Establishment of a mitochondrial DNA sequence database for the identification of fish species commercially available in South Africa

The limitations intrinsic to morphology-based identification systems have created an urgent need for reliable genetic methods that enable the unequivocal recognition of fish species, particularly those that are prone to overexploitation and/or market substitution. The aim of this study was to develop a comprehensive reference library of DNA sequence data to allow the explicit identification of 53 commercially available fish species in South Africa, most of which were locally caught marine species. Sequences of approximately 655 base pairs were generated for all species from the cytochrome c oxidase I (COI) gene, the region widely adopted for DNA barcoding. Specimens of the genus Thunnus were examined in further detail, employing additional mitochondrial DNA control region sequencing. Cumulative analysis of the sequences from the COI region revealed mean conspecific, congeneric and confamilial Kimura 2-parameter distances of 0.10%, 4.58% and 15.43%, respectively. The results showed that the vast majority (98%) of fish species examined could be readily differentiated by their COI barcodes, but that supplementary control region sequencing was more useful for the discrimination of three Thunnus species. Additionally, the analysis of COI data raised the prospect that Thyrsites atun (snoek) could constitute a species pair. The present study has established the necessary genetic information to permit the unambiguous identification of 53 commonly marketed fish species in South Africa, the applications of which hold a plethora of benefits relating to ecology research, fisheries management and control of commercial practices.     

The campaign to DNA barcode all fishes, FISH-BOL

FISH-BOL, the Fish Barcode of Life campaign, is an international research collaboration that is assembling a standardized reference DNA sequence library for all fishes. Analysis is targeting a 648 base pair region of the mitochondrial cytochrome c oxidase I (COI) gene. More than 5000 species have already been DNA barcoded, with an average of five specimens per species, typically vouchers with authoritative identifications. The barcode sequence from any fish, fillet, fin, egg or larva can be matched against these reference sequences using BOLD; the Barcode of Life Data System ( http://www.barcodinglife.org ). The benefits of barcoding fishes include facilitating species identification, highlighting cases of range expansion for known species, flagging previously overlooked species and enabling identifications where traditional methods cannot be applied. Results thus far indicate that barcodes separate c. 98 and 93% of already described marine and freshwater fish species, respectively. Several specimens with divergent barcode sequences have been confirmed by integrative taxonomic analysis as new species. Past concerns in relation to the use of fish barcoding for species discrimination are discussed. These include hybridization, recent radiations, regional differentiation in barcode sequences and nuclear copies of the barcode region. However, current results indicate these issues are of little concern for the great majority of specimens.     

DNA barcoding commercially important fish species of Turkey

DNA barcoding was used in the identification of 89 commercially important freshwater and marine fish species found in Turkish ichthyofauna. A total of 1765 DNA barcodes using a 654‐bp‐long fragment of the mitochondrial cytochrome c oxidase subunit I gene were generated for 89 commercially important freshwater and marine fish species found in Turkish ichthyofauna. These species belong to 70 genera, 40 families and 19 orders from class Actinopterygii, and all were associated with a distinct DNA barcode. Nine and 12 of the COI barcode clusters represent the first species records submitted to the BOLD and GenBank databases, respectively. All COI barcodes (except sequences of first species records) were matched with reference sequences of expected species, according to morphological identification. Average nucleotide frequencies of the data set were calculated as T = 29.7%, C = 28.2%, A = 23.6% and G = 18.6%. Average pairwise genetic distance among individuals were estimated as 0.32%, 9.62%, 17,90% and 22.40% for conspecific, congeneric, confamilial and within order, respectively. Kimura 2‐parameter genetic distance values were found to increase with taxonomic level. For most of the species analysed in our data set, there is a barcoding gap, and an overlap in the barcoding gap exists for only two genera. Neighbour‐joining trees were drawn based on DNA barcodes and all the specimens clustered in agreement with their taxonomic classification at species level. Results of this study supported DNA barcoding as an efficient molecular tool for a better monitoring, conservation and management of fisheries.     

Patterns of DNA Barcode Variation in Canadian Marine Molluscs

Background

Molluscs are the most diverse marine phylum and this high diversity has resulted in considerable taxonomic problems. Because the number of species in Canadian oceans remains uncertain, there is a need to incorporate molecular methods into species identifications. A 648 base pair segment of the cytochrome c oxidase subunit I gene has proven useful for the identification and discovery of species in many animal lineages. While the utility of DNA barcoding in molluscs has been demonstrated in other studies, this is the first effort to construct a DNA barcode registry for marine molluscs across such a large geographic area.

