Cytochrome Oxidase Subunit I Sequences Research Articles

A genetic approach for authentication on morphological differences of cultivated pompano species in Indonesia

Abstract. Hakim AA, Dikrurahman, Muhlis S, Astuti RP, Sari DW, Kamal MM. 2022. A genetic approach for authentication on morphological differences of cultivated pompano species in Indonesia. Biodiversitas 23: 4561-4569. Due to the increasing demand for animal protein, the Batam Marine Cultivation Fisheries Center (BPBL Batam) developed pompano (Trachinotus spp.) for aquaculture. Because of the different external characteristics, local communities recognized three types of pompano, i.e., bintang, emas, andhibridpompano. The study aimed to prove the authenticity of cultivated pompano (Trachinotus spp.) with morphological differences based on a DNA barcoding approach with 16S ribosomal RNA and cytochrome oxidase subunit I (COI) partial genesequences. A total of 30 barcode sequences from COI and 16S rRNA genes were obtained from three types of pompano, and all samples were validated as Trachinotus blochii with percent identity of 99.85-100% by COI and 100% by 16S rRNA. Emas, bintang, and hibrid pompano also have the same or very similar GC content both based on COI and 16S rRNA sequences. The average genetic distances calculated between species in the genusTrachinotus were 0.09 and 0.02 among COI and 16S rRNA sequences, respectively. Phylogenetic analysis constructed from COI and 16S rRNA gene sequences of all types of pompano and related species segregated genus into one clade (T. blochii) and the outgroup (other species) as another clade. This study contributed essential genetic data to the NCBI database both COI and 16S rRNA gene sequences. Despite having morphological differences, all types of pompano were successfully authenticated into the same species because they have identical nucleotide base arrangements and can be presumed from the same ancestor.

Identification of Phalacronotus bleekeri (Günther, 1864) (Siluridae) From the Mekong Delta, Vietnam and Use of Morphological Analysis to Separate Populations

This study identified Phalacronotus to species level and examined its morphometric variation among three collection localities, and between sexes, in the Mekong Delta, Vietnam. Specimens were collected from local markets and fishers, including An Giang (n = 36) and Can Tho (n = 36) in the Hau River and Tien Giang (n = 30) in the Tien River for morphological measurements and DNA barcoding analysis. Eight mitochondrial cytochrome oxidase subunit I (COI) sequences of Phalacronotus sp. shared 99.6 ± 0.23 % similarity with five GenBank (GB) P. bleekeri sequences and differed by 7.6 % to 13.3 % from seven GB P. apogon sequences. The Vietnamese specimens of Phalacronotus were similar to P. bleekeri in number of anal fin rays (75–80) and the shape of the upper jaw teeth. Thus, the COI sequences and these morphological characteristics indicate that Phalacronotus sp. samples collected in the Mekong Delta are Phalacronotus bleekeri (Günther, 1864). Morphometric characteristics differed between populations in 15 of 19 indices (P < 0.05) and between females and males in three characteristics relating to the belly. Head characteristics, body depth, and caudal peduncle depth were the main parameters contributing to the inter-population variation. Based on size-adjusted data, 96.7 % of specimens were correctly classified from the Tien Giang population, while 27.8 % and 33.3 % were misclassified between the Can Tho and An Giang populations, respectively. These results indicate that morphometric parameters of P. bleekeri varied mainly between populations in the two Mekong River tributaries.

Phylogeography of the veined squid, Loligo forbesii, in European waters

The veined squid, Loligo forbesii Steenstrup, 1856, occurs at the European Shelf areas including the Azores and represents a valuable resource for the European commercial fishery in the North East Atlantic. However, very little is known about its population structure and phylogeography. This lack of knowledge also impedes the development of sustainable fishery management for this species. The present study combined the use of two types of markers that retrieve patterns of gene flow in different time spans; the analysis of 16 nuclear microsatellites and sequencing of the mitochondrial cytochrome oxidase subunit I (COI). Whereas the high mutation rate of microsatellites allows the description of recent patterns of connectivity in species, the lower mutation rate of COI provides phylogeographic patterns on a longer timescale. A total of 347 individuals of L. forbesii were investigated from nearly the entire distribution range of the species, including the North East Atlantic Shelf, the Azores and the Mediterranean. Individuals from the Western and Eastern Mediterranean Sea have never been included in a genetic study before. We were able to analyse COI sequences from all 12 sampling areas and define three clades of L. forbesii. Due to our large sampling area, we are presenting 13 COI-haplotypes that were previously unknown. The microsatellite analysis does not include the Azores but three main clades could be identified at the remaining 11 sampling sites. Low FST values indicate gene flow over large geographical distances. However, the genetically significant differences and an additional slight grouping in the microsatellite structure reveal that geographical barriers seem to influence the population structure and reduce gene flow. Furthermore, both markers provide strong evidence that the observed phylogeographic pattern reflects the geographical history of the Azores and the Mediterranean Sea.

