Cytochrome Oxidase Subunit I Barcodes Research Articles

Declining populations of Astacus astacus drag European Branchiobdella parasita (Annelida: Clitellata) ectosymbionts to serious biodiversity loss

Branchiobdella parasita is specific ectosymbiont of European freshwater crayfish, including endangered noble crayfish (Astacus astacus). Although numerous information on distribution, biology or ecology were reported, genetic structure of B. parasita in European crayfish populations was not studied truthfully. The available molecular data include only cytochrome oxidase subunit I (COI) barcodes of B. parasita coexisting with noble crayfish in Alpine freshwaters in Austria, as well as stone crayfish (Austropotamobius torrentium) and white-clawed crayfish (Austropotamobius pallipes) in Balkan region. Here we report first genetic diversity and haplotype richness analysis of B. parasita populations on a pan-European scale. Specimens (n = 41) were collected from female noble crayfish from three lakes in northwest Poland (Kołtki Małe, Rosko Duże, Skrzynki Małe) and Elkenrother Weiher Lake in southwest Germany, high-quality COI barcodes were obtained and analysed against existing genetic data. The analysed populations were haplotype rich (h = 23) and had high COI haplotype diversity (Hd = 0.8988, I = 1.4554). Our results indicate that diversification rate of European B. parasita populations highly depends on habitat quality, its connection with surface waters and hosts species and mobility. We revealed that Central European populations inhabiting noble crayfish show significant genetic relationship, presenting common ancestral haplotypes and at the same time high genetic divergence from Balkan populations. Based on our results we suggest that ‘parasita-complex’ of phylogenetically related species with similar body shape and structure of chitin jaws, but with differentiating anatomical structure details are present across the continent.

DNA barcoding Clithon sp. (Gastropoda: Neritidae) from Badur Beach, Madura, Indonesia, based on COI gene molecular marker

Abstract. Djoemharsjah IS, Ambarwati R, Rahayu DA. 2023. DNA barcoding Clithonsp. (Gastropoda: Neritidae) from Badur Beach, Madura, Indonesia, based on COI gene molecular marker. Biodiversitas 24: xxxx. Neritidae is polymorphic with various shell colors and patterns; therefore, they were doubts about the identifying species from the genus Clithon, namely Clithon sp. from Badur Beach of Madura Island, Sumenep District, East Java Province, Indonesia. In addition, DNA barcoding could provide genetic information using short DNA sequences to quickly and precisely identify species. This study aimed to identify the genus Clithon sp. from Badur Beach, based on Cytochrome Oxidase subunit I(COI) genes and analysis of phylogenetic relationships. The research methods include sampling, sample preservation, morphological identification of species, DNA isolation, amplification, electrophoresis, and sequencing using the Sanger method with genetic analysis using bioinformatics software. The results of COI barcode identification obtained a DNA sequence length of 490bp with a similarity value of the three Clithon sp. samples between 96.75 to 98.97%. The identification with the Barcode of Life Data System consisted of three variations of nucleotide bases, and the average value of the genetic distance with the in-group was 1.74% as Clithon sp. The Phylogenetic tree Clithon sp. from Badur Beach, was in the same clade as Clithon sowerbianum Récluz 1843 and Clithon mertonianum Récluz 1843 with the Neighbor-Joining Tree and Maximum Likelihood methods with bootstrap values between 96-100. Therefore, the COI barcode DNA markers analysis successfully identified Clithon sp. from Badur Beach, Madura, Indonesia as C. sowerbianum.

A new species of Fusigobius (Teleostei: Gobiidae) from the Red Sea and Gulf of Aden

The gobiid species, Fusigobius humerosus sp. nov., is described based on 12 type and 18 non-type specimens collected from the Red Sea and Gulf of Aden. The new species can be distinguished from congeners by a combination of meristic and morphometric characters. The new species was formerly misidentified with F. humeralis: both are characterised by a semitranslucent body; head and body with numerous small dusky orange-yellow spots; a round black spot in humeral region just above base of pectoral fin; and a black spot subequal to pupil diameter at midbase of the caudal fin. However, Fusigobius humerosus sp. nov. differs from F. humeralis by scales on side of nape not extending forward to above posterior margin of preopercle (vs. scales variably extending forward to between above posterior preopercular margin and orbit); first dorsal-fin spine longest (vs. second and third dorsal-fin spines longest); shorter upper jaw; shorter anal-fin spine; and posterior nostril about halfway between orbit and anterior nostril (vs. posterior nostril closer to orbit). The most complete description of the genus Fusigobius is provided. In phylogenetic analyses of publicly available sequences of the barcoding portion of the mitochondrial cytochrome oxidase subunit I (COI) gene, sequences derived from the new species form a separate and well-divergent monophyletic lineage. The resulting COI gene tree further suggests that the new Fusigobius species is phylogenetically most closely related to F. humeralis which forms its sister species in the maximum likelihood tree. Molecular species delimitation of available Fusigobius COI barcodes shows that 19 or 20 hypothetical divergent evolutionary lineages can be deduced depending on the analytical approach (ABGD = 19 and bPTP = 20), indicating a potentially higher taxonomic richness than the presently acknowledged 11 valid species. However, the assignment of available species names for some lineages remains uncertain, highlighting the need for an additional integrative taxonomic research on this genus.

