Kimura 2-parameter Genetic Distances Research Articles

Morphological Description and Phylogenetic Analyses of a New Species of Callistoctopus (Cephalopoda, Octopodidae) from China

A new octopus species, Callistoctopus tenuipes sp. nov., was formally described from the southeastern coastal waters of China using morphological description and molecular analysis methods. C. tenuipes sp. nov. is a small- to moderate-sized octopus, which is characterized by very narrow and long arms. Although it was previously misidentified as the juvenile of Octopus minor (Sasaki, 1920), it can be recognised by spots, gill lamellae count, funnel organ shape, enlarged suckers, and ligula shape. C. tenuipes sp. nov. differs from the small-sized octopus Callistoctopus xiaohongxu, mainly in the gill lamellae count, funnel organ shape, and arm-length index. In the molecular analysis, sequences obtained from the cytochrome c-oxidase subunit I (COI) gene of eight specimens were 590 bp in length. The pairwise Kimura 2-parameter (K2P) genetic distances between Octopodidae species ranged from 8.58 to 23.79% based on the COI gene. The phylogenetic analyses suggested that C. tenuipes sp. nov. belonged to the Callistoctopus clade and may have a close affinity with C. xiaohongxu and O. minor. Moreover, three species delimitation methods all strongly supported C. tenuipes as a separate species.

The preliminary DNA barcoding dataset of fishes from Chinese coastal waters

DNA barcoding is a molecular method for species identification based on a short standardized DNA sequence derived from the 5’ region of the cytochrome c oxidase subunit I (COI) gene, which is widely applied in marine fish identification. In this study, DNA barcodes were generated for 1,671 specimens representing 309 morphological marine fish species belonging to 223 genera and covering 101 families from Chinese coastal waters. The mean Kimura 2-parameter (K2P) genetic distances within species, genera, families, and orders were 0.20%, 20.94%, 24.45%, and 25.34%, respectively. As expected, the mean interspecific distance was higher than the mean intraspecific distance. Phylogenetic trees based on Bayesian inference (BI) and neighbor-joining (NJ) methods agreed with the taxonomic classifications. A total of 306 operational taxonomic units (OTUs) were reidentified via automatic barcode gap discovery (ABGD) analysis. An estimated nucleotide substitution matrices showed a transversion bias compared to transitional mutation. The amino acid variations of the barcoding region were also examined, and 13 amino acid sites exhibited high variability. Overall, our findings confirmed that DNA barcoding is a highly reliable mean of identifying fish species in Chinese coastal waters, and that the barcode library established here would facilitate the monitoring and management of the marine fishery resources in China.

Triplophysadaryoae, a new nemacheilid loach species (Teleostei, Nemacheilidae) from the Syr Darya River basin, Central Asia.

Triplophysadaryoae, new species, is described from the Sokh River, a former tributary of Syr Darya that today fails to reach the river, in the Sokh District, an exclave of Uzbekistan, surrounded by Kyrgyzstan. Triplophysadaryoae is distinguished from other species of Triplophysa in Central Asia by a truncate caudal fin with 13 or 14 branched rays, body without obvious mottling, dorsal-fin origin opposite to pelvic-fin insertion, and absence of the posterior chamber of the air bladder. Molecular data suggest that Triplophysadaryoae is closely related to T.ferganaensis from the Shakhimardan stream, a small tributary of Syr Darya in the Yordon village, another exclave of Uzbekistan in Kyrgyzstan. The two species were separated by a Kimura 2-parameter genetic distance of 2.8% in the mitochondrial DNA cytochrome c oxidase subunit I barcode region; they are also distinguished morphologically. A key to the species of Triplophysa in the Syr Darya basin and adjacent regions is provided.

Johnius sasakii, a new species of croaker (Perciformes: Sciaenidae) with a key to Johnius from East Malaysia, Borneo.

