DNA barcoding of the ichthyofauna of Taal Lake, Philippines

This study represents the first molecular survey of the ichthyofauna of Taal Lake and the first DNA barcoding attempt in Philippine fishes. Taal Lake, the third largest lake in the Philippines, is considered a very important fisheries resource and is home to the world's only freshwater sardine, Sardinella tawilis. However, overexploitation and introduction of exotic fishes have caused a massive decline in the diversity of native species as well as in overall productivity of the lake. In this study, 118 individuals of 23 native, endemic and introduced fishes of Taal Lake were barcoded using the partial DNA sequence of the mitochondrial cytochrome c oxidase subunit I (COI) gene. These species belong to 21 genera, 17 families and 9 orders. Divergence of sequences within and between species was determined using Kimura 2-parameter (K2P) distance model, and a neighbour-joining tree was generated with 1000 bootstrap replications using the K2P model. All COI sequences for each of the 23 species were clearly discriminated among genera. The average within species, within genus, within family and within order percent genetic divergence was 0.60%, 11.07%, 17.67% and 24.08%, respectively. Our results provide evidence that COI DNA barcodes are effective for the rapid and accurate identification of fishes and for identifying certain species that need further taxonomic investigation.     

DNA barcode divergence among species and genera of birds and fishes

COI DNA barcoding is increasingly recognized as a significant new tool for the recognition and identification of animal species. Here, publicly available barcode data are compiled and analysed for birds (657 species) and fishes (1088 species). The proportion of species that cannot be barcode-distinguished by this marker is approximately 6.4% for birds and 2.1-2.5% for fishes. At all hierarchical taxonomic levels (species, genera, family, order, class), fish show greater mean COI divergence than birds. If two samples are barcode-identical, then for both birds and fishes, the probability that they are from the same species is 98-99%. The probability of conspecificity rapidly drops as divergence increases. At 2% COI divergence, this probability approximates to 1% for birds and 3% for fishes. The apparent difference between birds and fishes might partially reflect currently unrecognized cryptic species complexes in the latter. These probability estimates derive from pooled samples of birds and pooled samples of fishes, and will not apply in all situations. Recently evolved species complexes will have higher proportions of species that are barcode-identical. As barcode data accumulate, more refined statistical analyses will become possible.     

Probing diversity in freshwater fishes from Mexico and Guatemala with DNA barcodes

The freshwater fish fauna of Mexico and Guatemala is exceptionally diverse with >600 species, many endemic. In this study, patterns of sequence divergence were analysed in representatives of this fauna using cytochrome c oxidase subunit 1 (COI) DNA barcodes for 61 species in 36 genera. The average divergence among conspecific individuals was 0·45%, while congeneric taxa showed 5·1% divergence. Three species of Poblana , each occupying a different crater lake in the arid regions of Central Mexico, have had a controversial taxonomic history but are usually regarded as endemics to a single lake. They possess identical COI barcodes, suggesting a very recent history of isolation. Representatives of the Cichlidae, a complex and poorly understood family, were well discriminated by barcodes. Many species of Characidae seem to be young, with low divergence values (<2%), but nevertheless, clear barcode clusters were apparent in the Bramocharax – Astyanax complex. The symbranchid, Opisthernon aenigmaticum , has been regarded as a single species ranging from Guatemala to Mexico, but it includes two deeply divergent barcode lineages, one a possible new endemic species. Aside from these special cases, the results confirm that DNA barcodes will be highly effective in discriminating freshwater fishes from Central America and that a comprehensive analysis will provide new important insights for understanding diversity of this fauna.     

