the gradual accumulation of biomolecular damage in cells leading to the loss of cellular function and ultimately death. Cellular senescence is one such pathway that leads to aging. The accumulation of nucleic acid damage and genetic alterations that activate permanent cell-cycle arrest triggers the process of senescence. Cellular senescence can result from telomere erosion and ribosomal DNA instability. In this review, we summarize the molecular mechanisms of telomere length homeostasis and ribosomal DNA stability, and describe how these mechanisms are linked to cellular senescence and longevity through lessons learned from budding yeast.
In Malaysia, harmful algal blooms often occur along the coastal waters of west Sabah, where one of the causative organisms is the toxin-producing dinoflagellate, Pyrodinium bahamense var. compressum. A total of five P. bahamense var. compressum isolates were obtained from four locations and were cultured in f/2 medium. A Polymerase Chain Reaction (PCR) based technique was developed and used to screen for the presence of the dinoflagellate, P. bahamense var. compressum. A dinoflagellate-specific primer pair was designed based on sequences of P. bahamense var. compressum to amplify the 18S small subunit ribosomal DNA (rDNA) sequences. The rDNA of the P. bahamense var. compressum isolates were obtained. A species-specific primer pair was designed to target a 600 bp rDNA sequence of the target dinoflagellate. The primer pair targeting P. bahamense var. compressum did not yield any product with the fifteen algae cultures used as negative controls, but only amplified the rDNA of P. bahamense var. compressum cultures. The PCR method for identification of P. bahamense var. compressum was also applied on twenty field samples collected with plankton net. P. bahamense var. compressum cells were detected by PCR in five field samples and were confirmed by direct sequencing. From this study, a species-specific primer pair was obtained to identify the target species, P. bahamense var. compressum, among the natural complex communities of seawater.
In this study, inter- and intraspecific genetic diversity within the marine harmful dinoflagellate genus Coolia Meunier was evaluated using isolates obtained from the tropics to subtropics in both Pacific and Atlantic Ocean basins. The aim was to assess the phylogeographic history of the genus and to clarify the validity of established species including Coolia malayensis. Phylogenetic analysis of the D1-D2 LSU rDNA sequences identified six major lineages (L1-L6) corresponding to the morphospecies Coolia malayensis (L1), C. monotis (L2), C. santacroce (L3), C. palmyrensis (L4), C. tropicalis (L5), and C. canariensis (L6). A median joining network (MJN) of C. malayensis ITS2 rDNA sequences revealed a total of 16 haplotypes; however, no spatial genetic differentiation among populations was observed. These MJN results in conjunction with CBC analysis, rDNA phylogenies and geographical distribution analyses confirm C. malayensis as a distinct species which is globally distributed in the tropical to warm-temperate regions. A molecular clock analysis using ITS2 rDNA revealed the evolutionary history of Coolia dated back to the Mesozoic, and supports the hypothesis that historical vicariant events in the early Cenozoic drove the allopatric differentiation of C. malayensis and C. monotis.
Two DNA extraction methods, the Zirmil-beating cell disruption method (ZBC) and the QIAamp fast DNA stool mini kit (QIA), were used to extract DNA from the intestinal flora of the penaeid shrimp Litopenaeus vannamei, and their microbial communities were analyzed using 16S rDNA high-throughput sequencing. Results were obtained in terms of the number of reads, alpha diversity indexes, beta diversity indexes and taxonomic composition. The alpha diversity indexes of the community, according to the ZBC method, were higher than those according to the QIA method. Furthermore, results from the three samples using the ZBC method were less consistent than those where the QIA method was used. Further, using the latter method led to substantive clustering. It is suggested that the QIA method is more stable and repeatable than the ZBC method. Although the two extraction methods shared the major abundant microflora based on 16S rDNA high-throughput sequencing, bias associated with diversity analysis indexes and certain species was observed.
Bacillus pumilis was isolated and identified from limestone and the ability towards carbon dioxide (CO) sequestration was demonstrated. B . pumilus (S3 SC_1), isolated from Gua Tempurung, Gopeng, Perak was able to form calcite in the presence of calcium ions. B. pumilus was successfully characterized by using conventional biochemical characterization and 16s rDNA sequencing. Three types of experimental systems with B. pumilus, without B. pumilus and without continuous supply of CO2 with the presence of B. pumilus which could produce extracellular carbonic were studied to determine the effects of bacterially produced carbonic anhydrase (CA) by B. pumilus in removing CO2 as calcite. Through our current study, CO2 sequestration ability of B . pumilus was proven.