A new sciaenid fish, Johnius sasakii sp. nov. from the East Malaysian coastal waters of Sabah and Sarawak, Borneo is described herein. Johnius sasakii sp. nov. can be separated from the close congeners, Johnius heterolepis and Johnius carouna by having less gill rakers on the lower limb of the first gill arch (mode number 9 vs. 10 for J. heterolepis; 11 for J. carouna). It can be distinguished from J. heterolepis with the higher modal number of gill rakers on the upper limb of first gill arch (5 vs. 4), shorter anal-fin base length (9% vs. 10% of SL) and first anal-fin ray length (1012% vs. 1213% of SL) respectively. Furthermore, J. sasakii sp. nov. can be further differentiated from J. carouna by its shorter second anal-fin spine length 710% SL (usually 810%) vs. 1114% SL (usually 1112%) and slightly deeper body depth 2529% SL (usually 2628%) vs. 2328% SL (usually 2426%). Meanwhile, J. sasakii sp. nov. can be easily differentiated from J. macrorhynus by a present of molariform teeth on inner rows of jaws and stumpy gill rakers. Johnius (Johnius) sasakii sp. nov. can be clearly distinguished from species of the subgenus J. (Johnieops) by the lack of enlarged outer row teeth on upper jaw. A Kimura 2-parameter genetic distance comparison of 450 bp sequences of mitochondrial 16S rRNA and 488 bp nuclear gene S7 showed that J. sasakii sp. nov. are highly differentiated from J. heterolepis (16S: 10.4%, S7: 5.8%), J. carouna (16S:19.3%, S7:8.4%); and J. macrorhynus (16S: 16.7%, S7: 8.1%) respectively. The study highlights that the genetic approach from mtDNA and nDNA can contribute to the confirmation of taxonomic status of sympatric species in genus Johnius.

DNA Barcoding of Mosquitoes from the Pantanos de Centla Biosphere Reserve, Southeastern Mexico.

Accurate identification of mosquito species is essential to support programs that involve the study of distribution and mosquito control. Numerous mosquito species are difficult to identify based only on morphological characteristics, due to the morphological similarities in different life stages and large numbers of some species that are members of morphologically similar species complexes. In the present study, the mosquitoes collected in the Pantanos de Centla Biosphere Reserve, southeastern Mexico, were evaluated using a combination of morphological and molecular approaches (mitochondrial cytochrome c oxidase subunit I [COI] DNA barcode). A total of 1,576 specimens of 10 genera and 35 species, mostly adult stages, were collected. A total of 225 COI DNA barcode sequences were analyzed; most species formed well-supported groups in the neighbor joining, maximum likelihood, and Bayesian inference trees. The intraspecific Kimura 2-parameter (K2P) genetic distance averaged 1.52%. An intraspecific K2P distance of 6.20% was observed in Anopheles crucians s.l., while a deep split was identified in Culex erraticus and Cx. conspirator. This study showed that COI DNA barcodes offer a reliable approach to support mosquito species identification in Mexico.

Molecular identification and phylogenetic analysis of Papaver based on ITS2 barcoding.

In forensic cases suspected to involve Papaver somniferum, species identification is key to the investigation. To accurately detect and identify P.somniferum as well as common adulterants of the same genus, 19 internal transcribed spacer 2 (ITS2) sequences of P.somniferum (256bp), Papaver canescens (254bp), Papaver nudicaule (254bp), Papaver pavoninum (250bp), Papaver radicatum (254bp), and Papaver rhoeas (256bp) were obtained. Based on the ITS2 sequence, similarity analysis via BLAST, the nearest Kimura-2-parameter (K2P) genetic distances were calculated, and a phylogenetic tree was constructed using MEGA X software for the identification of six species of Papaver. Finally, differences in the ITS2 secondary structure between species were analyzed. The best matches of the P.somniferum ITS2 sequence were of other P.somniferum from different sources. The nearest K2P genetic distances between P.somniferum and its counterparts from other sources were zero, which was the smallest pairwise genetic distance among distances from the other five Papaver species. Various sources of P.somniferum clustered into an independent branch in the phylogenetic tree. The secondary structures of P.somniferum and P.rhoeas were significantly different from those of the other four species of Papaver. In summary, P.somniferum can be effectively distinguished from five closely related plants of the same genus by using ITS2 as a DNA barcode.

