DNA Barcoding Approach Research Articles

DNA barcoding and species delimitation of the genus Oxynoemacheilus (Teleostei: Nemacheilidae) in Anatolia.

The DNA barcoding approach was used for the determination of evolutionary relationships and species delimitation of the genus Oxynoemacheilus (Teleostei: Nemacheilidae). The COI barcode region (615 bp amplicon) was used to barcode 444 individuals from 64 morphologically identified species in the genus Oxynoemacheilus and 189 haplotypes were identified. The average of the interspecific p distance (9.59%) was about 21-fold higher than the average intraspecific distance (0.44%). A general genetic threshold of 1.46% sequence divergence was defined for species delimitation. The multiple species delimitation methods (BCM, GMYC, bPTP and TCS) revealed a total of 62 molecular operational taxonomic units for 64 morphospecies with a new loach species from the BuyukMelen River. Neighbour-joining, maximum likelihood and Bayesian inference analyses indicated that all haplotypes were clustered into 62 clades, which corresponded to Oxynoemacheilus species, with strong bootstrap support (≥95%). Furthermore, all samples grouped in concurrence with the taxonomic status of the species except for species groups (O. germencicus-O. cinicus-O. mesudae and O. leontinae-O. namiri) that were showed intraspecific overlap in genetic diversity for COI-based barcodes. In conclusion, our analyses indicate that COI-based barcodes provide reliable species discrimination. Therefore, we currently recommend COI barcodes as the suitable barcode forgenus Oxynoemacheilus.

Plant DNA barcoding and metabolomics for comprehensive discrimination of German Chamomile from its poisonous adulterants for food safety

German Chamomile (Matricaria recutita L.) is one of the most consumed herbal teas in the world. Deliberate and undeliberate adulteration in German Chamomile raw material represents serious concerns for consumers’ safety. Authentication of Matricaria recutita L. samples was performed using an integrative approach of DNA barcoding and chemical profiling. Discrimination of M. recutita from three morphologically related species namely Anthemis cotula L., Senecio desfontainei Druce and Senecio vulgaris L. was accomplished. Three plastid loci (mat K, rbcL and psbA-trnH) and two nuclear spacers (ITS and ITS2) were utilized for the DNA barcoding of M. recutita. The Chemical profiling of M. recutita was achieved using untargeted HPTLC-image analysis. Among the tested barcoding loci, ITS2 revealed the maximum genetic diversity among the studied plants. ITS2 showed adequate polymorphic sites to detect interspecific variation with high amplification and sequencing success. Chemical profiling showed that flavonoids, coumarins, sesquiterpene lactones and phenolic acids are the main secondary metabolite classes of the candidate plants. While, flavonoids and phenolic acids served as quality markers, sesquiterpene lactone (anthecotulide) and pyrrolizidine alkaloid (senecionine) accounted as toxicity markers for the adulterants. Notably, coumarin accumulation pattern could be utilized to discriminate M. recutita from its adulterants due to the absence of hernarin and umbelliferone from Senecio species. The results of this study suggest that, integrating genetic and metabolomic fingerprinting could serve as an effective tool, with 100% success rate, in authenticating M. recutita and discriminating its samples from the morphologically related toxic adulterants. This offers a comprehensive tool for the quality control of M. recutita samples in food industry.

Unveiling the Mycodrosophila projectans (Diptera, Drosophilidae) species complex: Insights into the evolution of three Neotropical cryptic and syntopic species.

The Zygothrica genus group has been shown to be speciose, with a high number of cryptic species. DNA barcoding approaches have been a valuable tool to uncover cryptic diversity in this lineage, as recently suggested for the Neotropical Mycodrosophila projectans complex, which seems to comprise at least three different species. The aim of this study was to confirm the subdivision of the M. projectans complex while shedding some light on the patterns and processes related to its diversification. In this sense, the use of single and multi-locus datasets under phylogenetic, distance, coalescence, and diagnostic nucleotide approaches confirmed the presence of at least three species under the general morphotype previously described as M. projectans. Only a few subtle morphological differences were found for the three species in terms of aedeagus morphology and abdominal color patterns. Ecologically, sympatry and syntopy seem to be recurrent for these three cryptic species, which present widely overlapping niches, implying niche conservatism. This morphological and ecological similarity has persisted though cladogenesis within the complex, which dates back to the Miocene, providing an interesting example of morphological conservation despite ancient divergence. These results, in addition to contrasting patterns of past demographic fluctuations, allowed us to hypothesize patterns of allopatric or parapatric diversification with secondary contact in Southern Brazil. Nevertheless, genetic diversity was generally high within species, suggesting that migration may encompass an adaptive response to the restrictions imposed by the ephemerality of resources.