A DNA macroarray was previously developed to detect major fungal and oomycete pathogens of solanaceous crops. To provide a convenient alternative for researchers with no access to X-ray film-developing facilities, specific CCD cameras or Chemidoc XRS systems, a chromogenic detection method with sensitivity comparable with chemiluminescent detection, has been developed. A fungal (Stemphylium solani) and an oomycete (Phytophthora capsici) pathogen were used to develop the protocol using digoxigenin (DIG)-labeled targets. The internal transcribed spacer (ITS) region of the nuclear ribosomal DNA (rDNA), including ITS1, 5.8S rDNA, and ITS2, was used as the target gene and polymerase chain reaction amplified as in the previous protocol. Various amounts of species-specific oligonucleotides on the array, quantities of DIG-labeled ITS amplicon, and hybridization temperatures were tested. The optimal conditions for hybridization were 55°C for 2 h using at least 10 pmol of each species-specific oligonucleotide and labeled target at 10 ng/ml of hybridization buffer. Incubation of the hybridized array with anti-DIG conjugated alkaline phosphatase substrates, NBT/BCIP, produced visible target signals between 1 and 3 h compared with 1 h in chemiluminescent detection. Samples from pure cultures, soil, and artificially inoculated plants were also used to compare the detection using chemiluminescent and chromogenic methods. Chromogenic detection was shown to yield similar results compared with chemiluminescent detection in regard to signal specificity, duration of hybridization between the array and targets, and cost, though it takes 1 to 2 h longer for the visualization process, thus providing a convenient alternative for researchers who lack darkroom facilities. To our knowledge, this is the first report of DNA macroarray detection of plant pathogens using a chromogenic method.
Boesenbergia rotunda (Zingiberaceae), is a high-value culinary and ethno-medicinal plant of Southeast Asia. The rhizomes of this herb have a high flavanone and chalcone content. Here we report the genome analysis of B. rotunda together with a complete genome sequence as a hybrid assembly. B. rotunda has an estimated genome size of 2.4 Gb which is assembled as 27,491 contigs with an N50 size of 12.386 Mb. The highly heterozygous genome encodes 71,072 protein-coding genes and has a 72% repeat content, with class I TEs occupying ~67% of the assembled genome. Fluorescence in situ hybridization of the 18 chromosome pairs at the metaphase showed six sites of 45S rDNA and two sites of 5S rDNA. An SSR analysis identified 238,441 gSSRs and 4604 EST-SSRs with 49 SSR markers common among related species. Genome-wide methylation percentages ranged from 73% CpG, 36% CHG and 34% CHH in the leaf to 53% CpG, 18% CHG and 25% CHH in the embryogenic callus. Panduratin A biosynthetic unigenes were most highly expressed in the watery callus. B rotunda has a relatively large genome with a high heterozygosity and TE content. This assembly and data (PRJNA71294) comprise a source for further research on the functional genomics of B. rotunda, the evolution of the ginger plant family and the potential genetic selection or improvement of gingers.
The genus Lentinus (Polyporaceae, Basidiomycota) is widely documented from tropical and temperate forests and is taxonomically controversial. Here we studied the relationships between Lentinus subg. Lentinus sensu Pegler (i.e. sections Lentinus, Tigrini, Dicholamellatae, Rigidi, Lentodiellum and Pleuroti and polypores that share similar morphological characters). We generated sequences of internal transcribed spacers (ITS) and partial 28S regions of nuc rDNA and genes encoding the largest subunit of RNA polymerase II (RPB1), focusing on Lentinus subg. Lentinus sensu Pegler and the Neofavolus group, combined these data with sequences from GenBank (including RPB2 gene sequences) and performed phylogenetic analyses with maximum likelihood and Bayesian methods. We also evaluated the transition in hymenophore morphology between Lentinus, Neofavolus and related polypores with ancestral state reconstruction. Single-gene phylogenies and phylogenies combining ITS and 28S with RPB1 and RPB2 genes all support existence of a Lentinus/Polyporellus clade and a separate Neofavolus clade. Polyporellus (represented by P. arcularius, P. ciliatus, P. brumalis) forms a clade with species representing Lentinus subg. Lentinus sensu Pegler (1983), excluding L. suavissimus. Lentinus tigrinus appears as the sister group of Polyporellus in the four-gene phylogeny, but this placement was weakly supported. All three multigene analyses and the single-gene analysis using ITS strongly supported Polyporus tricholoma as the sister group of the Lentinus/Polyporellus clade; only the 28S rRNA phylogeny failed to support this placement. Under parsimony the ancestral hymenophoral configuration for the Lentinus/Polyporellus clade is estimated to be circular pores, with independent transitions to angular pores and lamellae. The ancestral state for the Neofavolus clade is estimated to be angular pores, with a single transition to lamellae in L. suavissimus. We propose that Lentinus suavissimus (section Pleuroti) should be reclassified as Neofavolus suavissimus comb. nov.