Molecular phylogeny of the freshwater sponges in Lake Baikal

The phylogenetic relationship of the freshwater sponges (Porifera) in Lake Baikal is not well understood. A polyphyletic and/or monophyletic origin have been proposed. The (endemic) Baikalian sponges have been subdivided into two families: endemic Lubomirskiidae and cosmopolitan Spongillidae. In the present study, two new approaches have been made to resolve the phylogenetic relationship of Baikalian sponges; analysis of (1) nucleotide sequences from one mitochondrial gene, the cytochrome oxidase subunit I (COI) and of (2) one selected intron from the tubulin gene. Specimens from the following endemic Baikalian sponge species have been studied; Lubomirskia baicalensis , Baikalospongia intermedia, Baikalospongia recta , Baikalospongia bacillifera and Swartschewskia papyracea . They are all grouped to the family of Lubomirskiidae. Sequence comparisons were performed with the ubiquitously distributed freshwater sponge Spongilla lacustris (family Spongillidae) as well as with one marine sponge, Suberites domuncula . A sequence comparison * * The sequences reported here are being deposited in the EMBL data base. of the mitochondrial COI gene revealed a monophyletic grouping of the endemic Baikalian sponges with S. lacustris as the most related species to the common ancestor. The sequences of the COI gene from B. recta , B. intermedia , B. bacillifera and L. baicalensis were found to be identical and separated from those of S. lacustris and S. papyracea . In a second approach, the exon/intron sequences framing the intron‐2 of the sponge tubulin gene were chosen for the phylogenetic analysis. The intron sequences were aligned and used for construction of a phylogenetic tree. This analysis revealed again a monophyletic grouping with S. lacustris as the closest related species to the common ancestor. It is concluded that the Baikalian sponges, which have been studied here, are of monophyletic origin. Furthermore, the data suggest that the endemic species S. papyracea is the phylogenetically oldest, extant, endemic Baikalian sponge species.     

Genetic barcoding of commercial Bêche-de-mer species (Echinodermata: Holothuroidea)

There are more than 47 species of holothurians used for bêche-de-mer production, many of which are locally overfished. With three exceptions, all bêche-de-mer species are Aspidochirotida and species identification of many of these is difficult. We analysed available genetic information and newly generated sequences to determine if genetic barcoding with the mitochondrial COI gene can be used to identify bêche-de-mer species. Although genetic data were available for ～50% of bêche-de-mer species, sufficient information and within-species replication were only available for six species. We generated 96 new COI sequences extending the existing database to cover most common species. COI unambiguously identified most bêche-de-mer species providing a genetic barcode for the identification of known species. In addition, conspecific (1.3%) variation and congeneric (16.9%) divergence were well separated ('barcoding-gap') albeit with a small overlap, which may lead to some error if genetic sampling alone was applied for species discovery. In addition to identification of adults, COI sequences were useful to identify juveniles that are often morphologically different. Sequence data showed that large (deep) and small (shallow) morphotypes of Holothuria atra are the same species, but suggested potential cryptic species within this taxon. For bêche-de-mer, the COI barcode proved useful in species clarification and discovery, but further genetic and taxonomic work is essential for several species. Some bêche-de-mer clades were problematic with morphologically disparate specimens sharing the same barcode. Our study indicated the presence of undescribed species (Bohadschia sp.) and species that constitute separate species in the Indian and Pacific Ocean (e.g. Holothuria fuscogilva).     

Effect of marker choice and thermal cycling protocol on zooplankton DNA metabarcoding studies

DNA metabarcoding is a promising approach for rapidly surveying biodiversity and is likely to become an important tool for measuring ecosystem responses to environmental change. Metabarcoding markers need sufficient taxonomic coverage to detect groups of interest, sufficient sequence divergence to resolve species, and will ideally indicate relative abundance of taxa present. We characterized zooplankton assemblages with three different metabarcoding markers (nuclear 18S rDNA, mitochondrial COI, and mitochondrial 16S rDNA) to compare their performance in terms of taxonomic coverage, taxonomic resolution, and correspondence between morphology‐ and DNA‐based identification. COI amplicons sequenced on separate runs showed that operational taxonomic units representing >0.1% of reads per sample were highly reproducible, although slightly more taxa were detected using a lower annealing temperature. Mitochondrial COI and nuclear 18S showed similar taxonomic coverage across zooplankton phyla. However, mitochondrial COI resolved up to threefold more taxa to species compared to 18S. All markers revealed similar patterns of beta‐diversity, although different taxa were identified as the greatest contributors to these patterns for 18S. For calanoid copepod families, all markers displayed a positive relationship between biomass and sequence reads, although the relationship was typically strongest for 18S. The use of COI for metabarcoding has been questioned due to lack of conserved primer‐binding sites. However, our results show the taxonomic coverage and resolution provided by degenerate COI primers, combined with a comparatively well‐developed reference sequence database, make them valuable metabarcoding markers for biodiversity assessment.     

