DNA Barcoding Research Articles

Species Composition of a Small Mammal Community and Prevalence of Echinococcus spp. in the Alpine Pastoral Area of the Eastern Tibetan Plateau.

We aimed to investigate the species composition of a small mammal community and the prevalence of Echinococcus spp. in a typical endemic area of the Tibetan Plateau. One pika and five rodent species were identified based on the morphological characteristics of 1278 small mammal specimens collected during 2014-2019. Detection of Echinococcus DNA in tissue samples from small mammal specimens revealed that Ochotona curzoniae (pika, total prevalence: 6.02%, 26/432), Neodon fuscus (5.91%, 38/643), N. leucurus (2.50%, 3/120), and Alexandromys limnophilus (21.74%, 10/46) were infected by both E. multilocularis and E. shiquicus; Cricetulus longicaudatus (16.67%, 1/6) was infected by E. shiquicus; and no infection was detected in N. irene (0/15). Neodon fuscus and O. curzoniae were the two most abundant small mammal species. There was no significant difference in the prevalence of pika and the overall rodent species assemblage (6.26%, 53/846); however, the larger rodent populations suggested that more attention should be paid to their role in the transmission of echinococcosis in the wildlife reservoir, which has long been underestimated. Moreover, although DNA barcoding provides a more efficient method than traditional morphological methods for identifying large numbers of small mammal samples, commonly used barcodes failed to distinguish the three Neodon species in this study. The close genetic relationships between these species suggest the need to develop more powerful molecular taxonomic tools.

PsbA-trnH Intergenic Spacer profile of Wax Apple (Syzygium samarangense (Blume) Merr. & L.M. Perry) Cultivars

The fruit known as wax apple, scientifically named Syzygium samarangense (Blume) Merr. & L.M. Perry, is a well-liked agricultural product originating from Demak Regency in the Central Java Province. When it is difficult to distinguish between different forms of an organism, using DNA barcoding to authenticate the identity of species based on their DNA is an important way. The psbA-trnH intergenic spacer is a commonly used molecular approach to analyse the genetic characteristics of plant species. This study investigated the psbA-trnH intergenic spacer profile of wax apple cultivars from Demak regency. The investigation revealed that the psbA-trnH intergenic spacer sequences of the S. samarangense cultivars, namely Madu Thailand and Madu Deli Hijau, had lengths of 535 and 492 base pairs, respectively. Both cultivars demonstrate a greater nucleotide composition of deoxyadenylic acid (A) and deoxythymidylic acid (T) in comparison to deoxycytidilic acid (C) and deoxyguanylic (G). The genetic distance between S. samarangense ‘Madu Thailand’ and ‘Madu Deli Hijau’ indicates a very tight relationship, with a value of 0.000. The psbA-trnH intergenic spacer proved to be insufficient in differentiating the S. samarangense cultivars from Demak regency, mostly because to its low capacity to discern between the wax apple cultivars. Keywords: Demak, psbA-trnH intergenic spacer, Syzygium samarangense, wax apple

Delimiting species, revealing cryptic diversity, and population divergence in Qinghai-Tibet Plateau weevils through DNA barcoding.