A Rapid and Cost-Effective Identification of Invertebrate Pests at the Borders Using MinION Sequencing of DNA Barcodes.

The rapid and accurate identification of invertebrate pests detected at the border is a challenging task. Current diagnostic methods used at the borders are mainly based on time consuming visual and microscopic examinations. Here, we demonstrate a rapid in-house workflow for DNA extraction, PCR amplification of the barcode region of the mitochondrial cytochrome oxidase subunit I (COI) gene and Oxford Nanopore Technologies (ONT) MinION sequencing of amplified products multiplexed after barcoding on ONT Flongle flow cells. A side-by-side comparison was conducted of DNA barcode sequencing-based identification and morphological identification of both large (>0.5 mm in length) and small (<0.5 mm in length) invertebrate specimens intercepted at the Australian border. DNA barcode sequencing results supported the morphological identification in most cases and enabled immature stages of invertebrates and their eggs to be identified more confidently. Results also showed that sequencing the COI barcode region using the ONT rapid sequencing principle is a cost-effective and field-adaptable approach for the rapid and accurate identification of invertebrate pests. Overall, the results suggest that MinION sequencing of DNA barcodes offers a complementary tool to the existing morphological diagnostic approaches and provides rapid, accurate, reliable and defendable evidence for identifying invertebrate pests at the border.

Morphological and Molecular Characterization of Several Neogastropod Species (Mollusca: Gastropoda) From Coastal Waters of Bangladesh With One New Record

The present study reports the morphological and molecular characterization of marine neogastropods collected from the South-East to the South-West coasts of Bangladesh. A total of 21 species under 13 families were identified morphologically, of which 7 species were barcoded successfully using partial sequence of mitochondrial cytochrome oxidase subunit-I (COI) gene. The average nucleotide frequencies of these species were G (guanine) = 20.57%, C (cytosine) = 18.44%, A (adenine) = 23.65%, T (thymine) = 37.35% and the GC content was 39.01%. The average Kimura two-parameter (K2P) distances of the COI barcode sequences within species, genera, and families were 20.7%, 22.0%, and 22.0%, respectively. The average inter-species genetic divergence was calculated as 21.0%. In addition, the COI barcode sequence of Nassarius stolatus was identified and submitted to the GenBank for the first time. The study also reports the new record of a species, Indothais rufotincta Tan &amp; Sigurdsson, 1996 from Bangladesh. This finding greatly extends the distributional range of this species from the West coast to the East coast of the Bay of Bengal. J. Bio-Sci. 29(1): 79-91, 2021 (June)

Phylogenetic Network of Mitochondrial COI Gene Sequences Distinguishes 10 Taxa Within the Neotropical Albitarsis Group (Diptera: Culicidae), Confirming the Separate Species Status of Anopheles albitarsis H (Diptera: Culicidae) and Revealing a Novel Lineage, Anopheles albitarsis J.

The Neotropical Albitarsis Group is a complex assemblage of essentially isomorphic species which currently comprises eight recognized species-five formally described (Anopheles albitarsis Lynch-Arribalzaga, An. deaneorum Rosa-Freitas, An. janconnae Wilkerson and Sallum, An. marajoara Galvao and Damasceno, An. oryzalimnetes Wilkerson and Motoki) and three molecularly assigned (An. albitarsis F, G & I)-and one mitochondrial lineage (An. albitarsis H). To further explore species recognition within this important group, 658 base pairs of the mitochondrial DNA cytochrome oxidase subunit I (COI) were analyzed from 988 specimens from South America. We conducted statistical parsimony network analysis, generated estimates of haplotype, nucleotide, genetic differentiation, divergence time, and tested the effect of isolation by distance (IBD). Ten clusters were identified, which confirmed the validity of the eight previously determined species, and confirmed the specific status of the previous mitochondrial lineage An. albitarsis H. High levels of diversity were highlighted in two samples from Pará (= An. albitarsis J), which needs further exploration through additional sampling, but which may indicate another cryptic species. The highest intra-specific nucleotide diversity was observed in An. deaneorum, and the lowest in An. marajoara. Significant correlation between genetic and geographical distance was observed only in An. oryzalimnetes and An. albitarsis F. Divergence time within the Albitarsis Group was estimated at 0.58-2.25 Mya, during the Pleistocene. The COI barcode region was considered an effective marker for species recognition within the Albitarsis Group and a network approach was an analytical method to discriminate among species of this group.