DNA barcoding of Cuban freshwater fishes: evidence for cryptic species and taxonomic conflicts

Despite ongoing efforts to protect species and ecosystems in Cuba, habitat degradation, overuse and introduction of alien species have posed serious challenges to native freshwater fish species. In spite of the accumulated knowledge on the systematics of this freshwater ichthyofauna, recent results suggested that we are far from having a complete picture of the Cuban freshwater fish diversity. It is estimated that 40% of freshwater Cuban fish are endemic; however, this number may be even higher. Partial sequences (652 bp) of the mitochondrial gene COI (cytochrome c oxidase subunit I) were used to barcode 126 individuals, representing 27 taxonomically recognized species in 17 genera and 10 families. Analysis was based on Kimura 2-parameter genetic distances, and for four genera a character-based analysis (population aggregation analysis) was also used. The mean conspecific, congeneric and confamiliar genetic distances were 0.6%, 9.1% and 20.2% respectively. Molecular species identification was in concordance with current taxonomical classification in 96.4% of cases, and based on the neighbour-joining trees, in all but one instance, members of a given genera clustered within the same clade. Within the genus Gambusia, genetic divergence analysis suggests that there may be at least four cryptic species. In contrast, low genetic divergence and a lack of diagnostic sites suggest that Rivulus insulaepinorum may be conspecific with Rivulus cylindraceus. Distance and character-based analysis were completely concordant, suggesting that they complement species identification. Overall, the results evidenced the usefulness of the DNA barcodes for cataloguing Cuban freshwater fish species and for identifying those groups that deserve further taxonomic attention.     

DNA barcoding Korean birds

DNA barcoding, an inventory of DNA sequences from a standardized genomic region, provides a bio-barcode for identifying and discovering species. Several recent studies suggest that the sequence diversity in a 648 bp region of the mitochondrial gene for cytochrome c oxi- dase I (COI) might serve as a DNA barcode for identify- ing animal species such as North American birds, in- sects and fishes. The present study tested the effective- ness of a COI barcode in discriminating Korean bird species. We determined the 5' terminus of the COI bar- code for 92 species of Korean birds and found that spe- cies identification was unambiguous; the genetic differ- ences between closely related species were, on average, 25 times higher than the differences within species. We identified only one misidentified species out of 239 specimens in a genetic resource bank, so confirming the accuracy of species identification in the banking system. We also identified two potential composite species, calling for further investigation using more samples. The finding of large COI sequence differences between species confirms the effectiveness of COI barcodes for identifying Korean bird species. To bring greater reliability to the identification of species, increased in- tra- and interspecies sampling, as well as supplementa- tion of the mitochondrial barcodes with nuclear ones, is needed.     

中国蛛缘蝽科物种DNA条形码研究(英文)

【目的】本研究旨在探讨DNA条形码对中国蛛缘蝽科(半翅目：缘蝽总科)物种界定的适用性。【方法】对中国蛛缘蝽科13属23种207个样本的线粒体COI基因DNA条形码序列进行扩增,并扩增稻缘蝽属Leptocorisa 3个物种的31条内转录间隔区1(ITS-1)序列作为辅助标记。使用MEGA 11软件计算种间和种内遗传距离(Kimura 2-parameter, K2P);采用邻接法(neighbor-joining, NJ)进行物种聚类分析;利用中介邻接网络算法构建单倍型网络图。【结果】基于线粒体COI DNA条形码序列得出测试的中国蛛缘蝽科所有23个种的种内平均K2P距离在2%以下,种间K2P距离在0.98%~23.98%之间(平均17.50%)。多数物种彼此能够被较好地分开,且支持率较高。其中,中稻缘蝽Leptocorisa chinensis和大稻缘蝽L. oratoria共享部分COI单倍型,造成COI条形码无法区分二者,可通过ITS-1序列在单倍型网络分析中将二者区分。【结论】本研究得出的中国蛛缘蝽科中绝大部分物种的DNA条形码数据分析结果与基于形态特征的分类单元一致。然而,对于其中亲缘关系极近的物种,单靠线粒体数据尤其是COI条形码序列无法进行准确界定,需引入其他DNA序列或其他类型数据进行区分。     

DNA Barcode Identification of Freshwater Snails in the Family Bithyniidae from Thailand