DNA barcodes provide evidence for the independent origin of the cultivated silkwormsSamia ricini andSamia cynthia

Samia ricini (Wm. Jones) andSamia cynthia (Drury) (Lepidoptera: Saturniidae) have been used as traditional sources of food as well as silk-producing insects. However, the phylogenetic relationship between the two silkworms remains to be addressed. In this study, the mitochondrial cytochrome c oxidase subunit 1 (COI) gene sequences corresponding to DNA barcodes from 13Samia species were analysed, and a DNA barcode-based phylogenetic framework for theseSamia species was provided. Phylogenetic analysis showed that multiple individuals of a species could be clustered together. Our analysis revealed a close relationship amongSamia yayukae Paukstadt, Peigler and Paukstadt,Samia abrerai Naumann and Peigler,Samia kohlli Naumann and Peigler,Samia naessigi Naumann and Peigler,Samia naumanni Paukstadt, Peigler and Paukstadt, andSamia kalimantanensis Paukstadt and Paukstadt. The mixed clustering relationship and low Kimura-2-parameter (K2P) genetic distance (0.006) between individuals ofS. ricini andSamia canningi (Hutton) indicated that the cultivated silkwormS. ricini was derived from the non-cultivated silkwormS. canningi. The remote phylogenetic relationship and high K2P genetic distance (0.039) indicated thatS. ricini andS. cynthia are distinct species, thus providing solid molecular evidence that they had entirely independent origins. The relationships betweenS. kalimantanensis andS. naumanni and betweenS. cynthia andSamia wangi Naumann and Peigler, as well as the potential cryptic species withinS. abrerai were also discussed. This is the first study to assess the DNA barcodes of the genusSamia, which supplements the knowledge of species identification and provides the first molecular phylogenetic framework forSamia species.

A multilocus assessment reveals two new synonymies for East Asian Cyclommatus stag beetles (Coleoptera, Lucanidae).

Cyclommatus scutellaris Möllenkamp, 1912, Cyclommatus elsae Kriesche, 1921 and Cyclommatus tamdaoensis Fujita, 2010 are East Asian stag beetle species with long-debated taxonomic relationships due to high intraspecific morphological variability. In this study, we applied multilocus phylogenetic analyses to reassess their relationships. Two mitochondrial genes (16S rDNA, COI) and two nuclear genes (28S rDNA, Wingless) were used to reconstruct the phylogeny through the Bayesian inference (BI) and Maximum Likelihood (ML) methods. Both topologies supported two clades: the clade C. scutellaris was sister to the clade (C. elsae + C. tamdaoensis) with the subclade C. tamdaoensis embedded in the subclade C. elsae. The Kimura 2-parameter (K2P) genetic distance analysis yielded a low mean value (≤0.035) among the three taxa, which was well below the minimum mean value between other Cyclommatus species (≥0.122). We also compared the accuracy and efficiency of two approaches, GMYC and ABGD, in delimitating the three lineages. The result shows that ABGD is a better approach than GMYC. Our molecular data recognizes the three species as different populations of a single species, ranging from Taiwan Island to the continent. Therefore, we propose two new junior synonyms for C. scutellaris: C. tamdaoensis, syn. nov. and C. elsaesyn. nov.