Phylogenetic Analysis of the Dominant Non-biting Midge Forcipomyia bikanni (Diptera: Ceratopogonidae) around West Lake, Hangzhou, China

Abstract The nonbiting midges are tiny insects with a wide distribution around West Lake, China. In this study, the seasonal dynamics of the local dominant midge species was investigated. Phylogenetic trees of this species also were analyzed to discover its evolutionary history. Ultraviolet (UV) light traps were used for surveillance around West Lake in the period from April 2017 to November 2018. External morphological identification was performed using a stereomicroscope. Two genes of adult female midges that encoded the cytochrome oxidase subunit I (COI) and internal transcribed spacer 1 (ITS-1) were sequenced and aligned online using GenBank nucleotide database and the Molecular Evolutionary Genetics Analysis (MEGA) software 10.1.7. According to the field surveillance in 2017–2018, Forcipomyia bikanni Chan and LeRoux (Diptera: Ceratopogonidae) is the representative dominant species of West Lake, with its activity peaking in August and October. The COI and ITS-1 gene sequences of F. bikanni (XH01-5) were 523 bp and 315 bp, respectively. The final phylogenetic tree of the COI implied that F. bikanni (XH01-5) has 97.9% significant similarity with conspecific F. bikanni (LC015045) from the adjacent Fujian Province, China (E-value ≤ 0.1%). Furthermore, the ITS-1 sequences with 98% likelihood showed the highest linkage between F. bikanni (XH01-5) and F. townsvillensis (Taylor) (HM775502-HM775504) with different branches. Thus, the final unrooted phylogenetic trees of COI and ITS-1 are essential tools for reconstructing the evolutionary histories of nonbiting midges.

Two new pseudocryptic species in the medium-sized common European land snails, Fruticicola Held, 1838; as a result of phylogeographic analysis of Fruticicola fruticum (O. F. Müller, 1774) (Gastropoda: Helicoidea: Camaenidae)

Fruticicola fruticum (O. F. Müller, 1774), a medium-sized helicoid snail in the Camaenidae, has a wide range in Europe, reaching from the Urals and the Caucasus to the Balkans, and from the southern part of Scandinavia, through Central Europe to eastern and central France and northern Italy. There are numerous studies on its distribution, biology, life cycle, etc., but little is known about the genetic diversity of this taxon. Here, we studied the phylogeny and phylogeography of F. fruticum using two mitochondrial markers: cytochrome oxidase subunit I (COI) and 16S ribosomal RNA (16S); and four nuclear markers: 18S ribosomal RNA (18S), 28S ribosomal RNA (28S), internal transcribed spacer (ITS-2), and histone 3 (H3). The study was based on 59 populations sampled across the range. Whereas nuclear markers showed little differentiation, phylogenetic analysis of COI sequences clearly confirmed the distinctness of the European Fruticicola and Asian Bradybaena (p-distance 0.229). Within Fruticicola 54 haplotypes were detected, haplotype diversity (Hd) = 0.973 ± 0.006, nucleotide diversity (π) = 0.137 ± 0.005. ABGD and PTP delimitation analyzes distinguished eight mOTUs. Two sequences (our mOTU C) from Russia were published in the GenBank as two distinct species: F. schrenckii and F. transbaicalia. Seven further mOTUs identified in our study formed three distinct lineages, regarded as species. The first (mOTU A and mOTU B), represented by 40 populations, occupies a wide range across northern and central Europe, extending east to Ukraine and south to northern Croatia (mOTU B). It encompasses the type locality of F. fruticum, and can be recognized as F. fruticum sensu stricto. Another lineage (mOTU D and mOTU E), represented by six populations in central Romania, appears to form another species. Both mOTUs were found together in one population. A third lineage, containing mOTUs F, G and H, represented by 14 populations, was distributed across the Balkans from N.E. Croatia to Bulgaria. p-distances between the three species ranged from 0.172 to 0.219, and between all the mOTUs, pooled together, from 0.172 to 0.258. The highest genetic diversity was found in species 3 (0.112) and the lowest in species 1 (0.025), despite its largest geographic distribution. Pairwise p-distances, Tamura 3-parameter distances, composite likelihood distances, as well as the coancestry coefficient FST, calculated for all populations pooled together were significantly associated with geographic distance, but this was not the case within each of these three species. The significant association for all populations reflected high diversity between the species coupled with high geographic distances between their populations, not the character of intraspecies diversity. With a few exceptions, there hold a rather infinite island model with low migration. AMOVA detected 78% of the variance between the three species, 18% among populations within the species, and only 3.6% within the populations. The low genetic diversity of widespread F. fruticum s. stricto, compared with much higher diversity of two narrowly distributed newly found species of Fruticicola, may reflect the rapid spread of the former into previously uninhabitable regions, while the latter were able to maintain populations in glacial refugia. The estimated time of divergence between the three species, 1.7–2.19 mya, suggests their ancestors’ isolation in southern European refugia during the lower Pleistocene, the Gelasian/Calabrian. There was no clear association of variation in shell morphology and lineage or mOTU identity; on external characters, these species are semicryptic, subtle differences in reproductive anatomy among them were found.