DNA barcoding analysis and phylogenetic relationships of Indian wild coffee species

Wild coffee species are the reservoirs of genetic diversity and could play a critical role in the genetic improvement of coffee. However, the conservation and genetic assessment of wild coffee species has been largely neglected. In the present study, DNA barcoding approaches were employed to assess the phylogenetic relationships between five indigenous wild coffee species from India and compared with two cultivated, and four wild coffee species of African origin. The efficacy of three barcoding loci namely matK,rbcL, and trnL-trnF was investigated using PCR amplification and sequence characterization. The intergenic spacer trnL-trnF is the highly polymorphic loci followed by matK and rbcL chloroplast gene. Among the three barcoding loci, the matK locus has the maximum number of parsimony informative sites, whereas the trnL-trnF locus contains maximum singleton variable sites. Although all the three loci contain a few unique fixed nucleotides (UFNs), no individual barcode locus has the critical nucleotide sequence tags for all the five Indian wild coffee species that help in species discrimination. However, the multilocus combinations are efficient in discriminating the species due to the presence of SNPs and specific sequence tags. The phylogenetic tree constructed using the maximum likelihood analysis of the combined barcoding loci separated all the Indian wild coffee species from African wild coffee species compared to phylogeny inferred using individual barcoding loci. Our study supports the utility of DNA barcoding as a useful tool for coffee species identification, which can be used for conservation purposes.

Sorting out the plants responsible for a contamination with pyrrolizidine alkaloids in spice seeds by means of LC-MS/MS and DNA barcoding: Proof of principle with cumin and anise spice seeds

High value commodities such as spices suffer from occasional contaminations of both chemical and biological origin. Consequently, quality control and safety monitoring has become a pressing issue for the spice industry. Two recent independent studies showed that at least one third of the analyzed cumin and green anise spice seeds samples surpassed the by the European Union recently established threshold value for toxic pyrrolizidine alkaloids (PAs) and their corresponding N-oxides (PANOs). These heterocyclic secondary plant metabolites are produced by a large number of different plant families. In those spice seeds, it was found by means of DNA metabarcoding, that predominant contamination was due to the presence of herbal material from the Heliotropium genus (Boraginaceae). Unfortunately, the use of this specific type of DNA-based identification remains controversial for the majority of the official instances and preference is still given to the use of more tangible classical approaches, including microscopy and chemical analysis. However, these methodologies often suffer from inherent drawbacks. Here we demonstrate that at least for spice seeds, a combinatory approach of microscopy, chemical analysis and classical DNA barcoding of the isolated contaminants using the matK and trnH-psbA loci, provides qualitative and quantitative information on the amount of plant material responsible for the contaminations and the extent of the contamination. The generated data also demonstrates that the presence of a very limited number of Heliotropium sp. seeds in a standard commercially available canister is sufficient to surpass the allowed threshold value, illustrating once more the importance of weed control.

Identifikasi dan Analisis Filogenetik Ikan Ekonomis Penting Oreochromis sp. dengan Pendekatan DNA Barcoding

Species identification is one of the most basic and important factors in fisheries management. Oreochromis sp., such as tilapia (Oreochromis mosambicus) and tilapia (Oreochromis niloticus) are introduced species of fish originating from outside Merauke which are now the dominant fish and are commonly found in inland waters of Merauke. The purpose of this study was to identify the economically important fish species Oreochromis sp. which landed at Merauke Traditional Market with DNA Barcoding approach. DNA amplification with the COI gene resulted in a DNA sequence length of 655 bp with the identification result being Tilapia (Oreochromis niloticus). Based on the phylogenetic tree data and genetic distance, the DNA sequences of the samples formed the same clade as the Oreochromis niloticus sequences from GeneBank with 0% genetic distance and it was estimated that these species came from the same parents. Identification with DNA Barcoding makes it easy to identify species quickly and accurately.