Methodology/Principal Findings

This study examines patterns of DNA barcode variation in 227 species of Canadian marine molluscs. Intraspecific sequence divergences ranged from 0–26.4% and a barcode gap existed for most taxa. Eleven cases of relatively deep (>2%) intraspecific divergence were detected, suggesting the possible presence of overlooked species. Structural variation was detected in COI with indels found in 37 species, mostly bivalves. Some indels were present in divergent lineages, primarily in the region of the first external loop, suggesting certain areas are hotspots for change. Lastly, mean GC content varied substantially among orders (24.5%–46.5%), and showed a significant positive correlation with nearest neighbour distances.

Conclusions/Significance

DNA barcoding is an effective tool for the identification of Canadian marine molluscs and for revealing possible cases of overlooked species. Some species with deep intraspecific divergence showed a biogeographic partition between lineages on the Atlantic, Arctic and Pacific coasts, suggesting the role of Pleistocene glaciations in the subdivision of their populations. Indels were prevalent in the barcode region of the COI gene in bivalves and gastropods. This study highlights the efficacy of DNA barcoding for providing insights into sequence variation across a broad taxonomic group on a large geographic scale.     

Integrative taxonomy of Peniculus,Metapeniculus, and Trifur (Siphonostomatoida: Pennellidae), copepod parasites of marine fishes from Chile: species delimitation analyses using DNA barcoding and morphological evidence

Pennellidae is a family of copepod parasites of widely distributed marine fishes. The pennellid species are usually morphologically differentiated by cephalothorax, neck, trunk, and abdomen shape. These characters, however, show high polymorphism and therefore using only this type of data, delimitation at species level of this genus is difficult. In this study, we explored the genetic distances calculated from sequences of a DNA barcoding marker (COI mt) (678 base pairs). We also explored the genetic distances of 25 Peniculus specimens associated within nine marine fish species, four Metapeniculus specimens associated within one marine fish species, and four Trifur specimens associated within one marine fish species. All specimens were collected in Antofagasta Bay, Chile and were calculated from sequences of a DNA barcoding marker (COI mt) (678 base pairs). The genetic distance among the Peniculus specimens was 0.95% from the different host species, the Metapeniculus specimens distance was 0.44% and the Trifur specimens was 2.25%. Genetic difference between Peniculus and Metapeniculus was 17.86% and Peniculus differ from T. tortuosus by 18.16%. We analysed the barcoding gene fragment using Bayesian Inference (BI) for phylogenetic reconstruction using three outgroups. Based on the phylogenetic analysis an ultrametric tree was built and a general mixed Yule-coalescent (bGMYC) model was conducted for species delimitation. Morphometrics analyses were made with Bayesian statistics. Mean and credibility limit (95%) for each parameter was calculated. Results show that based on morphology the individuals collected can be assigned to P. cf. fistula von Nordmann, 1832, Metapeniculus antofagastensis Castro-Romero & Baeza-Kuroki, 1985, and Trifur cf. tortuosus Wilson, 1917. High morphological polymorphism was observed for the lineage of Peniculus associated to several host species of marine fishes. Similar results were obtained for Trifur cf. tortuosus parasites on Chilean marine fishes.     

Molecular characterization of marine and coastal fishes of Bangladesh through DNA barcodes

This study describes the molecular characterization of marine and coastal fishes of Bangladesh based on the mitochondrial cytochrome c oxidase subunit I (COI) gene as a marker. A total of 376 mitochondrial COI barcode sequences were obtained from 185 species belonging to 146 genera, 74 families, 21 orders, and two classes of fishes. The mean length of the sequences was 652 base pairs. In Elasmobranchii (Sharks and rays), the average Kimura two parameter (K2P) distances within species, genera, families, and orders were 1.20%, 6.07%, 11.08%, and 14.68%, respectively, and for Actinopterygii, the average K2P distances within species, genera, families, and orders were 0.40%, 6.36%, 14.10%, and 24.07%, respectively. The mean interspecies distance was 16‐fold higher than the mean intraspecies distance. The K2P neighbor‐joining (NJ) trees based on the sequences generally clustered species in accordance with their taxonomic position. A total of 21 species were newly recorded in Bangladesh. High efficiency and fidelity in species identification and discrimination were demonstrated in the present study by DNA barcoding, and we conclude that COI sequencing can be used as an authentic identification marker for Bangladesh marine fish species.     