New records of the occurrence of Megaleporinus macrocephalus () (Characiformes, Anostomidae) from the basins of the Itapecuru and Mearim rivers in Maranhão, Northeastern Brazil.

The "piaussu", Megaleporinus macrocephalus is an anostomatid fish species native to the basin of the Paraguay River, in the Pantanal biome of western Brazil. However, this species has now been recorded in a number of other drainages, including those of the upper Paraná, Uruguay, Jacuí, Doce, Mucuri, and Paraíba do Sulrivers. This study presents two new records of the occurrence of M. macrocephalus, in the basins of the Itapecuru and Mearim rivers in the state of Maranhão, in the Brazilian Northeast. The piaussu is a large-bodied fish of commercial interest that is widely raised on fish farms, and its occurrence in the Itapecuru and Mearim rivers is likely the result of individuals escaping from fish tanks when they overflow during the rainy season. Morphological analyses and sequences of the Cytochrome Oxidase Subunit I (COI) gene confirmed the taxonomic identification of the specimens as M. macrocephalus. The COI sequences were 99.66% similar to those of M. macrocephalus deposited in the BOLDSystems database. These records extend the known distribution of M. macrocephalus to the basins of the Itapecuru and Mearim rivers in the Brazilian Northeast, highlighting a new case of introduction of exotic fish species into Brazilian river basins.

The first genetic characterization of Setaria marshalli (Nematoda, Spirurida) with reliable DNA barcoding based on a mitochondrial genetic marker.

Setaria marshalli is a mosquito-borne filarial nematode that causes infection in calves younger than two years old. In the present study, nematodes were obtained from a calf in Japan and morphologically identified as S. marshalli. Additionally, the partial cytochrome oxidase subunit I (COI) region (596 bp) was analyzed for the first time to establish a reliable DNA barcode. Nucleotide sequences of COI were identical among the seven worms obtained. The COI region can be a useful marker for species discrimination in the case of S. marshalli since nucleotide variations observed between the closest congener, Setaria cervi (51/596 bp), were sufficient to allow species discrimination. However, the phylogenetic relationship of S. marshalli with its congeners was unclear in a maximum likelihood tree. We found that the partial COI sequence of S. marshalli analyzed in the present study matched a relevant section of the complete mitochondrial genome of S. labiatopapillosa that was deposited in the International Nucleotide Sequence Database. This finding suggests that S. marshalli was misdiagnosed as S. labiatopapillosa in a previous study. It is crucial to conduct accurate morphological analyses to obtain reliable molecular information regarding Setaria nematodes.

Morphological and Molecular Evidence Reveals the Longnose Skate Zearaja brevicaudata (Marini, 1933) to be a Senior Synonym of Dipturus lamillai Concha, Caira, Ebert & Pompert 2019.