Historical DNA from Museum Shell Collections: Evaluating the Suitability of Dried Micromollusks for Molecular Systematics

Natural history collections are an invaluable resource that can inform systematic studies and biodiversity discovery, and also contribute to understanding changes in species abundance and distributions over time. The decline in abundance and diversity of Pacific Island land snails has been a major conservation concern for more than six decades, but only the largest and most colorful snails are protected under the US Endangered Species act, and few are listed as critically endangered on the IUCN Red List. Like most invertebrates, the conservation status of many Hawaiian land snails still need assessment. Molecular data are highly informative for revising species limits and understanding evolutionary patterns and processes, but with as much as 70% of Hawaiian land snails already extinct, few fresh samples are available from which to extract DNA. To overcome the lack of material suitable for DNA barcoding, we test whether short DNA fragments of 225 to 355 bp can be sequenced from museum snail shells containing dried tissues collected more than 50 years ago. Short DNA sequences (225 bp) were obtained from 66.7% of lots, while longer DNA sequences (355 bp) were successfully sequenced from 24.2% of lots. Snail specimens stored in natural history cabinets for more than 100 years were successfully sequenced, supporting the inclusion of these materials for modern biodiversity studies. Molecular data from this study represents a small proportion of Hawaiian microsnail species housed among the millions of specimens in the Bishop Museum in Hawaii and other natural history collections. Additional resources and focused efforts are needed to scale this approach to incorporate many more of the hundreds of snail species in need of assessment in Hawaii. More broadly, there are large representative collections of endangered Pacific Island non-marine snails in many natural history museums that may be suitable for molecular work, either with DNA barcoding or other genomic approaches.

Diversity of sponge-associated fungi from a mangrove forest in Kemujan Island, Karimunjawa National Park, Indonesia

Abstract. Sibero MT, Pribadi R, Larasati SJH, Calabon MS, Sabdono A, Subagiyo S, Frederick EH. 2021. Diversity of sponge-associated fungi from a mangrove forest in Kemujan Island, Karimunjawa National Park, Indonesia. Biodiversitas 22: 5695-5605. Plenty of studies have reported interesting biological properties and bioactive compounds from sponge-associated fungi in Indonesia. However, only a few studies consider their biodiversity. Most of the studies collected the sponge from the coral reef ecosystem and barely reported from the mangrove ecosystem. This study aimed to discover the biodiversity of sponge-associated fungi from a mangrove ecosystem using a DNA barcoding approach. Five mangrove-associated sponges were collected from a tidal mangrove forest in Kemujan Island, Karimunjawa National Park, Indonesia. In total, we isolated 56 sponge-associated fungi, which consisted of seven genera in the phylum Ascomycota (77.78%), one genus in the phylum Basidiomycota (11.11%), and one genus in the phylum Mucoromycota (11.11%). Trichoderma (n: 15, 26.79%), Aspergillus (n: 13, 23.21%) and Fusarium (n: 12, 21.45%) were the top three dominant genera. In addition, based on the association of fungi to its sponge hosts, Aspergillus, Aureobasidium, Fusarium, and Trichoderma are sponge-generalists; Absidia and Pestalotiopsis are sponge-associates; while Lasiodiplodia, Montagnula, and Wallemia are sponge-specialist.

Wing Morphometric and DNA Barcoding Analysis of Two Different Public Health Important Anopheles Mosquito Species

Accurate identification and bio-ecology of mosquito vectors is paramount importance in several respects including the development of species-specific vector control strategies. In the current study, we explored two different advance tools such as Geometric morphometric (GM) and DNA barcoding analysis, to supplement morpho-taxonomy based identification of malaria vectors. In landmark-based GM analysis, the pattern of wing shape was examined and we noticed a clear variation in their shape among different Anopheles species. Similarly, in the DNA barcoding studies, the phylogenetic analysis based upon the marker genes such as COI, Cytb and D3 region of 28s rDNA gene sequences were carried out. The neighbour joining tree was formed by distinctive conspecific clusters illustrating genetic variation among different group of Anopheles species. Further, genetic exchange among different Anopheline populations was analysed by considering the COI gene sequences. In the gene flow study, the frequent genetic divergence as well as gene exchange among Anopheles stephensi mosquito populations was observed. Our findings suggested that GM analysis of the wing shape along with DNA barcoding approach can effectively be used together for accurate identification of mosquito vectors including Anopheles stephensi and Anopheles barbirostris.