DNA barcoding is a useful tool for the identification of marine fishes from Japan

In this study, 229 DNA sequences of cytochrome oxidase subunit I gene (COI) from 158 marine fishes of Japan were employed to test the efficacy of species identification by DNA barcoding. The average genetic distance was 60-fold higher between species than within species, as Kimura two parameter (K2P) genetic distances averaged 17.6% among congeners and only 0.3% among conspecifics. There were no overlaps between intraspecific and interspecific K2P distances, and all sequences formed species units in the neighbor-joining dendrogram. Hybridization phenomena in two species (Kyphosus vaigiensis and Pterocaesio digramma) were also detected through searches in Barcode of Life Data Systems (BOLD). DNA barcoding provides a new way for fish identification.     

DNA Barcoding for Species Assignment: The Case of Mediterranean Marine Fishes

Background

DNA barcoding enhances the prospects for species-level identifications globally using a standardized and authenticated DNA-based approach. Reference libraries comprising validated DNA barcodes (COI) constitute robust datasets for testing query sequences, providing considerable utility to identify marine fish and other organisms. Here we test the feasibility of using DNA barcoding to assign species to tissue samples from fish collected in the central Mediterranean Sea, a major contributor to the European marine ichthyofaunal diversity.

Methodology/Principal Findings

A dataset of 1278 DNA barcodes, representing 218 marine fish species, was used to test the utility of DNA barcodes to assign species from query sequences. We tested query sequences against 1) a reference library of ranked DNA barcodes from the neighbouring North East Atlantic, and 2) the public databases BOLD and GenBank. In the first case, a reference library comprising DNA barcodes with reliability grades for 146 fish species was used as diagnostic dataset to screen 486 query DNA sequences from fish specimens collected in the central basin of the Mediterranean Sea. Of all query sequences suitable for comparisons 98% were unambiguously confirmed through complete match with reference DNA barcodes. In the second case, it was possible to assign species to 83% (BOLD-IDS) and 72% (GenBank) of the sequences from the Mediterranean. Relatively high intraspecific genetic distances were found in 7 species (2.2%–18.74%), most of them of high commercial relevance, suggesting possible cryptic species.

Conclusion/Significance

We emphasize the discriminatory power of COI barcodes and their application to cases requiring species level resolution starting from query sequences. Results highlight the value of public reference libraries of reliability grade-annotated DNA barcodes, to identify species from different geographical origins. The ability to assign species with high precision from DNA samples of disparate quality and origin has major utility in several fields, from fisheries and conservation programs to control of fish products authenticity.     

Pay Attention to the Overlooked Cryptic Diversity in Existing Barcoding Data: the Case of Mollusca with Character-Based DNA Barcoding

With the global biodiversity crisis, DNA barcoding aims for fast species identification and cryptic species diversity revelation. For more than 10 years, large amounts of DNA barcode data have been accumulating in publicly available databases, most of which were conducted by distance or tree-building methods that have often been argued, especially for cryptic species revelation. In this context, overlooked cryptic diversity may exist in the available barcoding data. The character-based DNA barcoding, however, has a good chance for detecting the overlooked cryptic diversity. In this study, marine mollusk was as the ideal case for detecting the overlooked potential cryptic species from existing cytochrome c oxidase I (COI) sequences with character-based DNA barcode. A total of 1081 COI sequences of mollusks, belonging to 176 species of 25 families of Gastropoda, Cephalopoda, and Lamellibranchia, were conducted by character analysis. As a whole, the character-based barcoding results were consistent with previous distance and tree-building analysis for species discrimination. More importantly, quite a number of species analyzed were divided into distinct clades with unique diagnostical characters. Based on the concept of cryptic species revelation of character-based barcoding, these species divided into separate taxonomic groups might be potential cryptic species. The detection of the overlooked potential cryptic diversity proves that the character-based barcoding mode possesses more advantages of revealing cryptic biodiversity. With the development of DNA barcoding, making the best use of barcoding data is worthy of our attention for species conservation.     