Longnose skates have great economic importance in South American fisheries, and in order to preserve them, it is important to have a well-defined taxonomic status of their species. Dipturus lamillai was recently described for Malvinas Islands waters based on morphological and molecular comparisons with Zearaja chilensis. Although D. lamillai has been compared with several congeneric species, it was not properly compared with the morphologically similar Zearaja brevicaudata, the most abundant longnose skate in the Southwest Atlantic. Here, these species were compared by morphological and molecular analyses in order to evaluate their conspecificity. Linear morphometric variables of holotype and paratypes of D. lamillai and 69 specimens of Z. brevicaudata were compared and investigated using Principal Component Analysis. In addition, thorn patterns, denticle distributions, color, and clasper morphology were compared. No body proportions or other single character that could differentiate D. lamillai from Z. brevicaudata were found. Molecular analyses comprised of the comparison of the Cytochrome oxidase subunit I (COI) and the NADH dehydrogenase subunit 2. The results of the Maximum Likelihood (ML) carried out for each molecular marker showed that sequences from D. lamillai clustered together with those of Z. brevicaudata, and the molecular distance determined by Kimura two-parameter were lower than the expected for different species. Additionally, the Automatic Barcode Gap Discovery method and the Bayesian implementation of the Poisson tree processes were carried out with COI sequences to explore species limits, and their results were consistent with ML analyses. In summary, the results obtained showed that there are no morphological or molecular differences between these nominal species of the valid skate genus Zearaja, leading to the conclusion that they are conspecific. Therefore, we designated Z. brevicaudata as a senior synonym of D. lamillai.

Elucidating species diversity of genus Betta from Aceh waters Indonesia using morphometric and genetic data

Species of the genus Betta are very similar morphologically which could lead to inaccuracy in taxonomic identification. Therefore, morphological data should also be complemented with other approaches such as genetics to validate the taxonomic status of member species. The objective of this study was to validate the taxonomic status of the genus Betta from Aceh waters using morphometric and genetic data. Sampling was conducted from January to September 2020 at 12 locations within Aceh province. Based on initial assessment identification of morphology, there were four presumed species of Betta in Aceh waters, namely; Betta rubra, Betta dennisyongi, Betta splendens, Betta imbellis. A total of 50 samples from each presumed species were assessed for traditional and truss morphometric analysis, while 5 specimens were randomly selected from each morphospecies for genetic analysis based on the cytochrome oxidase subunit I (COI) gene. The traditional morphometric data divided the four Betta species into two group with B. rubra and B. dennisyongi in same group (group I), while B. splendens and B. imbellis in different group (group II). While, the truss morphometric data divided the four Betta species into three groups with B. rubra and B. dennisyongi in group 1, B. splendens in group 2, while B. imbellis form the other group. The head depth of the fishes was an important characteristic to distinguish members of the four species. In parallel, the genetic analysis supported the occurrence of four valid species with intraspecific values of 0.0–1.59% and interspecific values ranging from 3.3 to 24.7%, the same clustering relationship as in the morphometric data. While B. rubra, B. splendens, and B. imbellis sequences are already available in GenBank, this study reports the first record of B. dennisyongi COI sequences.

Molecular characterization and phylogenetic analysis of collected mosquitoes (Diptera: Culicidae) from Northcentral Nigeria using mitochondrial COI and ribosomal IGS gene regions