DNA Barcoding of Fish in Mischief Reef—Fish Diversity of a Reef Fish Community From Nansha Islands

Development of effective conservation and management strategies requires assessments of ecosystem biodiversity status, especially in understudied hotspots of global fish diversity. Coral reefs are important habitats for fishes, with biodiversity hotspots known globally. We present the first data on molecular diversity of fishes of Mischief Reef, the largest atoll in the Nansha Islands. Partial sequences (650 bp) of mitochondrial COI gene (Cytochrome c oxidase subunit I) are used to identify 209 individuals, representing 101 species, referable to 62 genera, 27 families, 8 orders, and 1 class. The most abundant orders are the Perciformes (176 specimens, 84.21%), Tetraodontiformes (13 specimens, 6.22%), and Beryciformes (13 specimens, 6.22%). Mean Kimura 2-Parameter genetic distances within genera, families, and orders are 4.51, 13.90, and 17.63%, respectively. We record Monotaxis heterodon from this region for the first time—a species that may previously have been misidentified as M. grandoculis. In addition, we recognized possible cryptic species of Lethrinus olivaceus based on significantly diverging barcode sequences. Barcode data provide new insights into fish diversity of Mischief Reef, important for developing further researches on this fauna, and for its conservation.

DNA barcoding of coral reef fishes from Chuuk State, Micronesia

The fish diversity of Chuuk Micronesia is currently under threat due to rapid changes in the coral reef ecosystem. Thus, accurate fish identification using DNA barcodes is fundamental for exploring species biodiversity and resource protection. In this study, we analyzed 162 fish mitochondrial DNA cytochrome c oxidase I (COI) barcodes from Chuuk Micronesia. Consequently, we identified 95 species from 53 genera in 26 families and seven orders. The average Kimura 2-parameter genetic distances within species, genera, families, and orders were calculated as 0.17%, 11.78%, 15.63%, and 21.90%, respectively. Also, we have utilized DNA barcodes to perform genetic divergence and phylogenetic analysis of families recognized as dominant groups in Chuuk State. Our findings confirm that DNA barcodes using COI are an effective approach in identifying coral reef fish species. We anticipate that the results of this study will provide baseline data for the protection of coral reef fish biodiversity at Chuuk Micronesia.

Phylogenetic relationships of closely-related phlebotomine sand flies (Diptera: Psychodidae) of Nyssomyia genus and Lutzomyia subgenus.

BACKGROUND The Nyssomyia genus and Lutzomyia subgenus include medical important species that are Latin American leishmaniases vectors. Little is known about the phylogenetic relationships of closely-related species in each of these taxonomic groups that are morphologically indistinguishable or differentiated by very subtle details. OBJECTIVES We inferred the phylogenetic relationships of closely-related species within both the Nyssomyia genus and the Lutzomyia subgenus using a cytochrome c oxidase subunit I (COI) fragment.METHODS The sampling was carried out from 11 Argentinean localities. For genetic analyses, we used GenBank sequences in addition to our sequences from Argentina. Kimura 2-parameter (K2P) genetic distance and nucleotide divergence (Da) was calculated between closely-related species of Nyssomyia genus, Lutzomyia subgenus and between clades of Lutzomyia longipalpis complex.FINDINGS The K2P and Da values within species of Nyssomyia genus and Lutzomyia subgenus were lower than the divergence detected between clades of Lu. longipalpis complex. The haplotype network analyses within Lutzomyia subgenus showed shared haplotypes between species, contrary to Nyssomyia genus with none haplotype shared. Bayesian inference within Nyssomyia genus presented structuring by species.MAIN CONCLUSIONS This study evidences the phylogenetic proximity among closely-related species within Nyssomyia genus and Lutzomyia subgenus. The COI sequences of Nyssomyia neivai derived from the present study are the first available in GenBank.

Caridina biyiga sp. nov., a new freshwater shrimp (Crustacea: Decapoda: Atyidae) from Leichhardt Springs, Kakadu National Park, Australia, based on morphological and molecular data, with a preliminary illustrated key to Northern Territory Caridina.