The Leptomias group represents one of the most diverse taxonomic group of weevils in the Qinghai-Tibet Plateau and its adjacent areas. Despite the potential of hidden diversity, relatively few comprehensive studies have been conducted on species diversity in this taxonomic group. In this study, we performed DNA barcoding analysis for species of the Leptomias group using a comprehensive DNA barcode dataset that included 476 sequences representing 54 morphospecies. Within the dataset, our laboratory contributed 474 sequences, and 390 sequences were newly generated for this study. The average Kimura 2-parameter distances among morphospecies and genera were 0.76% and 19.15%, respectively. In 94.4% of the species, the minimum interspecific distances exceeded the maximum intraspecific distances, indicating the presence of barcode gaps in most species of Leptomias group. The application of Automatic Barcode Gap Discovery, Assemble Species by Automatic Partitioning, Barcode Index Number, Bayesian Poisson tree processes, jMOTU, and Neighbor-joining tree methods revealed 45, 45, 63, 54, and 55 distinct clusters representing single species, respectively. Additionally, a total of four morphospecies, Leptomias kangmarensis, L. midlineatus, L. siahus, and L. sp.9RL, were found to be assigned to multiple subclade each, indicating the geographical divergences and the presence of cryptic diversity. Our findings of this study demonstrate that Qinghai-Tibet Plateau exhibits a higher species diversity of the Leptomias group, and it is imperative to investigate cryptic species within certain morphospecies using integrative taxonomic approaches in future studies. Moreover, the construction of a DNA barcode reference library presented herein establishes a robust foundational dataset to support forthcoming research on weevil taxonomy, phylogenetics, ecology, and evolution.

Amplifiable protein identification via residue-resolved barcoding and composition code counting.

Ultrasensitive protein identification is of paramount importance in basic research and clinical diagnostics but remains extremely challenging. A key bottleneck in preventing single-molecule protein sequencing is that, unlike the revolutionary nucleic acid sequencing methods that rely on the polymerase chain reaction (PCR) to amplify DNA and RNA molecules, protein molecules cannot be directly amplified. Decoding the proteins via amplification of certain fingerprints rather than the intact protein sequence thus represents an appealing alternative choice to address this formidable challenge. Herein, we report a proof-of-concept method that relies on residue-resolved DNA barcoding and composition code counting for amplifiable protein fingerprinting (AmproCode). In AmproCode, selective types of residues on peptides or proteins are chemically labeled with a DNA barcode, which can be amplified and quantified via quantitative PCR. The operation generates a relative ratio as the residue-resolved 'composition code' for each target protein that can be utilized as the fingerprint to determine its identity from the proteome database. We developed a database searching algorithm and applied it to assess the coverage of the whole proteome and secretome via computational simulations, proving the theoretical feasibility of AmproCode. We then designed the residue-specific DNA barcoding and amplification workflow, and identified different synthetic model peptides found in the secretome at as low as the fmol/L level for demonstration. These results build the foundation for an unprecedented amplifiable protein fingerprinting method. We believe that, in the future, AmproCode could ultimately realize single-molecule amplifiable identification of trace complex samples without further purification, and it may open a new avenue in the development of next-generation protein sequencing techniques.

Genotypic Identification of Trees Using DNA Barcodes and Microbiome Analysis of Rhizosphere Microbial Communities.

DNA barcodes can provide accurate identification of plants. We used previously reported DNA primers targeting the internal transcribed spacer (ITS1) region of the nuclear ribosomal cistron, internal transcribed spacer (ITS2), and chloroplast trnL (UAA) intron to identify four trees at Bergen Community College. Two of the four trees were identified as Acer rubrum and Fagus sylvatica. However, Quercus was only identified at the genus level, and the fourth tree did not show similar identification between barcodes. Next-generation sequencing of 16S rRNA genes showed that the predominant bacterial communities in the rhizosphere mainly consisted of the Pseudomonadota, Actinomycetota, Bacteroidota, and Acidobacteriota. A. rubrum showed the most diverse bacterial community while F. sylvatica was less diverse. The genus Rhodoplanes showed the highest relative bacterial abundance in all trees. Fungal ITS sequence analysis demonstrated that the communities predominantly consisted of the Ascomycota and Basidiomycota. Quercus showed the highest fungi diversity while F. sylvatica showed the lowest. Russula showed the highest abundance of fungi genera. Average similarity values in the rhizosphere for fungi communities at the phylum level were higher than for bacteria. However, at the genus level, bacterial communities showed higher similarities than fungi. Similarity values decreased at lower taxonomical levels for both bacteria and fungi, indicating each tree has selected for specific bacterial and fungal communities. This study confirmed the distinctiveness of the microbial communities in the rhizosphere of each tree and their importance in sustaining and supporting viability and growth but also demonstrating the limitations of DNA barcoding with the primers used in this study to identify genus and species for some of the trees. The optimization of DNA barcoding will require additional DNA sequences to enhance the resolution and identification of trees at the study site.