BackgroundMosquitoes are important vectors of disease-causing organisms such as filarial worms, malaria parasites, and arboviruses endemic to sub-Saharan Africa including Nigeria. Malaria is a disease caused by a plasmodium parasite, transmitted by the bite of infected mosquitoes and is no doubt a public health concern. There is limited information on the genetic diversity of mosquitoes in Nigeria. This is necessary because information about the genetic diversity of mosquitoes is a very important step towards vector control and management with the aim to mitigate or eliminate burden resulting from malaria and other diseases caused by mosquitoes. In the present study, we investigated the genetic variability and relatedness of mosquitoes based on the DNA sequences of the mitochondrial cytochrome oxidase subunit I (COI) and ribosomal intergenic non-coding spacer gene regions (IGS). Mosquitoes were collected from five different states in Northcentral Nigeria, they were morphologically identified using standard keys and genomic DNA was extracted. The specific regions of interests were amplified, and the PCR products were then sequenced.ResultsPCR was able to successfully amplify the expected amplicon sizes of COI and IGS sequences (710 and 169) base pairs, respectively. For COI sequence, pairwise genetic distance between mosquito species ranged from 0.00 to 0.17 in the COI sequences. The pairwise genetic distance among Culex, Aedes and Anopheles species in the IGS sequences ranged from 0.000 to 0.118. Phylogenetic analysis of the sequences of the mitochondrial cytochrome oxidase subunit showed that there was genetic diversity amongst the different mosquito species sampled. It effectively showed marked differences between Culicine and Anopheline mosquitoes.ConclusionsThe ribosomal IGS primers used for this study only amplified Anopheles spp. However, it revealed that there is diversity among the Anopheles gambiae and Anopheles arabiensis samples collected. This study concludes that the mitochondrial COI and ribosomal IGS gene regions are reliable markers for mosquito genetic diversity study and will surely yield a reliable result for molecular diversity assessment of mosquito species.

DNA Barcoding of Penaeidae (Decapoda; Crustacea): Non-Distance-Based Species Delimitation of the Most Economically Important Shrimp Family

The Penaeidae family includes some of the most economic and ecological important marine shrimp, comprising hundreds of species. Despite this importance and diversity, the taxonomic classification for penaeid shrimp has constantly been revised, and issues related to the species identification are common. In this study, we implemented DNA barcoding analyses in addition to single-gene species delimitation analyses in order to identify molecular operational taxonomy units (MOTUs) and to generate robust molecular information for penaeid shrimp based on the cytochrome oxidase subunit I (COI) mitochondrial gene. Our final data set includes COI sequences from 112 taxa distributed in 23 genera of penaeids. We employed the general mixed Yule coalescent (GMYC) model, the Poisson tree processes (PTP), and the Bayesian PTP model (bPTP) for MOTUs delimitation. Intraspecific and interspecific genetic distances were also calculated. Our findings evidenced a high level of hidden diversity, showing 143 MOTUs, with 27 nominal species not agreeing with the genetic delimitation obtained here. These data represent potential new species or highly structured populations, showing the importance of including a non-distance-based species delimitation approach in biodiversity studies. The results raised by this study shed light on the Penaeidae biodiversity, addressing important issues about taxonomy and mislabeling in databases and contributing to a better comprehension of the group, which can certainly help management policies for shrimp fishery activity in addition to conservation programs.

DNA barcoding of lamp shells (Brachiopoda: Lingula anatina) from Probolinggo, East Java, Indonesia

Abstract. Ambarwati R, Rahayu DA, Rachmadiarti F, Khaleyla F. 2021. DNA barcoding of lamp shells (Brachiopoda: Lingula anatina) from Probolinggo, East Java, Indonesia. Biodiversitas 22: 1764-1774. Lingula anatina is one of brachiopods found in tropical regions, however, the reports on this species from South East Asia is currently limited. This study, therefore, aims to identify the Cytochrome Oxidase subunit I (COI) sequence and phylogenetic of lamp shells from Probolinggo, Indonesia. A total of five samples of lamp shells were collected from Probolinggo, Indonesia, then identified based on morphological characters, and the identification was confirmed using molecular data. Subsequently, molecular characterization and identification were conducted based on 657 bp of COI gene similarity, sequence variation, genetic distance, phylogenetic topology, and BOLD System. According to the results, the Lingula species found in Probolinggo Indonesia was Lingula anatina. The median-joining network and Automatic Barcode Gap Discovery (ABGD) analysis showed this Lingula anatina population from Probolinggo had relatives of 22 haplotypes and divided into six haplogroups. Furthermore, the population belonged to the same haplogroup but had different haplotypes. Thus, this study provides more information for Lingula anatina phylogenetic and haplotype studies.