DNA barcoding to characterize biodiversity of freshwater fishes of Egypt.

The current study represents the first molecular characterization of freshwater fish species in Egypt from two major fish resources; the River Nile and Lake Nasser. A total of 160 DNA barcodes using a 655-bp-long fragment of the mitochondrial cytochrome oxidase subunit I (COI) gene were generated from 37 species belonging to 32 genera that represent 15 families from nine orders. The studied species were identified using different molecular-based identification approaches, in addition to the morphological identification, including neighbor-joining (NJ) trees, Barcode Index Number, and Automatic Barcode Gap Discovery (ABGD). The average genetic divergence based on the Kimura two-parameter model (K2P) within orders, families, genera, and species were 0.175, 0.067, 0.02, and 0.008, respectively. The minimum and maximum K2P distance-based genetic divergences were 0.0 and 0.154, respectively. Nucleotide diversity (π) varied among families and ranged between 0.0% for families Malapteruridae, Auchenoglanididae, Schilbeidae, Anguillidae, Centropomidae and Tetraodontidae and 17% for family Cyprinidae. The current study cautions against the lack of species coverage at public databases which limits the accurate identification of newly surveyed species and recommends that multiple methods are encouraged for accurate species identification. The findings of the current study also support that COI barcode enabled effective fish species identification in River Nile and Lake Nasser. Moreover, the results of the current study will establish a comprehensive DNA barcode library for freshwater fishes along the River Nile in Egypt. Egyptian freshwater fish DNA barcodes will contribute substantially to future efforts in monitoring, conservation, and management of fisheries in Egypt.

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.

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.

Species identification of horse flies (Diptera: Tabanidae) in Thailand using DNA barcoding

Horse flies (Diptera: Tabanidae) are of medical and veterinary importance because they are known to transmit pathogens. Approximately 80 species of horse flies have been reported in Thailand. Monitoring the distribution of horse fly species is important to control the spread of diseases transmitted by them. Currently, the species identification of horse flies is based on their morphology; this requires considerable skills and taxonomic expertise, and it may be difficult to identify morphologically similar species. DNA-based identification methods are increasingly being developed for rapid and accurate identification of various insect species. In this study, we used mitochondrial cytochrome oxidase subunit I (COI) for species identification of horse flies in Thailand. A 658 bp fragment of COI was amplified from 145 adult horse flies belonging to 48 morphologically distinct species and sequenced. Sequence analysis revealed an intraspecific divergence of 0.0%–4.4% and an interspecific divergence of 0.0%–16.2%. Our results showed that COI barcodes were effective in discriminating a majority of horse flies in Thailand on the basis of the barcoding gap and phylogenetic analyses. However, COI barcodes were unable to distinguish among members of the Tabanus striatus complex and some species within the T. ceylonicus group.

Molecular differentiation and species composition of genus Culicoides biting midges (Diptera: Ceratopogonidae) in different habitats in southern China

Culicoides biting midges (Diptera:Ceratopogonidae) cause a significant biting nuisance to humans, livestock, which are the biological vectors of a range of risky pathogens. Accurate illustration of vector play a key role in arthropod borne diseases surveillance. However, few studies have focused on the Culicoides, which caused bluetongue disease in 29 provinces of China since 1979.In this study, we assessed cytochrome oxidase subunit I (COI) mtDNA molecular marker for identification of ten major vector species and analyzed the Culicoides species community and diversity in different habitats. A total of 20,795 Culicoides samples collected from 11 sample sites were identified as 23 species belonging to 7 subgenera. Sequences of COI gene worked well as barcodes for identifying all the determined specimen in this study and were comparable with the existing sequence data from GenBank. We first reported COI barcode sequences of C. morisitai, C. insignipennis and C. homotomus. Morphological identification of Culicoides spp. samples within southern China appears relatively robust and some unidentified species were required further study. Our study shows that the COI sequence data can be used as a tool to identify species of Culicoides in Jiangxi Province.In our sampled area, the most abundant species was C. arakawae (61.89%), followed by C. oxystoma (13.77%), C. punctatus (10.10%), C. nipponensis (8.82%), C. homotomus (3.19%) and C.morisitai (1.17%) in this study. C. punctatus was the dominant species of Park habitat (62.22%), C. arakawae was the predominant species of Chicken habitat (96.66%), Vegetable plot habitat (92.0%), and Peasant household habitat (83.21%), respectively. C. oxystoma was the abundant species of Residential area habitat (40.11%), Hospital habitat (56.65%), and Pig & Cow habitat (48.77%), respectively. Results also show that the potential Bluetongue virus vectors belong to the Obsoletus and Pulicaris groups, are also included and relatively abundant, notably: Culicoides punctatus. These findings expand the current knowledge of Culicoides population composition in the southern part of China.