DNA barcoding is currently unreliable for species identification in most crayfishes.

DNA barcoding is commonly used for species identification. Despite this, there has not been a comprehensive assessment of the utility of DNA barcoding in crayfishes (Decapoda: Astacidea). Here we examined the extent to which local barcoding gaps (used for species identification) and global barcoding gaps (used for species discovery) exist among crayfishes, and whether global gaps met a previously suggested 10× threshold (mean interspecific difference being 10× larger than mean intra specific difference). We examined barcoding gaps using publicly available mitochondrial COI sequence data from the National Center for Biotechnology Information's nucleotide database. We created two versions of the COI datasets used for downstream analyses: one focused on the number of unique haplotypes (N H) per species, and another that focused on total number of sequences (N S; i.e., including redundant haplotypes) per species. A total of 81 species were included, with 58 species and five genera from the family Cambaridae and 23 species from three genera from the family Parastacidae. Local barcoding gaps were present in only 30 species (20 Cambaridae and 10 Parastacidae species). We detected global barcoding gaps in only four genera (Cambarus, Cherax, Euastacus, and Tenuibranchiurus), which were all below (4.2× to 5.2×) the previously suggested 10× threshold. We propose that a ~5× threshold would be a more appropriate working hypothesis for species discovery. While the N H and N S datasets yielded largely similar results, there were some discrepant inferences. To understand why some species lacked a local barcoding gap, we performed species delimitation analyses for each genus using the N H dataset. These results suggest that current taxonomy in crayfishes may be inadequate for the majority of examined species, and that even species with local barcoding gaps present may be in need of taxonomic revisions. Currently, the utility of DNA barcoding for species identification and discovery in crayfish is quite limited, and caution should be exercised when mitochondrial-based approaches are used in place of taxonomic expertise. Assessment of the evidence for local and global barcoding gaps is important for understanding the reliability of molecular species identification and discovery, but outcomes are dependent on the current state of taxonomy. As this improves (e.g., via resolving species complexes, possibly elevating some subspecies to the species-level status, and redressing specimen misidentifications in natural history and other collections), so too will the utility of DNA barcoding.

DNA barcoding and morphological characterization of marine stingrays (genus Fontitrygon) from the African Gulf of Guinea

DNA barcoding and morphological characterization of marine stingrays (genus <i>Fontitrygon</i>) from the African Gulf of Guinea

Molecular quantification of herbs (Herb-Q): a pyrosequencing-based approach and its application in Pinellia ternata

Variations in herb dosage due to species adulteration and dosing inaccuracies can substantially affect clinical safety and efficacy. Accurate species quantification remains challenging, as current methods often yield inconsistent results. This study introduces a novel pyrosequencing-based technique, termed herb molecular quantification (Herb-Q), designed to precisely quantify herbal products. We evaluated its effectiveness using Pinellia ternata and five of its adulterants. Initially, we assessed commonly used DNA barcodes with sequences from a public database, identifying two candidate regions, Maturase K (matK) and internal transcribed spacer 2 (ITS2), for screening specific single nucleotide polymorphism (SNP) loci, allowing for species-specific identification. These loci were validated by amplifying and sequencing genomic material from collected samples. Our validation studies showed that Herb-Q demonstrated excellent linearity, accuracy, repeatability, and detection limits. We established quantitative standard curves with high R2 values (> 0.99) to enable precise species quantification, which were combined with external standards to provide clear and accurate visual quantification results. The average bias in quantifying the tuber of P. ternata was 2.38%, confirming that Herb-Q can accurately identify and quantify herbal product constituents. Moreover, the entire quantification process took less than 4 h. This study presents a novel, rapid method for accurately quantifying species in herbal products and advances the application of DNA barcoding from species identification to quantitative detection.