Caridina biyiga sp. nov. is described from Leichhardt Springs, Kakadu National Park, Northern Territory, Australia, based on morphological and molecular data. The new species is highly distinctive among its congeners in having the dactylus of pereiopod 5 similar to the dactyli of pereiopods 3-4 and lacking the comb-like row of spiniform setae typical of the genus. The branchial formula is also distinctive for the genus in lacking an arthrobranch at the base of pereiopod 1 and in having a vestigial epipod on maxilliped 1. Despite the distinctive morphology of the new species, the molecular data did not provide strong support for the recognition of a new genus. Phylogenetic analyses of Australian Caridina using the mitochondrial 16S rDNA gene fragment placed the new species in a well-supported clade containing at least one typical species of Caridina. Within this clade, referred to as the 'thermophila' group, Kimura 2-parameter (K2P) genetic distances of 16S rDNA between the new species and sister taxa ranged from 5.1-6.0%. Analyses using the mitochondrial 3' COI gene fragment from species of the 'thermophila' group yielded K2P genetic distances between the new species and its sister taxa ranging from 10.4-15.1%. A preliminary illustrated key to Northern Territory Caridina is provided. The conservation significance of Leichhardt Springs and its aquatic fauna are also briefly discussed.

DNA barcoding of coastal ray-finned fishes in Vietnam.

DNA barcoding based on a fragment of the cytochrome c oxidase subunit I (COI) gene is widely applied in species identification and biodiversity studies. The aim of this study was to establish a comprehensive barcoding database of coastal ray-finned fishes in Vietnam. A total of 3,638 specimens were collected from fish landing sites in northern, central and southern Vietnam. Seven hundred and sixty-five COI sequences of ray-finned fishes were generated, belonging to 458 species, 273 genera, 113 families and 43 orders. A total of 59 species were newly recorded in Vietnam and sequences of six species were new to the Genbank and BOLD online databases. Only 32 species cannot be annotated to species level because difficulty in morphological identifications and their Kimura-2-Parameter (K2P) genetic distances to most similar sequences were more than 2%. Moreover, intra-specific genetic distances in some species are also higher than 2%, implying the existence of putative cryptic species. The mean K2P genetic distances within species, genera, families, orders and classes were 0.34%, 12.14%, 17.39%, 21.42%, and 24.80, respectively. Species compositions are quite different with only 16 common species among northern, central and southern Vietnam. This may attribute to multiple habitats and environmental factors across the 3,260 km Vietnamese coastline. Our results confirmed that DNA barcoding is an efficient and reliable tool for coastal fish identification in Vietnam, and also established a reliable DNA barcode reference library for these fishes. DNA barcodes will contribute to future efforts to achieve better monitoring, conservation, and management of fisheries in Vietnam.

DNA barcode and minibarcode identification of freshwater fishes from Cerrado headwater streams in Central Brazil.

The extraordinary species diversity of the Neotropical freshwater fish fauna is world renown. Yet, despite rich species diversity, taxonomic and genetic resources for its Cerrado ichthyofauna remain poorly developed. We provide a reference library of 149 DNA barcodes for 39 species/lineages of Cerrado headwater stream fishes from the Brazilian Distrito Federal and nearby areas and test the utility of distance-based criteria, tree-based criteria and minibarcodes for specimen identification. Mean Kimura 2-parameter genetic distances within species to orders ranged 1·8-12·1%. However, mean intraspecific v. congeneric-interspecific distances (0·9-1·3%) overlapped extensively and distance-based barcoding failed to achieve correct identifications due to c. 4-12·1% error rates and 19·5% ambiguous identifications related to the presence of singletons. Overlap was reduced and best-match success rates improved drastically to 83·5% when Characidium barcodes representing potential misidentifications or undescribed species were removed. Tree-based monophyly criteria generally performed similarly to distance methods, correctly differentiating up to c. 85% of species/lineages despite neighbour-joining and Bayesian tree errors (random lineage-branching events, long-branch attraction). Five clusters (Ancistrus aguaboensis, Characidium spp., Eigenmannia trilineata, Hasemania hanseni and Hypostomus sp. 2) exhibited deep intraspecific divergences or para-/polyphyly and multiple Barcode Index Number assignments indicative of putative candidate species needing taxonomic re-examination. Sliding-window analyses also indicated that a 200 bp minibarcode region performed just as well at specimen identification as the entire barcode gene. Future DNA barcoding studies of Distrito Federal-Cerrado freshwater fishes will benefit from increased sampling coverage, as well as consideration of minibarcode targets for degraded samples and next-generation sequencing.