Bioprospecting of cow's ruminal microbiota from a slaughterhouse in Ambarawa, Central Java, Indonesia

Abstract. Murwani R, Sibero MT, Silitonga PRA, Ambariyanto A. 2021. Bioprospecting of cow's ruminal microbiota from a slaughterhouse in Ambarawa, Central Java, Indonesia. Biodiversitas 22: 5030-5038. Ruminal microorganisms play essential roles in maintaining ruminant health. However, most studies focused only on ruminal lactic acid bacteria (LAB), although other ruminal microorganisms might have biological properties for biotechnological purposes. Therefore, the current study aimed to isolate ruminal bacteria (LAB and non-LAB) and fungi from ruminal material and conducted a bioprospecting study to understand their ability to produce antibacterial compounds and polysaccharide-degrading enzymes. The ruminal bacteria were isolated on MRS and ISP4 agar, while PDA was used to isolate the different fungi. The antibacterial property was tested against multidrug-resistant Escherichia coli and Salmonella enterica ser. Typhi. The ability to produce agarase, alginate-lyase, and carrageenase was screened. Prospective isolates were identified using DNA barcoding approach. Twelve bacteria were isolated using MRS agar, six from ISP4 agar, and four fungi from PDA. Among twelve bacteria from MRS agar, eleven were considered LAB, which consisted of Lactobacillus plantarum and Pediococcus acidilactici. Several classes of bacteria such as actinobacteria, firmicutes, ?-proteobacteria, and ?-proteobacteria were isolated during this study. In addition, three fungal classes, namely Saccharomycetes, Eurotiomycetes, and Mucoromycetes were also isolated. All bacteria from MRS agar were suggested to have potential compounds with antimicrobial activity, while all ruminal fungi exhibited potential sources of polysaccharide-degrading enzymes.

Characterizing Billbug (Sphenophorus spp.) Seasonal Biology Using DNA Barcodes and a Simple Morphometric Analysis.

Simple SummaryBillbugs (Sphenophorus spp.) are a group of grass-feeding weevils considered to be one of the most important and widespread insect pests of turfgrass. However, our limited understanding of regional variation in billbug species composition and inability to identify the damaging larval stage to species level, has hindered our ability to resolve the seasonal biology of many billbug species and constrained development of effective management approaches. In this study, we developed a robust DNA barcoding approach for identification of morphologically cryptic billbug larvae. Using this molecular tool combined with larval head capsule measurements we characterized regional variation in billbug species and developed larval phenology charts. Our approach provides researchers with the molecular tools necessary to fill critical gaps in our understanding of billbug seasonal biology and will facilitate the development of improved turfgrass pest management programs.Billbugs (Sphenophorus spp.) are a complex of grass-feeding weevil species that reduce the aesthetic and functional qualities of turfgrass. Effective billbug monitoring and management programs rely on a clear understanding of their seasonal biology. However, our limited understanding of regional variation in the species compositions and seasonal biology of billbugs, stemming primarily from our inability to identify the damaging larval stage to species level, has hindered efforts to articulate efficient IPM strategies to growers. We used a combination of DNA barcoding methods and morphometric measures to begin filling critical gaps in our understanding of the seasonal biology of the billbug species complex across a broad geographic range. First, we developed a DNA barcoding reference library using cytochrome oxidase subunit 1 (COI) sequences from morphologically identified adult billbugs collected across Indiana, Missouri, Utah and Arizona. Next, we used our reference library for comparison and identification of unknown larval specimens collected across the growing season in Utah and Indiana. Finally, we combined our DNA barcoding approach with larval head capsule diameter, a proxy for developmental instar, to develop larval phenology charts. Adult COI sequences varied among billbug species, but variation was not influenced by geography, indicating that this locus alone was useful for resolving larval species identity. Overlaid with head capsule diameter data from specimens collected across the growing season, a better visualization of billbug species composition and seasonal biology emerged. This approach will provide researchers with the tools necessary to fill critical gaps in our understanding of billbug biology and facilitate the development of turfgrass pest management programs.