Species identification of livefood flightless fly (Torinido-shoujoubae) through DNA barcoding.

Torinido-shoujoubae, as it is called in Japanese, is a flightless Drosophila sp. that is sold commercially in Japan. This Drosophila sp. is often used as feeds for model organisms such as reptiles and spiders. There is no scientific name provided for the fruit fly that is known as Torinido-shoujoubae, as well as any historical background or data behind this species. There has been a previous study that was conducted through morphological characteristics analysis of the body as well as the male copulatory organ and has been estimated as Drosophila hydei. The objective of this study was to determine the species of this unidentified fly known as Torinido-shoujoubae based on a molecular evidence with a DNA barcoding. Samples were purchased from four separate suppliers to examine whether there are any differences between them. COI regions were amplified using PCR and the sequenced results were aligned against two databases, NCBI and BOLD. Torinido-shoujoubae samples provided from all suppliers were confirmed to be D. hydei.

DNA barcoding reveals a new cryptic species of Denisia Hübner, 1825 (Lepidoptera, Oecophoridae) from the south-western Alps

A new species of concealer moths (Oecophoridae), Denisia crypticasp. nov., is described from specimens collected in the south-western Alps (Italy, France). It differs from similar congeneric taxa of the Denisia stipella species-group by a combined pattern of colour and markings of the forewing upperside, the colour of the head, particularly the yellow second segment of the labial palpi, and by highly divergent DNA barcode (cytochrome c-oxidase subunit 1) sequences. Male and female genitalia are like several other species, with only subtle diagnostic characters. Adult and partially genitalia of D. crypticasp. nov. and the closest European species are shown for comparison. Finally, a complete checklist of European species of Denisia is added.

Population genetic diversity and structure of Tephritis angustipennis and Campiglossa loewiana (Diptera: Tephritidae) based on COI DNA barcodes in the three-river source region, China.

Tephritis angustipennis (Diptera: Tephritidae)and Campiglossa loewiana (Diptera: Tephritidae) are phytophagous pests in China. Their damage has significantly impacted the collection and cultivation of germplasm resources of native Asteraceae plants. However, the genetic characteristics and structure of their population are unclear. This study focused on the highly damaging species of T. angustipennis and C. loewiana collected from the three-river source region (TRSR). We amplified the mitochondrial cytochrome C oxidase subunit I (mtCOI) gene sequences of these pests collected from this area and compared them with COI sequences from GenBank. We also analyzed their genetic diversity and structure. In T. angustipennis, 5 haplotypes were identified from 5 geographic locations; the genetic differentiation between France population FRPY (from Nylandia, Uusimaa) and China populations GLJZ (from Dehe Longwa Village, Maqin County), GLDR (from Zhique Village, Dari County), and GLMQ (from Rijin Village, Maqin County) was the strongest. GLJZ exhibited strong genetic differentiation from GLDR and GLMQ, with relatively low gene flow. For C. loewiana, 11 haplotypes were identified from 5 geographic locations; the genetic differentiation between the Chinese population GLMQ-YY (from Yangyu Forest Farm, Maqin County) and Finnish population FDNL (from Nylandia, Uusimaa) was the strongest, with relatively low gene flow, possibly due to geographical barriers in the Qinghai-Tibet plateau. Only 1 haplotype was identified across GLDR, GLMQ, and GLBM. High gene flow between distant locations indicates that human activities or wind dispersal may facilitate the dispersal of fruit flies and across different geographic. Geostatistical analysis suggested a recent population expansion of these 2 species in TRSR. Our findings provide technical references for identifying pests in the TRSR region and theoretical support for managing resistance, monitoring pest occurrences, analyzing environmental adaptability, and formulating biological control strategies for Tephritidae pests on Asteraceae plants.