Quorum sensing (QS) is a mechanism adopted by bacteria to regulate expression of genes according to population density. N-acylhomoserine lactones (AHLs) are a type of QS signalling molecules commonly found in Gram-negative bacteria which have been reported to play a role in microbial spoilage of foods and pathogenesis. In this study, we isolated an AHL-producing Hafnia alvei strain (FB1) from spherical fish pastes. Analysis via high resolution triple quadrupole liquid chromatography/mass spectrometry (LC/MS) on extracts from the spent supernatant of H. alvei FB1 revealed the existence of two short chain AHLs: N-(3-oxohexanoyl) homoserine lactone (3-oxo-C6-HSL) and N-(3-oxo- octanoyl) homoserine lactone (3-oxo-C8-HSL). To our knowledge, this is the first report of the production of AHLs, especially 3-oxo-C8-HSL, by H. alvei.
This study provides a first description of the phylogeographic patterns and evolutionary history of two species of the mudskipper genus Periophthalmus. These amphibious gobies are distributed throughout the whole Indo-Pacific region and Atlantic coast of Africa, in peritidal habitats of soft-bottom coastal ecosystems. Three sequence datasets of two widely distributed species, Periophthalmus argentilineatus and P. kalolo, were obtained by amplifying and sequencing two mtDNA markers (D-loop and 16S rDNA) and the nDNA rag1 region. The three datasets were then used to perform phylogeographic, demographic and population genetic analyses. Our results indicate that tectonic events and past climatic oscillations strongly contributed to shape present genetic differentiation, phylogeographic and demographic patterns. We found support for the monophyly of P. kalolo, and only shallow genetic differentiation between East-African and Indo-Malayan populations of this species. However, our collections of the morphospecies P. argentilineatus include three molecularly distinct lineages, one of them more closely related to P. kalolo. The presence of Miocenic timings for the most recent common ancestors of some of these morphologically similar clades, suggests the presence of strong stabilising selection in mudskippers' habitats. At population level, demographic analyses and palaeoecological records of mangrove ecosystems suggest that Pleistocene bottlenecks and expansion plus secondary contact events of the studied species were associated with recurrent sea transgressions during interglacials, and sea regressions or stable regimes during glacials, respectively.
Fusarium species section Liseola namely F. fujikuroi, F. proliferatum, F. andiyazi, F. verticillioides, and F. sacchari are well-known plant pathogens on rice, sugarcane and maize. In the present study, restriction analysis of the intergenic spacer regions (IGS) was used to characterize the five Fusarium species isolated from rice, sugarcane and maize collected from various locations in Peninsular Malaysia. From the analysis, and based on restriction patterns generated by the six restriction enzymes, Bsu151, BsuRI, EcoRI, Hin6I, HinfI, and MspI, 53 haplotypes were recorded among 74 isolates. HinfI showed the most variable restriction patterns (with 11 patterns), while EcoRI showed only three patterns. Although a high level of variation was observed, it was possible to characterize closely related species and isolates from different species. UPGMA cluster analysis showed that the isolates of Fusarium from the same species were grouped together regardless of the hosts. We conclude that restriction analysis of the IGS regions can be used to characterize Fusarium species section Liseola and to discriminate closely related species as well as to clarify their taxonomic position.
Morphological identification of edible mushrooms can sometimes prove troublesome, because phenotypic variation in fungi can be affected by substrate and environmental factors. One of the most important problems for mushroom breeders is the lack of a systematic consensus tool to distinguish different species, which are sometimes morphologically identical. Basidiomycetes as one of the largest groups of edible mushrooms have become more important in recent times for their medicinal and nutritional properties. Partial rDNA sequences, including the Internal Transcribed Spacer I-5.8SrDNA-Internal Transcribed Spacer II, were used in this study for molecular identification and assessment of phylogenetic relationships between selected edible species of the Basidiomycetes. Phylogenetic trees showed five distinct clades; each clade belonging to a separate family group. The first clade included all the species belonging to the Pleurotaceae (Pleurotus spp.) family; similarly, the second, third, fourth, and fifth clades consist of species from the Agaricaceae (Agaricus sp.), Lyophllaceae (Hypsigygus sp.), Marasmiaceae (Lentinula edodes sp.) and Physalacriaceae (Flammulina velutipes sp.) families, respectively. Moreover, different species of each family were clearly placed in a distinct sub-cluster and a total of 13 species were taken for analysis. Species differentiation was re-confirmed by AMOVA analysis (among the populations: 99.67%; within: 0.33%), nucleotide divergence, haplotyping and P value. Polymorphism occurred throughout the ITS regions due to insertion-deletion and point mutations, and can be clearly differentiated within the families as well as genera. Moreover, this study proves that the sequence of the ITS region is a superior molecular DNA barcode for taxonomic identification of Basidiomycetes.