Using a DNA barcoding approach to facilitate biosecurity: Identifying invasive alien macrophytes traded within the South African aquarium and pond plant industry

South Africa's water bodies have been invaded multiple times by various alien macrophytes in the last 200 years and this trend is set to continue – posing a significant threat to the already fragile indigenous aquatic ecosystems and the services they provide. The aquarium and ornamental pond plant industry has been found to serve as a primary pathway for the continued introduction of high-risk taxa. Although regulations like the National Environmental Management: Biodiversity Act (NEM:BA) 10 of 2004 together with some national programmes are currently in place to ensure compliance by the different industry role players, adequate enforcement is hampered by challenges inherent to definitive species identification. In this study, the utility of a DNA barcoding approach to aid in addressing these issues was assessed. An extensive macrophyte core barcode (matK and rbcLa) reference library was compiled and tested to: (i) establish if a “barcode gap” was present, ii) determine the optimised threshold for species delimitation and (iii) assess identification efficiency using different distance based metrices. This reference library was subsequently used to identify plants collected during a survey of ten macrophyte retailer shops in and around the Johannesburg metropolitan area for which DNA barcodes could be generated. Results obtained indicate the presence of considerable interspecific and curbed intraspecific distance between the marker combinations tested and demonstrate high species identification rates. Additionally, multiple alien aquatic plants currently in trade under the following NEM:BA categories were recovered: 1a (invasive species), 1b (invasive species controlled by program) and species prohibited in terms of section 67(1). A surprisingly large number of first-time invasive alien macrophytes not currently on the NEM:BA lists were also identified. The dataset and findings presented in this study have important implications for informing pre-emptive invasive macrophyte management in South Africa e.g. tracking the country's commitments towards achieving the Convention on Biological Diversity's (CBD) Aichi Biodiversity Target 9 (as well as its inheritor post-2020) and providing recommendations for updates to the NEM:BA alien and invasive species lists. The library also presents opportunities for exploring the utility of spin-off applications that rely on robust molecular datasets (e.g. eDNA monitoring).

Molecular phylogenetics of Malesian Diospyros (Ebenaceae) based trnL-F spacer sequences

Abstract. Wanda IF, Djuita NR, Chikmawati T. 2021. Molecular phylogenetics of Malesian Diospyros (Ebenaceae) based trnL-F spacer sequences. Biodiversitas 22: 4106-4114. Diospyros is a genus composed of potential species as an economic commodity with high diversity. However, there is limited information on the phylogenetic relationship of this genus in the Malesian region. This study aimed to provide information on the species diversity through a DNA barcoding approach and revealing the phylogenetic information of Diospyros spp. in the Malesian region. This study used 20 Diospyros accessions from Bogor Botanical Garden collections, 40 Diospyros accessions, and four outgroup accessions obtained from the NCBI database. The DNA barcoding primer utilized comes from plastids, trnL-F intergenic spacer. The phylogenetic trees were constructed using the Maximum-Parsimony method. A total of 20 accessions of Diospyros were validated with sequence data on the genebank. The result showed that all accessions had relationships with 44 other Diospyros species globally. Here, we reported 10 new trnL-F intergenic spacer sequences of Malesian Diospyros species. A phylogenetic tree grouped 64 monophyletic Diospyros species into seven clades. The phylogenetic results supports the biogeographic hypothesis: the Malesian region, the Malesian-Caledonian Region, and Cosmopolite species in almost all bioregions.

The dark side of pseudoscorpion diversity: The German Barcode of Life campaign reveals high levels of undocumented diversity in European false scorpions.