DNA-encoded probe-based assay for profiling plant kinase activities.

Elucidating kinase-substrate relationships is pivotal for deciphering cellular signaling mechanisms, yet it remains challenging due to the complexity of kinase networks. Herein, we report the development of a versatile DNA-based kinase assay platform for high-throughput profiling of plant protein kinase activities and substrate preferences. Our approach employs DNA-linked peptide substrates, facilitating quantitative and specific kinase activity detection through next-generation DNA sequencing. Leveraging DNA barcodes as quantitative readouts, our approach establishes a high-throughput, sensitive, and specific platform for dissecting kinase-substrate networks in plants, representing a powerful tool for elucidating signaling mechanisms in plants.

Complete chloroplast genomes of the hemiparasitic genus Cymbaria: Insights into comparative analysis, development of molecular markers, and phylogenetic relationships.

The hemiparasitic tribe Cymbarieae (Orobanchaceae) plays a crucial role in elucidating the initial stage of the transition from autotrophism to heterotrophism. However, the complete chloroplast genome of the type genus Cymbaria has yet to be reported. In addition, the traditional Mongolian medicine Cymbaria daurica is frequently subjected to adulteration or substitution because of the minor morphological differences with Cymbaria mongolica. In this study, the complete chloroplast genomes of the two Cymbaria species were assembled and annotated, and those of other published 52 Orobanchaceae species were retrieved for comparative analyses. We found that the Cymbaria chloroplast genomes are characterized by pseudogenization or loss of stress-relevant genes (ndh) and a unique rbcL-matK inversion. Unlike the high variability observed in holoparasites, Cymbaria and other hemiparasites exhibit high similarity to autotrophs in genome size, guanine-cytosine (GC) content, and intact genes. Notably, four pairs of specific DNA barcodes were developed and validated to distinguish the medicinal herb from its adulterants. Phylogenetic analyses revealed that the genus Cymbaria and the Schwalbea-Siphonostegia clade are grouped into the tribe Cymbarieae, which forms a sister clade to the remaining Orobanchaceae parasitic lineages. Moreover, the diversification of monophyletic Cymbaria occurred during the late Miocene (6.72 Mya) in the Mongol-Chinese steppe region. Our findings provide valuable genetic resources for studying the phylogeny of Orobanchaceae and plant parasitism, and genetic tools to validate the authenticity of the traditional Mongolian medicine "Xinba.".

A molecular approach to unravel trophic interactions between parasitoids and hyperparasitoids associated with pecan aphids.

Advances in molecular ecology can overcome many challenges in understanding host-parasitoid interactions. Genetic characterization of the key-players in systems helps to confirm species and identify trophic linkages essential for ecological service delivery by biological control agents; however, relatively few agroecosystems have been explored using this approach. Pecan production consists of a large tree perennial system containing an assortment of seasonal pests and natural enemies. As a first step to characterizing host-parasitoid associations in pecan food webs, we focus on aphid species and their parasitoids. Based on DNA barcoding of field-collected and reared specimens, we confirmed the presence of 3 species of aphid, one family of primary parasitoids, and 5 species of hyperparasitoids. By applying metabarcoding to field-collected aphid mummies, we were able to identify multiple species within each aphid mummy to unravel a complex food web of 3 aphids, 2 primary parasitoids, and upward of 8 hyperparasitoid species. The results of this study demonstrate that multiple hyperparasitoid species attack a single primary parasitoid of pecan aphids, which may have negative consequences for successful aphid biological control. Although further research is needed on a broader spatial scale, our results suggest multiple species exist in this system and may suggest a complex set of interactions between parasitoids, hyperparasitoids, and the 3 aphid species. This was the first time that many of these species have been characterized and demonstrates the application of novel approaches to analyze the aphid-parasitoid food webs in pecans and other tree crop systems.