Characterization of marine eukaryotic biofilms at offshore wind farm sites: assessment of DNA extraction methods and marker gene used for metabarcoding approaches

Among marine lifestyles, biofilms are considered as diversified communities embedded in complex exopolymers whose development depends on several factors, related to both environmental conditions and physical-chemical characteristics of substrates (Antunes et al. 2019, Bellou et al. 2012). For the maritime industry, bio-colonization and its impact on human activities were well-described (Schultz et al. 2011). However, this phenomenon represents a new challenge in Renewable Marine Energies (RME) due to their specificities (materials, structures, localization…). In particular, macro-organism assemblages appeared to include a wide variety of eukaryotic groups but the literature is sparse considering the sequencing of eukaryotic diversity in comparison to those of bacterial communities (Briand et al. 2018, Dang and Lovell 2000, Salta et al. 2013). As a matter of fact, the very small size of some of the eukaryotes and/or their insufficient morphological discernible features appear to considerably limit their detection and identification, leading to underestimate their diversity (Carugati et al. 2015). When talking about molecular approaches, analysis of eukaryotes also represents a challenge because such organisms possess resilient cellular structures which can give poor DNA extraction yield (Hermans et al., 2018Hermans et al. 2018). In addition, SSU rRNA in eukaryotes fails to be as universal as for prokaryotes (Bik et al. 2012, Medinger et al. 2010). However, the use of marker genes from environmental DNA, when focused on the targeted eukaryotic community, remains critical to decoding the complexity of marine biofilms diversity. In this study, four extraction methods, including a preliminary mechanic cell lysis, both soil and biofilm kits, and global approaches, have been compared. We also examined the coverage and the identification capability of several primers to characterize eukaryotic communities colonizing three plastic surface types (polyvinyl chloride, HD polyethylene, and polyamide) which have been immersed in several locations along the French Mediterranean and Atlantic coasts. Sequence quality and number remain the same whatever the extraction method. However, the richness and community structure were clearly affected regardless of the sample type (Figure 1). Finally, two kits (PowerMaxSoil, and PowerBiofilm kits) evaluated in this study were considered as the most powerful overall. Secondly, we amplified and sequenced short fragments of two genes: one region of the mitochondrial Cytochrome Oxidase subunit I (COI) and five variable regions of the 18S small subunit ribosomal DNA (rDNA) gene (V1V2, V4TAR, V4UNI, V7, and V9). The Chao1 index was considerably lower for the CO1 gene compared to those of the 18S rDNA regions. The V4TAR and V7 regions showed a significant highest richness, followed closely by the V1V2 and V9 regions. The 18S rDNA gene sequences were dominated by microeukaryotes whereas the COI sequences were dominated by macro-organisms. Each of the 18rDNA primer pairs also exhibited dissimilar community structures although the dominant taxa seemed to be common. To conclude, our results provided a global assessment of tools dedicated to the description of the diversity of marine eukaryotes biofilms from three surfaces used in the design of RME. Among the four extraction methods described here, PowerMaxSoil and PowerBiofilm kits allowed recovering the highest diversity. COI and 18S rDNA gene sequencing covered different groups including at high taxonomic levels. Despite limitations, metabarcoding will help in the characterization of marine biofilms diversity on RME. Especially, it may be relevant to use primers targeting these two genes to better cover the eukaryotic diversity.

Population genetic structure of Semisulcospira gottschei: simultaneous examination of mtDNA and microsatellite markers.

Semisulcospira gottschei is an Asian endemic species inhabiting Korea and China. However, genetic structure analysis of the resource management of this species has not been performed. To investigate the genetic diversity among populations, microsatellites can be used to determine the geographic origins of marine and freshwater species. This study investigated the genetic structures of the Korean and Chinese populations of S. gottschei based on mitochondrial DNA (mtDNA) Cytochrome oxidase subunit I (COI) and polymorphic microsatellite loci developed from Semisulcospira coreana. Analysis of the mtDNA COI sequence revealed 43 haplotypes, which indicated no gene flow between the Korean and Chinese populations. To further elucidate the genetic structures of the Korean and Chinese populations, the population genetics of S. gottschei were analyzed using nine microsatellite markers. The genetic diversity analysis showed an average of 5.25 alleles per locus, with an average allelic richness of 4.02. Excessive homozygosity was found at all loci, which was expected to be due to the presence of null alleles at all loci. Populations of S. gottschei formed two separate clusters according to pairwise FST and AMOVA. Also, the UPGMA tree, PCA, STRUCTURE, and GeneClass indicated separation of the 11 populations into two clusters: Korea and China. These results have potential use in the management, restoration, and distinction of the origin country of populations.