DNA barcoding is particularly useful for identification and species delimitation in taxa with conserved morphology. Pseudoscorpions are arachnids with high prevalence of morphological crypsis. Here, we present the first comprehensive DNA barcode library for Central European Pseudoscorpiones, covering 70% of the German pseudoscorpion fauna (35 out of 50 species). For 21 species, we provide the first publicly available COI barcodes, including the rare Anthrenochernes stellae Lohmander, a species protected by the FFH Habitats Directive. The pattern of intraspecific COI variation and interspecific COI variation (i.e., presence of a barcode gap) generally allows application of the DNA barcoding approach, but revision of current taxonomic designations is indicated in several taxa. Sequences of 36 morphospecies were assigned to 74 BINs (barcode index numbers). This unusually high number of intraspecific BINs can be explained by the presence of overlooked cryptic species and by the accelerated substitution rate in the mitochondrial genome of pseudoscorpions, as known from previous studies. Therefore, BINs may not be an appropriate proxy for species numbers in pseudoscorpions, while partitions built with the ASAP algorithm (Assemble Species by Automatic Partitioning) correspond well with putative species. ASAP delineated 51 taxonomic units from our data, an increase of 42% compared with the present taxonomy. The Neobisium carcionoides complex, currently considered a polymorphic species, represents an outstanding example of cryptic diversity: 154 sequences from our dataset were allocated to 23 BINs and 12 ASAP units.

Non-invasive gene delivery across the blood-brain barrier: present and future perspectives.

Non-invasive gene delivery across the blood-brain barrier: present and future perspectives.

Grouper DNA barcoding studies in Indonesia: A short review

Indonesia is recognized as one of the territories that have the highest reef fish biodiversity in the world. One of the commercially valuable fish in this area is the groupers (locally name "kerapu"). At least 76 grouper species have been reported in Indonesian waters, with three species were categorized into "vulnerable", five species "Data Deficient", and 68 species under the "Least Concern" category based on IUCN classification. The increasing exploitations rate had been reported caused the grouper stocks in Indonesia to decrease and threatened extinction. However, only limited scientific data is available regarding the grouper in Indonesia, including their identification. In most fish landing sites across Indonesia, the groupers are morphologically identified and recorded as "kerapu" to replace their scientific species names. Accurate species identification is essential in designing appropriate and sustainable management of fisheries resources. One of the tools that have been used in fish identification is DNA barcoding. In the last two decades, this molecular method has been applied to identify many fish groups globally, including grouper fish. This study reviewed the DNA barcoding approach in grouper identification in Indonesia based on the available literature.Keywords:DNA barcodingGrouperMolecular TaxonomyFisheriesIndonesia

DNA Fingerprinting and Genetic Diversity Analysis of some Egyptian Rice Genotypes

A new rice variety takes about 12 years to release, the number of available distinct morphological features is very small, due to the continual pressure of releasing new varieties. The objectives of this study were: to estimate the genetic variation of Egyptian japonica, indica, and indica/japonica genotypes, and to identify, DNA fingerprinting with employing simple sequence repeat (SSR) markers to develop DNA barcode. This study includes 15 rice genotypes that were genetically evaluated using 32 microsatellites. DNA barcodes as well as the unique pattern of SSR polymorphism were produced. A total of 116 alleles was detected and the number of alleles ranged from 2 to 5, with an average of 3.625 alleles per locus. The polymorphism information content (PIC) introduced a mean of 0.566 and there was a positive correlation between the highest value of PIC and the highest number of detected alleles by SSRs. The principal coordinate analysis, specifically, PC1 and PC2, explained 54.72% and 12.96% of the total variability, respectively, for the 32 SSR. One set of multiplex assay with five markers each (RM307, RM317, RM470, RM412 and RM242) was developed for all the 15 rice genotypes. The findings revealed that increasing genetic diversity in the national rice breeding programme requires introducing germplasm from other places with various genetic backgrounds. Furthermore, the identification, DNA barcode approach and certification of genotypes utilizing microsatellite markers could be a decent supplement to existing agro-morphological features when genotypes are firmly related.