Morphological, Molecular Identification and Genetic Diversity Assessment of Three Terrestrial Isopods from the High-Altitude Region

Terrestrial specimens were collected from Ashshafa, a south-western highland area in Saudi Arabia. Three species, i.e., Porcellio laevis, Porcellionides pruinosus (Porcellionidae), and Armadillidium vulgare (Armadillidiidae), were identified in this study based on their morphological characteristics. Partial mitochondrial cytochrome C oxidase subunit I (COI) gene sequences were used for DNA barcoding and biodiversity assessments. A phylogenetic tree of 22 haplotypes from 35 specimens of the three isopod species was drawn from the most similar sequences obtained from BLAST with the associated accession numbers. The tree included two clades. The first clade included samples of P. laevis and P. pruinosus, whereas the second clade included samples of A. vulgare. Each identified species formed a distinct subclade within the main clade, along with similar sequences obtained from the NCBI database. The heat map of genetic distance among haplotypes shows the haplotype diversity (Hd) ranged from 0.590 to 0.933 (mean = 0.767) and total nucleotide diversity (πT) ranged from 0.001 to 0.089 (mean=0.049), with a similar trend observed for nucleotide diversity per site (θw) ranged from 0.001 to 0.80 (mean = 0.049). In contrast, synonymous nucleotide diversity (πs), mean=0.009, was low compared to nonsynonymous nucleotide diversity (πs), mean=0.060, across all species. In conclusion, the morphological identification of terrestrial isopods was confirmed using COI gene sequencing of mitochondrial DNA. These results will be helpful in developing a deeper isopod identification method.

DNA Barcode Characteristic of Dendrobium crumenatum based on ITS2

Dendrobium crumenatum, known as the pigeon orchid, is a type of orchid that is used as medicine by local people. Identification of D.crumenatum has limitations because it is similar to the orchid species D. heterocarpum. DNA barcoding is an alternative technique for identifying D.crumenatum using molecular markers. One of the molecular markers that is reliable and widely used in DNA barcoding is ITS2. This study aims to identify the ITS2 sequence as a molecular barcode effective for D.crumenatum. Genomic DNA isolation of Dendrobium crumenatum was carried out using the Cetyl Trimethyl Ammonium Bromide (CTAB) method and DNA amplification using PCR. The results showed that DNA barcoding research using the ITS2 sequence in D. crumenatum provides specific results to distinguish D. crumenatum from other Dendrobium species. The similarity in flower morphology in D. crumenatum and D. formosum proposed as the cause of close proximity shown in phylogenetic tree. Based on these results, the ITS2 sequence is highly recommended as a molecular marker for barcoding orchids, especially D. crumenatum since its capability for differentiating between species with morphological similarities. Key words: Dendrobium crumenatum, DNA Barcoding, ITS2

DNA BARCODING IN THE VALIDATION OF SCAD SPECIES IDENTIFICATION (GENUS: DECAPTERUS) IN AMBON

Limitations on the morphology-based identification system and the decreasing number of taxonomists have become a problem that requires a molecular approach toward the recognition and identification of species. DNA barcoding has been considered an efficient solution to solve a few of taxonomy’s problems. This research intended to identify several scads species which have been found in the waters surrounding Ambon morphologically and molecularly through DNA barcoding. Fish samples were collected from January to June 2018 at the Mardika fish market Ambon, and then brought to the Laboratory of the Maritime and Marine Science Centre of Excellence Pattimura University for further analysis. Based on morphological identification, six species of scad were discovered, being Decapterus macarellus, D. macarellus (suspected), D. macrosoma, D. macrosoma (suspected), D. russelli (suspected)and D. kurroides. Using DNA barcoding, only four species of scad were found, being D. macarellus, D. macrosoma, D. russelli and D. kurroides with identification percentage ranging from 99-100%. After validation, the species of scad which can be found in Ambon’s waters are D. macarellus, D. macrosoma, D. russelli and D. kurroides. DNA barcoding is a complementary alternative that can reinforce morphology-based identification quickly and accurately.