DNA barcoding of commercially important groupers (Epinephelidae) in Aceh, Indonesia

Here, we report for the first time a comprehensive survey of the DNA barcoding of commercially important groupers in Aceh waters, Indonesia using the cytochrome oxidase subunit I (COI) gene. The fish samples were collected from major fish landing sites and fish markets in nine districts in Aceh, that covered the western and eastern coastal ecosystems of Aceh. In total, the 144 COI sequences revealed 26 grouper species with most represented by multiple specimens. As expected, genetic distances increased from lower to higher taxonomic levels: 0 %–1.27 % within species, 3.79 %–19.96 % within genus and 13.84 %–24.15 % within family. Out of the total species, 23 (88.5 %), were categorised as Least Concern (LC) and three species (11.5 %) were Data Deficient (DD) under the IUCN Red List. Furthermore, four species were categorized under ‘decreasing population trend’ (Epinephelus bleekeri, E. coioides, Plectropomus leopardus, and Variola albimarginata). This study is a significant contribution to the DNA barcode library of groupers in this region and expansion of the Indonesian and the global barcode entries in general.

Identification of fungus-growing termites and mutualistic Termitomyces from two provinces in Thailand

Fungus growing termites is a unique group of termites that can grow mutualistic fungi from Basidiomycota: Termitomyces inside their nest. This group of termites has an important ecological role as a major decomposer. However, comprehensive study of fungus-growing termites is limited in Thailand and the complex termites-fungus relationship is a challenging issue to study. This research was aimed to identify the species of termites and fungal symbionts using DNA barcoding, to compare the molecular results with morphological identification, and to gain insights into the co-evolutionary relationship by constructing co-cladogenesis analysis from the termites and the fungal symbionts. Four termite species (Macrotermes annandalei, Odontotermes feae, Ancistrotermes pakistanicus and Ancistrotermes sp.) from four Sakaerat and two Chulabhorn colonies were identified based on the BLAST-N of cytochrome oxidase sub-unit I (COI) sequence and morphological identification. The partial COI sequence of O. feae identified in this study is the first record of this species deposited into the NCBI nucleotide database. Molecular identification of the fungi were conducted by using nuclear ribosomal internal transcribed spacer (ITS) region. The results showed that those six termite colonies grew specifically the fungal genus Termitomyces. Three genetic groups of Termitomyces i.e. Group 1, Group 2 (T. cylindricus), and Group 6, were present among the six colonies. Molecular identification was confidently supported by morphological identification of termite samples, at least up to the genus level. High host-symbionts specificity is considerably observed within some clades on paired termite-fungus phylogeny. However, more sampling is needed for the more confident result.

Study on Biometric Parameters and Genetic Diversity of Labeo rohita from Harike Wetland-A Ramsar Site

Background: Harike wetland (31°08¢ N to 31°23¢ N latitudes and 74°90¢ E to 75°12¢ E longitudes) is an internationally important Ramsar site, supports rare, vulnerable and endangered plants, fish and other faunal species. Weed infestation, pollution and encroachment are considered as important threats of its biota thus time series data with respect to fish diversity, catch composition and genetic variability has an utter importance to interpret changes over time. Cyprinidae family of freshwater fishes includes carps is the largest fish family found in Harike wetland comprising around 50% of total fish composition by weight basis and among carps, Rohu (Labeo rohita) is one of the commercially important food fish species available throughout the year and preferred by consumers. As river Beas and Sutlej, two major rivers of Indus river system confluence at Harike thus possibility of variation within fish stock is very high. With this background present study was carried out to evaluate the fish biodiversity and catch composition of Harike wetland. Study also emphasized on biometrics, length –weight relationship and genetic diversity based on mitochondrial marker genes of commercially important food fish Labeo rohita. Methods: Assessment of fish catch composition and diversity were conducted in landing centre adjacent to Harike wetland through Rapid Fisheries Assessment by Market Survey (RFAMS) technique. Fin tissue samples were collected for genetic diversity analysis of L. rohita by cytochrome oxidase subunit I (COI) gene sequencing, From the fish genomic DNA a partial fragment of approximately 655bp was PCR amplified by FishF1 (5'-TCAACCAACCACAAAGACATTGGCAC-3') and FishR1 (5'-TCGACTAATCATAAAGATATCGGC AC-3') primer pair. Calculation of intraspecific mean and pairwise distances was performed by MEGA 6.0 software using the K2P parameters Results: Total 30 species of fishes were recorded from Harike wetland and these belong to 14 families and 21 genera. In L. rohita average weight (Wt), total length (TL), standard length (SL) and forked length (FL) were recorded 2600±5.64g (1700-3600 g), 58.2±5.65 cm (51.3-67.6cm), 48.0±8.54 cm (42.5-55.5cm) and 46.0±0.25 cm (38.0-54.5cm), respectively. Biometric study revealed that sufficient numbers of mature L. rohita are available in wetland. L. rohita established negative algometric growth (b= 2.701); thus species became slender as it increased in length. The pairwise distances ranged from 0.00 to 18.49% with a mean ±S.E value of 4.70%±0.40. Total of 9 haplotypes were observed in L. rohita COI sequences. The diversity in haplotype and nucletide values were observed 0.848 and 0.024, respectively. The present study states that L. rohita stocks in Harike wetland are genetically diverse.