DNA barcoding for identifying synanthropic flesh flies (Diptera, Sarcophagidae) of Colombia

The first step for a successful use of any insect as indicator in forensic sciences is providing a precise taxonomic identification at species level. Due to morphology-based identification of Sarcophaginae flies (Diptera, Sarcophagidae) is often difficult and requires strong taxonomic expertise, their use as forensic indicators has been limited. Consequently, molecular-based approaches have been accepted as alternative means of identification. Thus, we aimed testing the efficiency of the barcode region of the mitochondrial cytochrome oxidase subunit I (COI) gene for identification of synanthropic flesh flies of several species of the genera Peckia, Oxysarcodexia, Ravinia, and Tricharaea collected in Colombia. The 645-bp fragment of COI was amplified and aligned (215 parsimoniously informative variable sites). We calculated Kimura two-parameter genetic distances and reconstruct a Neighbor-Joining phylogenetic tree. Our Neighbor-Joining tree recovered all species as monophyletic, and confirmed a new species of the genus Ravinia as also indicated by the interspecific genetic divergences and morphological observations. We obtained a 100% of identification success. Thus, the COI barcodes showed efficiency as an alternative mean of identification of species of flesh flies collected on decaying organic matter in Colombia.

Identification, Discrimination, and Discovery of Species of Marine Planktonic Ostracods Using DNA Barcodes.

The Ostracoda (Crustacea; Class Ostracoda) is a diverse, frequently abundant, and ecologically important component of the marine zooplankton assemblage. There are more than 200 described species of marine planktonic ostracods, many of which (especially conspecific species) can be identified only by microscopic examination and dissection of fragile morphological characters. Given the complexity of species identification and increasing lack of expert taxonomists, DNA barcodes (short DNA sequences for species discrimination and identification) are particularly useful and necessary. Results are reported from analysis of 210 specimens of 78 species of marine planktonic ostracods, including two novel species, and 51 species for which barcodes have not been previously published. Specimens were collected during 2006 to 2008 from the Atlantic, Indian, and Southern Oceans, Greenland Sea and Gulf of Alaska. Samples were collected from surface to 5,000 m using various collection devices. DNA sequence variation was analyzed for a 598 base-pair region of the mitochondrial cytochrome oxidase subunit I (COI) gene. Kimura-2-Parameter (K2P) genetic distances within described species (mean = 0.010 ± 0.017 SD) were significantly smaller than between species (0.260 + 0.080), excluding eight taxa hypothesized to comprise cryptic species due to morphological variation (especially different size forms) and/or collection from different geographic regions. These taxa showed similar K2P distance values within (0.014 + 0.026) and between (0.221 ± 0.068) species. All K2P distances > 0.1 resulted from comparisons between identified or cryptic species, with no overlap between intra- and interspecific genetic distances. A Neighbor Joining tree resolved nearly all described species analyzed, with multiple sequences forming monophyletic clusters with high bootstrap values (typically 99%). Based on taxonomically and geographically extensive sampling and analysis (albeit with small sample sizes), the COI barcode region was shown to be a valuable character for discrimination, recognition, identification, and discovery of species of marine planktonic ostracods.

Occurrence of Orthione griffenismarkham, 2004 (Isopoda: Bopyridae), parasite of the mud shrimp upogebia major(de Haan, 1841) in South Korean waters, and its implications with respect to marine bioinvasion

In recent years, extensive populations of burrowing mud shrimp have suddenly invaded the tidal flats in South Korea where clams, Ruditapes philippinarum, are harvested. These shrimp pose a serious threat to the clam industry along the west coast of South Korea. While conducting a mud shrimp monitoring program, we collected six female/male pairs of a pseudionine bopyrid isopod, Orthione griffenisMarkham, 2004, which in North America has been considered an introduced species from Asia that threatens northeastern Pacific estuarine ecosystems. We report the occurrence of this isopod as an inhabitant of the branchial chambers of the host shrimp, Upogebia major(de Haan, 1841), a very common species along the South Korean west coast, along with a morphological description and phylogenetic analysis based on COI barcodes. DNA analysis supported strongly that South Korean Orthioneis the same species of western Pacific (USA), O. griffenis. Two regionally disjunct isopods showed very low (0.8%) genetic variation in the mitochondrial COI (cytochrome oxidase subunit I) sequences. The implications of the occurrence of this species with respect to marine bioinvasion are also discussed.