Applications of DNA barcoding to fish landings: authentication and diversity assessment

DNA barcoding methodologies are being increasingly applied not only for scientific purposes but also for diverse real-life uses. Fisheries assessment is a potential niche for DNA barcoding, which serves for species authentication and may also be used for estimating within-population genetic diversity of exploited fish. Analysis of single-sequence barcodes has been proposed as a shortcut for measuring diversity in addition to the original purpose of species identification. Here we explore the relative utility of different mitochondrial sequences (12S rDNA, COI, cyt b, and D-Loop) for application as barcodes in fisheries sciences, using as case studies two marine and two freshwater catches of contrasting diversity levels. Ambiguous catch identification from COI and cyt b was observed. In some cases this could be attributed to duplicated names in databases, but in others it could be due to mitochondrial introgression between closely related species that may obscure species assignation from mtDNA. This last problem could be solved using a combination of mitochondrial and nuclear genes. We suggest to simultaneously analyze one conserved and one more polymorphic gene to identify species and assess diversity in fish catches.     

DNA barcoding and evaluation of genetic diversity in Cyprinidae fish in the midstream of the Yangtze River

The Yangtze River is the longest river in China and is divided into upstream and mid‐downstream regions by the Three Gorges (the natural barriers of the Yangtze River), resulting in a complex distribution of fish. Dramatic changes to habitat environments may ultimately threaten fish survival; thus, it is necessary to evaluate the genetic diversity and propose protective measures. Species identification is the most significant task in many fields of biological research and in conservation efforts. DNA barcoding, which constitutes the analysis of a short fragment of the mitochondrial cytochrome c oxidase subunit I (COI) sequence, has been widely used for species identification. In this study, we collected 561 COI barcode sequences from 35 fish from the midstream of the Yangtze River. The intraspecific distances of all species were below 2% (with the exception of Acheilognathus macropterus and Hemibarbus maculatus). Nevertheless, all species could be unambiguously identified from the trees, barcoding gaps and taxonomic resolution ratio values. Furthermore, the COI barcode diversity was found to be low (≤0.5%), with the exception of H. maculatus (0.87%), A. macropterus (2.02%) and Saurogobio dabryi (0.82%). No or few shared haplotypes were detected between the upstream and downstream populations for ten species with overall nucleotide diversities greater than 0.00%, which indicated the likelihood of significant population genetic structuring. Our analyses indicated that DNA barcoding is an effective tool for the identification of cyprinidae fish in the midstream of the Yangtze River. It is vital that some protective measures be taken immediately because of the low COI barcode diversity.     

Identification of larval fish in mangrove areas of Peninsular Malaysia using morphology and DNA barcoding methods

The identification of larval fish has been an important morphological issue in marine biology due to the dramatic transformations that most species undergo from early larval stages to adulthood. Insufficient morphological diagnostic characters in larval fishes made it easy to misidentify them and a difficult process to key to genus and species level. The experiment aims to find out, by applying DNA barcoding, how consistent the morphological identifications can be among larval fish. Larval fish were mainly collected using plankton nets around mangrove areas in Pendas (Johor), Setiu (Terengganu), Pekan (Pahang) and Matang (Perak) Malaysia between April 2015 and October 2015. A total of 354 samples were morphologically identified, mostly to the family level and a few to the genus level. Larval fish ranged from 1.5 mm to 31 mm of total length, with the most abundant individuals being <3 mm. Among them, a total of 177 individuals were selected for DNA barcoding analyses. Molecular works involved polymerase chain reaction (PCR) and sequencing of mitochondrial Cytochrome c Oxidase I (COI) gene fragment (655 base pairs) methods. DNA barcoding enabled all samples to be identified down to species level. The overall genetic identities ranged from 91% to 100%. Morphological identification classified the specimens into 19 families and 11 genera while DNA barcoding identified them into 19 families 33 genera and 40 species. A comparison between the two methods showed a mismatched identification of 42.6% where the accuracy percentage for morphological identification was moderate for the family level (67.8%) but was low for genus level identification (30%). The DNA barcoding method also managed to successfully identify 86.4% of the samples up to their species level where morphological method has failed to do so. The most misidentified families in the study were Blenniidae, Sparidae, Apogonidae Ambassidae and Monachantidae while almost all samples from the family Gobiidae and Engraulidae were correctly identified to family level because of their distinct morphology. In conclusion, taxonomic studies of larval fish should continue using combination of both morphology and DNA barcoding methods. Morphological identification should be more conservative i.e., when in doubt, it is better to key only to family and not to the genus and species level. DNA barcoding is a better method for deeper taxonomic levels identification with the existence of robust sequence reference libraries and should be able to validate the accuracy of traditional larval fish identification.     