Freshwater snails in the family Bithyniidae are the first intermediate host for Southeast Asian liver fluke (Opisthorchis viverrini), the causative agent of opisthorchiasis. Unfortunately, the subtle morphological characters that differentiate species in this group are not easily discerned by non-specialists. This is a serious matter because the identification of bithyniid species is a fundamental prerequisite for better understanding of the epidemiology of this disease. Because DNA barcoding, the analysis of sequence diversity in the 5’ region of the mitochondrial COI gene, has shown strong performance in other taxonomic groups, we decided to test its capacity to resolve 10 species/ subspecies of bithyniids from Thailand. Our analysis of 217 specimens indicated that COI sequences delivered species-level identification for 9 of 10 currently recognized species. The mean intraspecific divergence of COI was 2.3% (range 0-9.2 %), whereas sequence divergences between congeneric species averaged 8.7% (range 0-22.2 %). Although our results indicate that DNA barcoding can differentiate species of these medically-important snails, we also detected evidence for the presence of one overlooked species and one possible case of synonymy.     

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.     

Cryptic diversity in flathead fishes (Scorpaeniformes: Platycephalidae) across the Indo‐West Pacific uncovered by DNA barcoding

Identification of taxonomical units underpins most biological endeavours ranging from accurate biodiversity estimates to the effective management of sustainably harvested, protected or endangered species. Successful species identification is now frequently based on a combination of approaches including morphometrics and DNA markers. Sequencing of the mitochondrial COI gene is an established methodology with an international campaign directed at barcoding all fishes. We employed COI sequencing alongside traditional taxonomic identification methods and uncovered instances of deep intraspecific genetic divergences among flathead species. Sixty‐five operational taxonomic units (OTUs) were observed across the Indo‐West Pacific from just 48 currently recognized species. The most comprehensively sampled taxon, Platycephalus indicus, exhibited the highest levels of genetic diversity with eight lineages separated by up to 16.37% genetic distance. Our results clearly indicate a thorough reappraisal of the current taxonomy of P. indicus (and its three junior synonyms) is warranted in conjunction with detailed taxonomic work on the other additional Platycephalidae OTUs detected by DNA barcoding.     

Ancient phylogenetic separation between Pacific and Atlantic cephalochordates as revealed by mitochondrial genome analysis

The subphylum Cephalochordata (lancelets) is a relatively small taxonomic group in contrast to the subphyla Urochordata and Vertebrata. As an initial step to determine whether lancelets exhibit small genetic divergence in keeping with their conservative body organization or large genetic variation, four Branchiostoma species from the Pacific (B. belcheri and B. malayanum) and Atlantic (B. floridae and B. lanceolatum) Oceans were genetically compared using partial mitochondrial DNA sequences of the cytochrome oxidase c subunit I (COI) and 16S ribosomal RNA (16S rRNA) genes. In both genes, large genetic differences were revealed between the Pacific and Atlantic species, as well as within the former. Two maximum-likelihood trees from the COI and 16S rRNA genes showed that the Pacific and Atlantic lancelets were reciprocally clustered into different clades. Furthermore, both gene trees consistently exhibited deep phylogenetic separation between the two oceans. The estimated divergence time suggested that differentiation may have followed the migration of ancestral lancelets from the Pacific to the Atlantic Oceans via the Tethys Sea.     

Complete mitochondrial genome of the <Emphasis Type="Italic">Pleuronichthys lighti</Emphasis> (Pleuronectiformes,Pleuronectidae) with phylogenetic consideration

Mitochondrial genome has been used to shed light on many fields of both basic and applied research, including the study of molecular evolution. The complete mitochondrial genome sequence of 17368 bp nucleotides from the Pleuronichthys lighti was determined. It was a circular double-stranded DNA molecule with identical set of 22 transfer RNA genes, 2 ribosomal RNA genes, 13 protein-coding genes as well as a non-coding control region. Stand asymmetry in the nucleotide composition was reflected in the codon usage of genes oriented in opposite directions. In the control region, we identified the extended termination associated sequence domain, the central conserved sequence block domain and the conserved sequence block domain, and two complete repeat region. They were “TTACAATA” and “TGTTGTAA”, respectively. All known 12 mitochondrial genomes of Pleuronectinae fishes were downloaded and analyzed; there were 5570 variable sites in the consensus sequences of 15241 base pairs, calculation of total sites were 35.5%. The highest sequence divergence was 50% (ATP8) and the Kimura-2-parameter genetic distance was 0.235 (ND6), whereas the COIII had the lowest sequence divergence (28.8%) and genetic distance (0.128); the protein coding genes were mainly acted by purifying selection which was detected by selection tests. Analysis of confidence and the information content for per nucleotide revealed ND5, ATP6, COI and ND4 genes were suitable molecular markers for phylogenetic study of Pleuronectinae fishes. Phylogenetic analysis using Bayesian computational algorithms based on COI genes provided support for the taxonomic status of P. lighti, which was consistent with the traditional taxonomy.     