New records and DNA barcoding of deer flies, Chrysops (Diptera: Tabanidae) in Thailand

Chrysops spp. or deer flies (Diptera: Tabanidae) are hematophagous flies of medical and veterinary importance and some species are important vectors of Trypanosoma evansi, the causative agent of surra in Thailand. However, data regarding deer fly species and their molecular identification are limited. Accurate species identification will indicate the appropriate control measures. In this study, an entomological survey of deer flies from different sites in Thailand between May 2018 and June 2019 were conducted. In addition, mitochondrial cytochrome oxidase subunit I (COI) barcoding region was used for species identification. A total of 82 females were collected and 6 species were identified. Of these, three species are new records for Thailand: C. designatus, C. fuscomarginalis and C. vanderwulpi bringing the species total found in Thailand to nine. The COI sequences revealed an intraspecific divergence of 0.0%–2.65% and an interspecific divergence of 7.03%–13.47%. Phylogenetic analysis showed that all deer fly species were clearly separated into distinct clusters according to morphologically identified species. These results indicated that COI barcodes were capable in discriminating between deer fly species on the basis of the barcoding gap and phylogenetic analysis. Therefore, DNA barcoding is a valuable tool for species identification of deer flies in Thailand.

First report of the fall armyworm, Spodoptera frugiperda (Smith, 1797) (Lepidoptera, Noctuidae), a new migratory pest in Korea.

The fall armyworm, Spodoptera frugiperda (Smith, 1797), originated from tropical and subtropical America is one of sporadic agricultural pests in the world. Since the moth has high migration capacity, it rapidly expanded the world distribution such as Africa in 2016, India in 2018, and East-Asian countries in 2019. In Korea, this species was firstly found at maize fields of Jeju Island, in early June 2019, and subsequently detected at many counties of Jeolla-do and Gyeongsang-do in June and July 2019. The first invaded populations of S. frugiperda in Korea were genetically confirmed as one species, S. frugiperda by using a mitochondrial cytochrome oxidase subunit I (COI) gene, and analyzed to be comprised of two haplotypes (hap-1 and hap-2) each belonging to different clades. Among 31 COI sequences, the hap-1 sequence was predominant, accounting for 93.5%.

Molecular identification of Cerithiidae (Mollusca: Gastropod) in Hainan island, China

A number of same species of Cerithiidae are morphologically unlike, whereas most of species in the same genus are morphologically similar and just exhibit subtle differences. It is difficult to identify them by morphological methods alone. DNA barcoding is a modern molecular technique that can be used to identify species accurately, and is particularly helpful when distinguishing morphologically similar species. In order to identify species of Cerithiidae using DNA barcoding technology based on mitochondrial cytochrome oxidase subunit I (COI) and 16S ribosomal RNA (16S rRNA) genes, this study calculated intraspecific and interspecific genetic distance and constructed the phylogenetic trees. A total of 80 COI and 16S rRNA barcode sequences were obtained from 10 species and 3 genera. Some unknown specimens were further identified and a cryptic species may exist in Cerithium traillii, showing that DNA barcoding technology has the potential to discover new species and cryptic species. The phylogenetic trees revealed that all of the cerithiids could converge upon a monophyly with high support values and two genera (Cerithium and Clypeomorus) maybe support the reclassification. It is necessary for traditional morphological methods to combine with the DNA barcoding for classification and identification of Cerithiidae.