Efficacy of using DNA barcoding to identify parasitoid wasps of the melon-cotton aphid (<Emphasis Type="Italic">Aphis gossypii</Emphasis>) in watermelon cropping system

Parasitoid wasps have received a great deal of attention in the biological control of melon-cotton aphid (Aphis gossypii Glover). The species of parasitoids are often difficult to identify because of their small body size and profound diversity. DNA barcoding offers scientists who are not expert taxonomists a powerful tool to render their field studies more accurate. Using DNA barcodes to identify aphid parasitoid wasps in specific cropping systems may provide valuable information for biological control. Here, we report the use of DNA barcoding to confirm the morphological identification of 14 species (belonging to 13 genera of 7 families) of parasitoid wasps from two-year field samples in a watermelon cropping system. We generated DNA sequences from the mitochondrial COI gene and the nuclear D2 region of 28S rDNA to assess the genetic variation within and between parasitoid species. Automatic Barcode Gap Discovery (ABGD) supported the presence of 14 genetically distinct groups in the dataset. Among the COI sequences, we found no overlap between the maximum K2P distance within species (0.49%) and minimum distance between species (6.85%). The 28S sequences also showed greater interspecific distance than intraspecific distance. DNA barcoding confirmed the morphological identification. However, inconsistency and ambiguity of taxonomic information available in the online databases has limited the successful use of DNA barcoding. Only five species matched those in the BOLD and GenBank. Four species did not match the entries in GenBank and five species showed ambiguous results in BOLD due to confusing nomenclature. We suggested that species identification based on DNA barcodes should be performed using both COI and other genes. Nonetheless, we demonstrate the potential of the DNA barcoding approach to confirm field identifications and to provide a foundation for studies aimed at improving the understanding of the biocontrol services provided by parasitoids in the melon ecosystem.     

Improved COI barcoding primers for Southeast Asian perching birds (Aves: Passeriformes)

The All Birds Barcoding Initiative aims to assemble a DNA barcode database for all bird species, but the 648-bp 'barcoding' region of cytochrome c oxidase subunit I (COI) can be difficult to amplify in Southeast Asian perching birds (Aves: Passeriformes). Using COI sequences from complete mitochondrial genomes, we designed a primer pair that more reliably amplifies and sequences the COI barcoding region of Southeast Asian passerine birds. The 655-bp region amplified with these primers overlaps the COI region amplified with other barcoding primer pairs, enabling direct comparison of sequences with previously published DNA barcodes.     

DNA barcoding of Caenogastropoda along coast of China based on the COI gene

DNA barcoding provides an efficient method for species-level identifications. In this study, we have amplified partial sequences of mitochondrial cytochrome c oxidase I (COI) gene from 110 specimens of 45 species of Caenogastropoda collected from the coast along China to evaluate whether DNA barcodes can distinguish these species accurately. The average Kimura 2-parameter (K2P) distances within species, genera and families were 0.44%, 13.96% and 22.27%, respectively. Both the neighbour-joining tree and the Bayesian tree showed a clear discrimination of all the species in our study with highly supported clades. These results proved that the species of Caenogastropoda can be efficiently and accurately identified by DNA barcoding based on the COI gene.     

DNA barcodes identify marine fishes of São Paulo State,Brazil

Anthropogenic impacts are an increasing threat to the diversity of fishes, especially in areas around large urban centres, and many effective conservation actions depend on accurate species identification. Considering the utility of DNA barcoding as a global system for species identification and discovery, this study aims to assemble a DNA barcode reference sequence library for marine fishes from the coastal region of São Paulo State, Brazil. The standard 652 bp ‘barcode’ fragment of the cytochrome c oxidase subunit I (COI) gene was PCR amplified and bidirectionally sequenced from 678 individuals belonging to 135 species. A neighbour‐joining analysis revealed that this approach can unambiguously discriminate 97% of the species surveyed. Most species exhibited low intraspecific genetic distances (0.31%), about 43‐fold less than the distance among species within a genus. Four species showed higher intraspecific divergences ranging from 2.2% to 7.6%, suggesting overlooked diversity. Notably, just one species‐pair exhibited barcode divergences of <1%. This library is a first step to better know the molecular diversity of marine fish species from São Paulo, providing a basis for further studies of this fauna – extending the ability to identify these species from all life stages and even fragmentary remains, setting the stage for a better understanding of interactions among species, calibrating the estimations about species composition and richness in an ecosystem, and providing tools for authenticating bioproducts and monitoring illegal species exploitation.     

