Debar: A sequence-by-sequence denoiser for COI-5P DNA barcode data.

BackgroundDNA barcoding is a valuable taxonomic tool for rapid and accurate species identification and cryptic species discovery in black flies. Indonesia has 143 nominal species of black flies, but information on their biological aspects, including vectorial capacity and biting habits, remains underreported, in part because of identification problems. The current study represents the first comprehensive DNA barcoding of Indonesian black flies using mitochondrial cytochrome c oxidase subunit I (COI) gene sequences.MethodsGenomic DNA of Indonesian black fly samples were extracted and sequenced, producing 86 COI sequences in total. Two hundred four COI sequences, including 118 GenBank sequences, were analysed. Maximum likelihood (ML) and Bayesian inference (BI) trees were constructed and species delimitation analyses, including ASAP, GMYC and single PTP, were performed to determine whether the species of Indonesian black flies could be delineated. Intra- and interspecific genetic distances were also calculated and the efficacy of COI sequences for species identification was tested.ResultsThe DNA barcodes successfully distinguished most morphologically distinct species (> 80% of sampled taxa). Nonetheless, high maximum intraspecific distances (3.32–13.94%) in 11 species suggested cryptic diversity. Notably, populations of the common taxa Simulium (Gomphostilbia) cheongi, S. (Gomphostilbia) sheilae, S. (Nevermannia) feuerborni and S. (Simulium) tani in the islands of Indonesia were genetically distinct from those on the Southeast Asian mainland (Malaysia and Thailand). Integrated morphological, cytogenetic and nuclear DNA studies are warranted to clarify the taxonomic status of these more complex taxa.ConclusionsThe findings showed that COI barcoding is a promising taxonomic tool for Indonesian black flies. The DNA barcodes will aid in correct identification and genetic study of Indonesian black flies, which will be helpful in the control and management of potential vector species.Graphical abstract

DNA barcoding is a novel concept for the taxonomic identification, in that it uses a specific short genetic marker in an organism’s DNA to discriminate species. In 2003, professor Paul D. N. Hebert, “the father of DNA barcoding”, of the University of Guelph, Ontario, Canada first proposed the idea to identify biological species using DNA barcode, where the mitochondrial gene cytochrome c oxidase subunit I (COI) was supposed to be the first candidate for animals (Hebert et al. 2003a). Their studies of COI profiling in both higher taxonomic categories and species-level assignment demonstrated that COI gene has significant resolutions across the animal kingdom except the phylum Cnidaria (Hebert et al. 2003b, Ward et al. 2005, Hajibabaei et al. 2006). From then on, a wide broad of taxonomic groups (i.e. birds, fish, butterflies, spiders, ants, etc) were examined by COI gene for its usability as the barcode (i.e. Hebert et al. 2004a, Hebert et al. 2004b, Greenstone et al. 2005, Smith et al. 2005, Barber and Boyce 2006, Meier et al. 2006, Kerr et al. 2007, Kumar et al. 2007, Pfenninger et al. 2007, Stahls and Savolainen 2008, Zhou et al. 2009). Meanwhile, other candidate genes, including Internal Transcribed Spacer (ITS), trnH-psbA intergenic spacer (trnH-psbA), Ribulose-bisphosphate carboxylase (rbcL) and Maturase K (matK) were analysed by different research groups (Jaklitsch et al. 2006, Evans et al. 2007, Ran et al. 2010, de Groot et al. 2011, Liu et al. 2011, Piredda et al. 2011, Yesson et al. 2011). Till recently, there are about 30 DNA barcode candidates are tested, and 4 to 8 of them are widely used for the identification of diversified taxonomic groups with a relatively good resolution. It has been estimated that there are 10 to 100 million species of living creatures in the earth, while what we know is very limited. Knowing the biodiversity is one of the crucial biological issues of ecology, evolutionary biology, bio-security, agro-biotechnology, bioresources and many other areas. For very long period, taxonomists have provided a nomenclatural hierarchy and key prerequisites for the society. However, the needs for species identification requested by non-taxonomists require the knowledge held by taxonomists. Therefore, a standardized, rapid and inexpensive species identification approach is needed to establish for the non-specialists. There had some attempts on the molecular identification systems based on polymerase chain reaction (PCR), especially in bacterial studies (Woese 1996, Zhou et al. 1997, Maiden et al. 1998, Wirth et al. 2006), but no successful solutions for broader scopes of eukaryotes (reviewed in Frezal and Leblois 2008). The DNA Barcode of Life project is another attempt to create a universal eukaryotic identification system based on molecular approaches. Following studies by Hebert et al.