DNA Barcoding Analysis of Betok Fish (Anabas testudineus) from Kampar, Riau Based on Cytochrome Oxidase Subunit I (COI)

Ikan betok (Anabas testudineus, Bloch 1792) is one of fishes which is a member of family Anabantidae. Betok is an endemic fish in Riau Province. The scientific study about DNA barcoding of betok fish is still rarely. This research aims to analyze the DNA barcode sequence of cytochrome c oxidase I (COI) on betok fish. Methods were used in this study such as sampling, total DNA extraction, PCR, electrophoresis, sequencing and data analysis. The COI sequence studied had size of 694 bp. The BLASTn analysis showed that the betok fish had the highest similarity of 99.7% with A. testudineus and the lowest 93.00% with A. cobojinus. There was one nucleotide that characterize the betok fish based on the COI sequence namely nucleotide number 309. This study may enrich the DNA barcode database of betok fish in GeneBank.

Decoding Genetic Signatures for Identification: A Molecular Investigation Approach in Pakistan via Cytochrome C Oxidase I (COI) Gene Sequencing

DNA barcoding is an efficient and rapid approach to the identification of species. A standard genetic marker is used to barcode the sequence of unidentified species. So, mitochondrial cytochrome c oxidase I (COI) genetic marker is mostly used for species molecular identification. Therefore, molecular identification and phylogeographic relationship of Cirrhinus reba (C. reba) were carried out in the present study. In the present study, we propose the barcoding analysis of the mitochondrial cytochrome c oxidase I (COI) gene of freshwater fish as different sequences were obtained from the sequencer and found (all &lt; 500) base pairs and successfully barcode with mitochondrial COI genetic marker for identification. BLAST analysis provides 100% identity match with reference sequences of GenBank database and confirmed the species as C. reba as the length of the sequence is found 496 base pairs. The present study successfully reported DNA barcoding for genetic diversity and identification contributing to covering sufficient information about molecular analysis to identify unknown species the best of our knowledge this is the first study reporting a genetic tool for identification of the commercial food fish of Pakistan. Moreover, DNA barcoding can play a very significant role in molecular identification and inference of relationships in diverse freshwater ecosystem.

Identifikasi Teripang Laut (Holothuroidea) dari Perairan Laut Pulau Kabaruan Berdasarkan Identifikasi Morfologi dan DNA Barcoding

Indonesia is known with its high biodiversity. One of them is sea cucumber. Various species of sea cucumbers are found in Indonesian sea in Talaud Regency at Kabaruan Island. Identification of sea cucumbers to reveal the biodiversity of sea cucumbers in Kabaruan Island, which is almost unknown. This research was to identify six samples of sea cucumber (Holothuroidea) in Kabaruan Island based on morphology and DNA barcoding using the COI gene. The differences between each species can be seen from the morphological characteristics, the length and width of sea cucumbers, papillae, pattern and color. The results showed that there were 4 species from 6 samples, which is Holothuria leucospilota, Holothuria whitmaei, Eleutherozoa sp., and Holothuria atra. That six samples with the code A1, A2, B1, C1, and C2 have a homology level of 99%-100% with the target nucleotide sequence from GenBank. Meanwhile, the sample’s code A3 has a homology level of 90% with the Eleutheroza sp. so there is a possibility that sample A3 is a new taxa that has the highest similarity to the Eleutheroza sp. From the six samples, it was found that samples A2 and B1 had the highest level of similarity which is 100%. Meanwhile, the samples with the lowest percentage values are samples A3 and B1 and samples A2 and A3, with a percentage value of 82.75%. There were 4 species from six samples found in Kabaruan Island. There is a possibility that Sample A3 is a new species that is similar to Eleutheroza sp.