Species composition and conservation status of shark from fishery landings and fish markets in Sri Lanka revealed by DNA barcoding

Sharks are considered to be top predators in marine ecosystems. Due to the high market value of shark fins, fishing pressure on these top predators has increased significantly and they are currently considered to be a threatened marine vertebrate group. Sri Lanka is an island nation with 21 major fish harbours in operation and one of the top 20 shark fishing countries. With an increasing local and global demand for shark meat and fins, the species consumed by Sri Lankans as well as species composition of the landing are in question because the identification of processed shark is difficult (e.g. fish filet) and there is a lack of species-specific landing data. In the present study, we applied a DNA barcoding approach to identify species composition of the shark catch in Sri Lanka. Shark tissue samples were collected in 2018 and 2019 from 10 fishing harbours and fish markets around Sri Lanka. In total, 330 out of 353 tissue samples were successfully barcoded and the results revealed 17 shark species corresponding to five families including Alopias superciliosus, Carcharhinus amblyrhynchoides, C. falciformis, C. leucas, C. limbatus, C. longimanus, C. melanopterus, C. sorrah, Chiloscyllium griseum, Hemipristis elongate, Loxodon macrorhinus, Paragaleus randalli, Prionace glauca, Rhizoprionodon oligolinx, R. acutus, Sphyrna lewini and Galeocerdo cuvier. Among them, C. falciformis (silky shark) is the most dominant (39.4 %). Variation in fishing area and fishing gear used could be the main determinants of species composition across localities. The results showed 62 % of shark species identified in this study are threatened globally based on IUCN categories. Surprisingly, two banned species, A. superciliosus and C. longimanus, were found with low abundance. The results of this study provide crucial information for improving the Sri Lankan shark management plan in the future. In addition, we suggest a regular assessment of shark landing species composition be implemented to monitor the efficiency of current management.

Evidence of cryptic diversity in freshwater Macrobrachium prawns from Indochinese riverine systems revealed by DNA barcode, species delimitation and phylogenetic approaches.

The diversity of Indochinese prawns in genus Macrobrachium is enormous due to the habitat diversification and broad tributary networks of two river basins: the Chao Phraya and the Mekong. Despite long-standing interest in SE-Asian decapod diversity, the subregional Macrobrachium fauna is still not yet comprehensively clarified in terms of taxonomic identification or genetic diversification. In this study, integrative taxonomic approaches including morphological examination, DNA barcoding, and molecular species delimitation were used to emphasize the broad scale systematics of Macrobrachium prawns in Indochina. Twenty-seven nominal species were successfully re-verified by traditional and molecular taxonomy. Barcode gap analysis supported broad overlapping of species boundaries. Taxonomic ambiguity of several deposited samples in the public database is related to inter- and intraspecific genetic divergence as indicated by BOLD discordance. Diagnostic nucleotide positions were found in six Macrobrachium species. Eighteen additional putative lineages are herein assigned using the consensus of species delimitation methods. Genetic divergence indicates the possible existence of cryptic species in four morphologically complex and wide-ranging species: M. lanchesteri, M. niphanae, M. sintangense, and some members of the M. pilimanus group. The geographical distribution of some species supports the connections and barriers attributed to paleo-historical events of SE-Asian rivers and land masses. Results of this study show explicitly the importance of freshwater ecosystems in Indochinese subregions, especially for the Mekong River Basin due to its high genetic diversity and species composition found throughout its tributaries.

DNA barcoding of gastropods Terebralia semistriata (Mörch, 1852 (Potamididae: Gastropoda)

Potamididae: Gastropods are grouped as one of family belongs to class gastropods which is found very abundant in tropical intertidal areas, especially in mangrove ecosystems. Apart from being self defense (protection), the shells of Potamididae gastropods are also useful as a key species for identification. This study aims to identify Potamididae gastropods through a barcoding DNA approach using JgLCO1490 and JgHCO2198 primers. The results of identification based on morphological characters, revealed that the Potamididae gastropods analyzed were Terebralia sp. Furthermore, identification using the barcoding DNA approach, show that the Potamididae gastropods were a type of Terebralia semistriata with a 98.93% similarity rate. The length of DNA sequence amplified with the COI gene was 655 bp. Phylogenetic analysis and genetic distance, the DNA sequences of Terebralia semistriata from Merauke form the same clade as Terebralia semistriata from Queensland, Australia with genetic distance of 0.1%. Identification species using the DNA barcoding approach makes identification of organisms easier, faster and more accurate.