Molecular Barcoding of Aquatic Oligochaetes: Implications for Biomonitoring

Aquatic oligochaetes are well recognized bioindicators of quality of sediments and water in watercourses and lakes. However, the difficult taxonomic determination based on morphological features compromises their more common use in eco-diagnostic analyses. To overcome this limitation, we investigated molecular barcodes as identification tool for broad range of taxa of aquatic oligochaetes. We report 185 COI and 52 ITS2 rDNA sequences for specimens collected in Switzerland and belonging to the families Naididae, Lumbriculidae, Enchytraeidae and Lumbricidae. Phylogenetic analyses allowed distinguishing 41 lineages separated by more than 10 % divergence in COI sequences. The lineage distinction was confirmed by Automatic Barcode Gap Discovery (ABGD) method and by ITS2 data. Our results showed that morphological identification underestimates the oligochaete diversity. Only 26 of the lineages could be assigned to morphospecies, of which seven were sequenced for the first time. Several cryptic species were detected within common morphospecies. Many juvenile specimens that could not be assigned morphologically have found their home after genetic analysis. Our study showed that COI barcodes performed very well as species identifiers in aquatic oligochaetes. Their easy amplification and good taxonomic resolution might help promoting aquatic oligochaetes as bioindicators for next generation environmental DNA biomonitoring of aquatic ecosystems.     

COI is better than 16S rRNA for DNA barcoding Asiatic salamanders (Amphibia: Caudata: Hynobiidae)

The 5' region of the mitochondrial DNA (mtDNA) gene cytochrome c oxidase I (COI) is the standard marker for DNA barcoding. However, because COI tends to be highly variable in amphibians, sequencing is often challenging. Consequently, another mtDNA gene, 16S rRNA gene, is often advocated for amphibian barcoding. Herein, we directly compare the usefulness of COI and 16S in discriminating species of hynobiid salamanders using 130 individuals. Species identification and classification of these animals, which are endemic to Asia, are often based on morphology only. Analysis of Kimura 2-parameter genetic distances (K2P) documents the mean intraspecific variation for COI and 16S rRNA genes to be 1.4% and 0.3%, respectively. Whereas COI can always identify species, sometimes 16S cannot. Intra- and interspecific genetic divergences occasionally overlap in both markers, thus reducing the value of a barcoding gap to identify genera. Regardless, COI is the better DNA barcoding marker for hynobiids. In addition to the comparison of two potential markers, high levels of intraspecific divergence in COI (>5%) suggest that both Onychodactylus fischeri and Salamandrella keyserlingii might be composites of cryptic species.     

青海省6种裂腹鱼COI基因作为DNA条形码的适用性初探

青海省裂腹鱼鱼类至少有20种,占土著鱼类的40%以上,具有重要的生态价值。由于生态环境的恶化和人为因素的干扰,很多物种群已濒临灭绝。快速和准确的物种鉴定对于这些物种的保护至关重要,而基于形态学的传统分类法很难满足这一需求。因此,本研究通过DNA条形码技术初步探讨了在青海省裂腹鱼物种鉴定中的适用性。本研究中,测序获得了青海26尾裂腹鱼(6个物种)的细胞色素c氧化酶亚基I (COI)基因,并经GenBank数据库进行比对;基于K2P模型分析COI序列变异;运用贝叶斯法(BI)和最大似然法(ML)构建系统发育树。结果显示,6种裂腹鱼COI序列检测得到15个单倍型,且各物种间无共享的单倍型。基于K2P模型,最大种内遗传距离和最小种间遗传距离分别为(0.621±0.297)%和(2.792±0.644)%。种间平均遗传距离为(12.205±1.307)%,约为种内平均遗传距离((0.327±0.162)%)的37倍,表明各物种的COI序列间已经形成明显"条形码间隙"。AMOVA分析显示,遗传变异主要来自种间,约占97.05%;FST=0.970 54,p<0.01,说明各物种间分化程度极高。此外,基于BI和ML方法构建的系统树具有一致的拓扑结构,分辨率较高;各物种均单独聚为一个发育枝,拓扑结构合理。以上结果表明,COI基因作为DNA条形码在青海裂腹鱼物种鉴定中具有较高的适用性。     