Identification of deer species (Cervidae, Cetartiodactyla) in China using mitochondrial cytochrome c oxidase subunit I (mtDNA COI)

In China, many deer species are threatened and their wild populations are decreasing. In this study, a segment of mitochondrial cytochrome oxidase subunit I (COI) was used as a DNA barcode to identify Cervidae species. COI sequences of 30 individuals from nine species were determined. Together with 148 sequences from BOLD and Genbank, a total of 178 sequences from 21 species of the family Cervidae were analyzed. The results showed that all species had unique COI sequences, and there was no barcode sharing among them. The mean K2P distances within species, genus, and family were 1.3%, 3.4%, and 8.9%, respectively. The neighbor-joining (NJ) tree was in most cases concordant with modern deer classification. Three species showed maximum intraspecific divergences higher than their minimum interspecific divergences. However, all species could be discriminated by their diagnostic characters in BLOG analysis. The present study confirmed that COI barcodes can effectively distinguish Cervidae species in China.

COI barcodes and phylogeny of doves (Columbidae family)

Cytochrome oxidase subunit I (COI) gene has been recognized as an authentic tool for species identification. Besides its potential barcoding capacity, COI sequences have also been used for inferring the phylogeny. Phylogenetic relationships among genera of Columbidae (pigeons and doves family) have not been fully resolved because of scarce sampling of taxa and limited availability of sequence data. In this study, we have evaluated the efficiency of COI barcodes for species identification and phylogenetic analysis of various doves. We sequenced the 693 bp region of COI gene of three species of doves including Oena capensis, Streptopelia decaocto, and Streptopelia senegalensis. After retrieving the relevant sequences from the GenBank, the entire data-set of 85 sequences represented 25 dove species from 11 different genera of the family Columbidae. The COI sequences of four species including Chalcophaps indica (two specimens), Columbina inca (five specimens), Geopelia striata (three specimens), and Macropygia phasianella (three specimens) were identical. The mean intraspecific base differences ranged from 0 to 37 while the P-distances ranged between 0 and 0.058. For most of the species, the P-distances were ≤ 0.008. Phylogenetic analysis differentiated the taxa into three major clusters. One of the clusters grouped five genera including Claravis, Columbina, Gallicolumba, Geopelia, and Geotrygon. The remaining two clusters grouped three genera each including Chalcophaps, Oena, and Turtur in one cluster and Macropygia, Streptopelia, and Zenaida in another cluster. Further sub-clustering clearly separated all the genera into individual clusters except two discrepancies for the genera Streptopelia and Turtur. Species-level cladistics clearly separated all the species into distinctive clades. In conclusion, COI barcoding is a powerful tool for species identification with added information on phylogenetic inference. The finding of this study will help to understand the complex phylogeny of the family Columbidae.

Barcoding of biting midges in the genus Culicoides: a tool for species determination

Biting midges of the genus Culicoides (Diptera: Ceratopogonidae) are insect vectors of economically important veterinary diseases such as African horse sickness virus and bluetongue virus. However, the identification of Culicoides based on morphological features is difficult. The sequencing of mitochondrial cytochrome oxidase subunit I (COI), referred to as DNA barcoding, has been proposed as a tool for rapid identification to species. Hence, a study was undertaken to establish DNA barcodes for all morphologically determined Culicoides species in Swedish collections. In total, 237 specimens of Culicoides representing 37 morphologically distinct species were used. The barcoding generated 37 supported clusters, 31 of which were in agreement with the morphological determination. However, two pairs of closely related species could not be separated using the DNA barcode approach. Moreover, Culicoides obsoletus Meigen and Culicoides newsteadi Austen showed relatively deep intraspecific divergence (more than 10 times the average), which led to the creation of two cryptic species within each of C. obsoletus and C. newsteadi. The use of COI barcodes as a tool for the species identification of biting midges can differentiate 95% of species studied. Identification of some closely related species should employ a less conserved region, such as a ribosomal internal transcribed spacer.