Patella ferrugineais an endangered marine gastropod, distributed on the western Mediterranean coasts, whose range has progressively contracted, due to intense human exploitation. A genetic analysis was performed on two unidentified young individuals belonging to the genusPatellafound attached to the shell of an adult ofP. ferruginea, with a twofold aim: (i) to achieve their correct taxonomic attribution by means of the DNA barcoding; and (ii) to shed some light on the hypothesized larval philopatry and/or juveniles phoresis inP. ferruginea. The survey was carried out comparing the sequences of the cytochrome c oxidase subunit I (COI) Folmer region obtained for the two juveniles with those obtained for adults ofP. ferruginea, P. caerulea, P. rustica, andP. ulyssiponensis, from different sites of the western Mediterranean, by means of maximum likelihood cluster analysis and a Bayesian-based assignment test. Results obtained evidenced that: (i) COI may be used with confidence as DNA barcoding in the genusPatella; and (ii) the two juveniles studied are not conspecific: one belonged toP. ferruginea, the other toP. rustica.The latter finding raises doubts about the juvenile phoresis and about the occurrence of larval philopatry inP. ferruginea, suggesting that an extensive use of a molecular approach for a better evaluation of the recruitment features of this endangered species should be adopted.

We evaluated the extent of intraspecific and interspecific genetic distances and the effectiveness of predefined threshold values using the main genes for estimates of biodiversity and specimens' identification in anurans. Partial sequences of the mitochondrial genes for small (12S) and large (16S) ribosomal subunits, cytochrome c oxidase subunit I (COI) and Cytochrome b (Cytb) of the family Hylidae were downloaded from GenBank and curated for length, coverage, and potential contaminations. We performed analyses for all sequences of each gene and the same species present in these datasets by distance and tree (monophyly)-based evaluations. We also evaluated the ability to identify specimens using these datasets applying "nearest neighbor" (NN), "best close match" (BCM) and "BOLD ID" tests. Genetic distance thresholds were generated by the function 'threshVal' and "localMinima" from SPIDER package and traditional threshold values (1%, 3%, 6% and 10%) were also evaluated. Coding genes, especially COI, had a better identification capacity than non-coding genes on barcoding gap and monophyly analysis and NN, BCM, BOLD ID tests. Considering the multiple factors involved in global DNA barcoding evaluations, we present a critical assessment of the use of these genes for biodiversity estimation and specimens' identification in anurans (e.g. hylids).

The taxonomy and phylogeny of the pufferfishes belonging to the family Tetraodontidae found in India are poorly understood. We investigated five species of freshwater and marine pufferfishes using partial sequences of 16S rRNA and cytochrome c oxidase subunit I (COI) of mitochondrial genes. The sequence alignment of 16S rRNA yielded 573 bp, whereas COI gene sequence alignment yielded 614 bp. The sequence analysis of the genes revealed two distinct groups of freshwater and marine origin, which are genetically distinct from each other and exhibit identical phylogenetic resolution. The partial sequences of both the genes provided sufficient phylogenetic resolution to distinguish all the five species of pufferfishes. The COI sequences could be used as DNA barcodes for identification of the pufferfishes.

Beetles (Coleoptera) represent one of the most diverse insect groups and play vital ecological roles, yet their accurate identification is often challenging due to morphological similarities among taxa. DNA barcoding has emerged as a powerful and reliable tool for species-level identification and biodiversity monitoring. In this study, we established a local DNA barcode reference database for beetles in the Liancheng Nature Reserve, Gansu Province, China. From May to August 2024, beetle specimens were collected and identified using both morphological traits and DNA barcoding. Three species delimitation methods—Automatic Barcode Gap Discovery (ABGD), Assemble Species by Automatic Partitioning (ASAP), and Bayesian Poisson Tree Processes (bPTP)—were employed as complementary analytical tools, and phylogenetic relationships were inferred from cytochrome c oxidase subunit I (COI) sequences. A total of 164 COI sequences (650 bp) were obtained, representing 126 beetle species from 95 genera and 20 families. DNA barcoding successfully resolved morphologically ambiguous taxa, with many sequences reported here for the first time. Phylogenetic analysis revealed that species within the same genus formed cohesive clades before clustering at the family level, confirming the species-level discriminative power of the COI gene. Collectively, these findings demonstrate that COI-based DNA barcoding is a powerful complement to traditional taxonomy. The establishment of this preliminary reference database provides a valuable molecular resource for beetle identification and a practical tool to support biodiversity conservation, resource management, and long-term monitoring in the Liancheng Nature Reserve.