Barcoding aphids (Hemiptera: Aphididae) of the Korean Peninsula: updating the global data set

DNA barcode (mitochondrial COI) sequences are provided for species identification of aphids from the Korean Peninsula. Most (98%) of the 154 species had distinct COI sequences (average 0.05% intraspecific pairwise divergence) relative to the degree of sequence divergence among species (average value 5.84%). For species in common with other regions, barcodes for Korean samples fell near or within known levels of variation. Based on these results, we conclude that DNA barcodes can provide an effective tool for identifying aphid species in such applications as pest management, monitoring and plant quarantine.     

Molecular diversity of Germany's freshwater fishes and lampreys assessed by DNA barcoding

This study represents the first comprehensive molecular assessment of freshwater fishes and lampreys from Germany. We analysed COI sequences for almost 80% of the species mentioned in the current German Red List. In total, 1056 DNA barcodes belonging to 92 species from all major drainages were used to (i) build a reliable DNA barcode reference library, (ii) test for phylogeographic patterns, (iii) check for the presence of barcode gaps between species and (iv) evaluate the performance of the barcode index number (BIN) system, available on the Barcode of Life Data Systems. For over 78% of all analysed species, DNA barcodes are a reliable means for identification, indicated by the presence of barcode gaps. An overlap between intra‐ and interspecific genetic distances was present in 19 species, six of which belong to the genus Coregonus. The Neighbour‐Joining phenogram showed 60 nonoverlapping species clusters and three singleton species, which were related to 63 separate BIN numbers. Furthermore, Barbatula barbatula, Leucaspius delineatus, Phoxinus phoxinus and Squalius cephalus exhibited remarkable levels of cryptic diversity. In contrast, 11 clusters showed haplotype sharing, or low levels of divergence between species, hindering reliable identification. The analysis of our barcode library together with public data resulted in 89 BINs, of which 56% showed taxonomic conflicts. Most of these conflicts were caused by the use of synonymies, inadequate taxonomy or misidentifications. Moreover, our study increased the number of potential alien species in Germany from 14 to 21 and is therefore a valuable groundwork for further faunistic investigations.     

Estimating diversity of crabs (Decapoda: Brachyura) in a no-take marine protected area of the SW Atlantic coast through DNA barcoding of larvae

DNA barcoding was used to identify crab larvae from the Marine Biological Reserve of Arvoredo, encompassing a coastal archipelago off the SW Atlantic coast (27°S, 48°W). Partial mitochondrial COI or 16S rRNA gene sequences were obtained for 488 larvae, leading to the identification of 20 species. The COI sequences generated 13 barcode index numbers (BINs) within Barcode of Life Data Systems (BOLD), among which 11 were concordant with single species. DNA from ～ 6% of the larvae did not amplify using the primers tested; based on external morphological characteristics, these larvae represented four possible additional operational taxonomic units (OTUs) at the family level. Intraspecific variation for the COI and 16S rRNA genes was found to be < 2.6% and < 2.1% respectively (Kimura 2-parameter distance), whereas interspecific divergence ranged from 7.9% to 21.5% and 6.4% to 14.5%, respectively. These results imply that both genes are suitable for use in species identification of brachyuran crabs of this area. Molecular identification of this group successfully enabled the diagnosis of larvae of closely related species, including congeners in Mithrax, Achelous and Callinectes. In addition, eight out of 20 species recognized represent new records for the reserve suggesting that the brachyuran fauna in the area has been underestimated based on traditional biodiversity measures. The availability of primers suited to the targeted species, and the development of a taxonomically comprehensive DNA barcoding database are the major recommendations to improve the accuracy and feasibility of using DNA barcoding for species identification of SW Atlantic brachyuran crabs.