DNA Barcodes of Rosy Tetras and Allied Species (Characiformes: Characidae: Hyphessobrycon) from the Brazilian Amazon Basin

DNA barcoding can be an effective tool for fast and accurate species-level identification based on sequencing of the mitochondrial cytochrome c oxidase subunit (COI) gene. The diversity of this fragment can be used to estimate the richness of the respective species. In this study, we explored the use of DNA barcoding in a group of ornamental freshwater fish of the genus Hyphessobrycon. We sequenced the COI from 10 species of Hyphessobrycon belonging to the “Rosy Tetra Clade” collected from the Amazon and Negro River basins and combined our results with published data. The average conspecific and congeneric Kimura 2-parameter distances were 2.3% and 19.3%, respectively. Six of the 10 species were easily distinguishable by DNA barcoding (H. bentosi, H. copelandi, H. eques, H. epicharis, H. pulchrippinis, and H. sweglesi), whereas the remaining species (H. erythrostigma, H. pyrrhonotus, H. rosaceus and H. socolofi) lacked reciprocal monophyly. Although the COI gene was not fully diagnostic, the discovery of distinct evolutionary units in certain Hyphessobrycon species under the same specific epithet as well as haplotype sharing between different species suggest that DNA barcoding is useful for species identification in this speciose genus.     

Comparative performance of the 16S rRNA gene in DNA barcoding of amphibians

BACKGROUND: Identifying species of organisms by short sequences of DNA has been in the center of ongoing discussions under the terms DNA barcoding or DNA taxonomy. A C-terminal fragment of the mitochondrial gene for cytochrome oxidase subunit I (COI) has been proposed as universal marker for this purpose among animals. RESULTS: Herein we present experimental evidence that the mitochondrial 16S rRNA gene fulfills the requirements for a universal DNA barcoding marker in amphibians. In terms of universality of priming sites and identification of major vertebrate clades the studied 16S fragment is superior to COI. Amplification success was 100% for 16S in a subset of fresh and well-preserved samples of Madagascan frogs, while various combination of COI primers had lower success rates.COI priming sites showed high variability among amphibians both at the level of groups and closely related species, whereas 16S priming sites were highly conserved among vertebrates. Interspecific pairwise 16S divergences in a test group of Madagascan frogs were at a level suitable for assignment of larval stages to species (1-17%), with low degrees of pairwise haplotype divergence within populations (0-1%). CONCLUSION: We strongly advocate the use of 16S rRNA as standard DNA barcoding marker for vertebrates to complement COI, especially if samples a priori could belong to various phylogenetically distant taxa and false negatives would constitute a major problem.     

COI barcoding of true bugs (Insecta, Heteroptera)

Several recent studies have proposed that partial DNA sequences of the cytochrome c oxidase I (COI) mitochondrial gene might serve as DNA barcodes for identifying and differentiating between animal species, such as birds, fish and insects. In this study, we tested the effectiveness of a COI barcode to identify true bugs from 139 species collected from Korea and adjacent regions (Japan, Northeastern China and Fareast Russia). All the species had a unique COI barcode sequence except for the genus Apolygus (Miridae), and the average interspecific genetic distance between closely related species was about 16 times higher than the average intraspecific genetic distance. DNA barcoding identified one probable new species of true bug and revealed identical or very recently divergent species that were clearly distinguished by morphological characteristics. Therefore, our results suggest that COI barcodes can reveal new cryptic true bug species and are able to contribute for the exact identification of the true bugs.     

DNA BARCODING: Barcoding corals: limited by interspecific divergence, not intraspecific variation

The expanding use of DNA barcoding as a tool to identify species and assess biodiversity has recently attracted much attention. An attractive aspect of a barcoding method to identify scleractinian species is that it can be utilized on any life stage (larva, juvenile or adult) and is not influenced by phenotypic plasticity unlike morphological methods of species identification. It has been unclear whether the standard DNA barcoding system, based on cytochrome c oxidase subunit 1 (COI), is suitable for species identification of scleractinian corals. Levels of intra- and interspecific genetic variation of the scleractinian COI gene were investigated to determine whether threshold values could be implemented to discriminate conspecifics from other taxa. Overlap between intraspecific variation and interspecific divergence due to low genetic divergence among species (0% in many cases), rather than high levels of intraspecific variation, resulted in the inability to establish appropriate threshold values specific for scleractinians; thus, it was impossible to discern most scleractinian species using this gene.