Objective To make up the limitations of traditional morphological classification methods, we identified vector fleas by DNA barcoding in Qinghai Province. Methods The mt DNA cytochrome c oxidase subunit Ⅰ(COⅠ) gene was amplified by PCR from 36 muscle tissues of fleas in 3 states, 2 cities and 5 counties of Qinghai Province, and the obtained COⅠ gene fragments were sequenced and aligned. The intra- and inter-species genetic distances were calculated with Mega 6 software using K2-P model and a phylogenetic tree was constructed with neighbor-joining (NJ) method. Results Totally 36 COⅠ gene sequences of 2 superfamilies, 4 genera and 6 kinds of vector fleas were measured, the average genetic distance was 0.119, and the intraspecific distance was 0.002 - 0.027, the interspecific distance was 0.039 - 0.207, and the interspecific genetic distance was significantly greater than the intraspecific genetic distance. NJ tree showed the same species had formed a single line with high support rate and interspecific branch was clear. Conclusion DNA barcoding is suitable for identification of vector fleas in Qinghai Province, may make up the limitations of traditional morphological classification methods. Key words: Siphonaptera; Cytochrome c oxidase subunit Ⅰ; DNA barcoding

A multiplex-PCR assay based on mitochondrial cytochrome c oxidase subunit I (COI) sequences was developed for identification of five members of the Barbirostris Complex which occur in Thailand: Anopheles barbirostris s.s., An. dissidens, An. saeungae, An. wejchoochotei and An. barbirostris species A3. Anopheles campestris was not included in the assay due to the lack of unequivocal sequences. Allele-specific primers were designed for specific nucleotide segments of COI sequences of each species. Mismatch method and addition of long GC tail were applied for some primers. The assay provided products of 706 bp for An. barbirostris s.s., 238 bp for An. dissidens, 611 bp for An. saeungae, 502 bp for An. wejchoochotei and 365 bp for An. barbirostris A3. The assay was tested using 111 wild-caught female mosquitoes from Bhutan, Cambodia, Indonesia (Sulawesi) and Thailand. The results of the multiplex PCR were in complete agreement with COI sequencing; however, one of three specimens from Bhutan and all 11 specimens from Indonesia were not amplifiable by the assay due to their distinct COI sequences. This, together with the distinct rDNA sequences of these specimens, suggests the presence of at least two additional new species in the Barbirostris Complex.

Genus Parapenaeopsis constitute an important fishery resource in Indian waters. Parapenaeopsis stylifera, being morphologically very similar to Parapenaeopsis coromandelica, mainly differentiated using telsonic armature and it is subject of controversy regarding the number of armature for the species. The present study was aimed to resolve the taxonomic ambiguity between P. stylifera and P. coromandelica and to study phylogenetic relationship among five species under the genus Parapenaeopsis using partial sequences of mitochondrial 16S ribosomal DNA (rDNA) and cytochrome c oxidase subunit I (COI) genes. Sequence analysis revealed an average genetic divergence value ranging from 7.65 to 20.3% for COI gene and 2.4 to 12.0% for 16S rDNA gene between five species under study. The result shows that P. stylifera and P. coromandelica Alcock, 1906 are genetically a distinct species with average divergence value of 7.6% based on COI sequence and 2.4% based on the 16S rDNA. However, systematic relationship between the P. stylifera and P. coromandelica were closer than other species, thus formed separate clade in phylogenetic tree. The species specific molecular signatures developed in the present study will be helpful in effective fisheries management of the species in Indian waters.

In wildlife forensics, precise species identification is essential, particularly in cases involving poaching, illegal wildlifetrade, and biodiversity conservation. Molecular technologies are necessary for accurate forensic analysis since traditionalmorphological methods frequently fail when biological specimens are processed, incomplete, or degraded. MitochondrialDNA (mtDNA) has proven particularly useful in these situations because of its large copy number, maternal mode ofinheritance, and increased stability in damaged tissues. The cytochrome c oxidase subunit I (COI) gene is one of the mostcommonly used mtDNA markers for DNA barcoding due to its high interspecies variability, evolutionary conservation,and inclusion in extensive international databases.This study examined the COI gene sequences of ten often encountered domestic and wild Indian species: Elephas maximus(elephant), Bos gaurus (gaur), Bos taurus (domestic cow), Axis axis (chital), Axis porcinus (hog deer), Macaca mulatta (rhesusmacaque), Pavo cristatus (Indian peacock), Sus scrofa (wild boar), and Panthera tigris (tiger) and Panthera pardus (leopard).COI sequences that are publicly accessible were obtained from NCBI GenBank database. BLAST was used for sequencevalidation, and Clustal Omega was used for multiple sequence alignment. Using MEGA12, a neighbor-joining tree wasconstructed and pairwise distance analysis was performed to evaluate genetic distance and phylogenetic relationships.Significant genetic divergence was found among the species under study, and the phylogenetic tree showed groupingpatterns that aligned with established taxonomic relationships. These results demonstrate that COI-based DNA barcodingis a reliable method for differentiating between closely related species.

Objective To apply the DNA barcoding technology for identification of rodent animals and to establish a rodent animal DNA barcode database in Gansu Province. Methods A total of 54 rodent animals were detected. DNA barcoding technology was used to analyze the DNA mitochondrial cytochrome C oxidase subunit I (CO I) gene sequence in Gansu Province. Results The intra-specific genetic distance was 0-2% while the inter-specific distance ranged from 18% to 30%. Eight major clusters were apparently showed on a Neighbor joining tree. Conclusion DNA barcoding technology could overcome the shortcomings of the morphological identification, so it could be used to identify the rodent animals and has important implications for disease control and prevention in the natural focus of Gansu Province. Key words: Rodentia; DNA barcoding; Mitochondrial cytochrome C oxidase subunit I; Species identification

Recently, sea cucumbers (Echinodermata: Holothuroidea) have been over-exploited in many areas of the world, including in the Ryukyu Islands, southern Japan, due to increases in their economic importance. Nevertheless, management and protection of sea cucumbers are insufficient worldwide. The black sea cucumber Holothuria (Halodeima) atra Jaeger, 1833, inhabits a large range across the Indo-West Pacific Ocean and is a widely harvested species. Here we conducted population genetic analyses on H. atra using partial mitochondrial DNA sequences of cytochrome c oxidase subunit I (COI) and 16S ribosomal RNA (16S) to examine 11 different populations around three island groups in the middle Ryukyus; Okinawajima Island, the Kerama Islands, and the Sakishima Islands, all within Okinawa Prefecture. We found 27 haplotypes for COI and 16 haplotypes for 16S. Locations within national and quasi-national parks (Zamami Island, Keramas, and Manza, Okinawajima; managed by the national Ministry of Environment and Okinawa Prefecture, respectively) had the highest number of haplotypes, whereas locations with less management and more anthropogenic pressure had lower numbers The mean of all samples' genetic diversity indices was moderate with regards to both haplotype and nucleotide diversity. According to our results, Zamami Ama was the most genetically diverse location based on both markers used, likely because it is located within Kerama-Shoto National Park with comparatively stricter regulations than most other locations. Based on our COI sequences, three-quarters of the locations with the highest haplotype diversity were found to be distant from Okinawajima Island, indicating that the genetic diversity of H. atra was reduced around Okinawajima Island. Our results possibly reflect negative impacts from anthropogenic pressures such as over-harvesting and coastal development, although future comprehensive research including sequences of nuclear loci is needed to confirm this hypothesis.

DNA barcoding is a molecular method that rapidly identifies an individual to a known taxon or its closest relative based on a 650-bp fragment of the cytochrome c oxidase subunit I (COI). In this study, DNA barcodes of members of the family Accipitridae, including Haliastur indus (brahminy kite), Haliaeetus leucogaster (white-bellied sea eagle), Ichthyophaga ichthyaetus (grey-headed fish eagle), Spilornis holospilus (crested serpent-eagle), Spizaetus philippensis (Philippine hawk-eagle), and Pithecophaga jefferyi (Philippine eagle), are reported for the first time. All individuals sampled are kept at the Philippine Eagle Center in Davao City, Philippines. Basic local alignment search tool results demonstrated that the COI sequences for these species were unique. The COI gene trees constructed using the maximum-likelihood and neighbour-joining (NJ) methods supported the monophyly of the booted eagles of the Aquilinae and the sea eagles of the Haliaeetinae but not the kites of the Milvinae.

Amphibians, a diverse and ecologically important group, are facing global declines due to various factors, including habitat loss and climate change. Accurate species identification is crucial for effective conservation efforts, and DNA barcoding has emerged as a powerful tool in this regard. This study compares the efficacy of two DNA barcoding primer sets, targeting the 16S ribosomal RNA gene and the Cytochrome Oxidase I (COI) gene, for identifying 20 amphibian species. While both primer sets successfully amplified sequences, the 16S rRNA gene region identified all 20 samples, whereas the COI region identified 14. The amplified sequences, approximately 550 base pairs for 16S rRNA and 658 base pairs for COI facilitated precise taxonomic placement within amphibian families using Neighbor-Joining phylogenetic trees. These findings enhance DNA barcoding methodology and aid in understanding amphibian diversity, crucial for effective conservation strategies amidst global declines drives by habitat loss, diseases, and climate change.

Potential Use of DNA Barcodes in Regulatory Science: Applications of the Regulatory Fish Encyclopedia