Insights into cryptic diversity and adaptive evolution of the clam Coelomactra antiquata (Spengler, 1802) from comparative transcriptomics

The clam Coelomactra antiquata (Spengler, 1802) is one of the most economically important marine bivalves in China. Previous genetic analysis shows that C. antiquata possibly consists of two cryptic species, a northern and a southern lineage. Some differences between the two lineages are observed in their morphology and fitness in aquaculture. However, little is known about the genetic basis of these differences. In this study, a comparative transcriptomics study was performed to provide additional information for delineating the cryptic species of C. antiquata, as well as insights into the adaptive evolution in the southern lineage of C. antiquata. A total of 4377 orthologous genes were identified using a phylogenetic tree-based approach. The Kimura 2-parameter genetic distances were 0.0385 between the two lineages of C. antiquata, and 0.006 within the northern lineage. The sequence divergences supports that the lineages represent two cryptic species. We identified 28 positively selected genes (PSGs) from the southern lineage of C. antiquata using a modified branch-site model. The PSGs were implicated in innate immune responses, metabolism, development, cell division, transmembrane transport, and signaling processes, which could be related to the higher disease resistance, growth rate, and adaptability of the southern lineage than its northern counterpart. The comparative transcriptome analysis provides a foundation for future investigations on the biological differences between the two lineages of C. antiquata.

Molecular identification of the Danzhou chicken breed in China using DNA barcoding

Mitochondrial cytochrome C oxidase subunit I (COI) has been used as a DNA barcode to identify population genetic diversity and distinguish animal species as it is variable enough to distinguish between species, yet suitably conserved. A new native chicken breed, named Danzhou chicken was discovered in Hainan, China in 2014, although identification is difficult by morphological examination alone. The mitochondrial COI genes of six chicken breeds, including four local and two imported breeds (Danzhou, Wenchang, Bawang, Beijing-You, Hy-Line Brown, and Ross) were compared and assessed in terms of their efficacy for DNA barcoding. The results showed that the number of COI gene variants in Danzhou chickens was less than those of other breeds, except Bawang and the genetic structure was relatively stable. The Kimura 2-parameter genetic distance between Danzhou chickens and the five other breeds was from ∼0.001 to 0.734. The genetic distance of the six breeds was ∼0.001–0.339, with that of Danzhou being the highest (0.339). Danzhou chickens clustered with Bawang and Wenchang chickens in the phylogenetic tree due to geographic closeness. Danzhou chickens could be identified more accurately using COI barcoding. Multiple molecular markers combined with morphological differences were more persuasive for identifying species.

Triplophysa sanduensis, a new loach species of nemacheilid (Teleostei: Cypriniformes) from South China.

A new species of Nemacheilidae, Triplophysa sanduensis sp. nov., is described here based on specimens collected from a small cave in Dengguang Village, Zhonghe Town, Sandu County, Guizhou Province, China. The new species can be distinguished from its congeners by the following combination of characters: branched dorsal-fin rays 8-9; branched pectoral-fin rays 8-9; branched pelvic-fin rays 5; branched anal-fin rays 5; vertebrae 4+37; snout length 45.5-50.1% of head length; postdorsal length 39.0-42.8% of standard length; caudal-peduncle depth/caudal-peduncle length range from 56.4% to 71.9%; the posterior chamber of the gas bladder degenerated; body elongated, covered by scattered scales; eyes present and normal in size, not reduced or absent; and a complete lateral line. A phylogenetic analysis based on the mitochondrial cytochrome oxidase subunit 1 (COI) gene resolved Triplophysa sanduensis as sister to Triplophysa longliensis. Kimura's 2-parameter genetic distance of the COI gene sequences between these two species was 1.9%.

DNA barcoding of fishes from Lake Lanao, Philippines

Lake Lanao, the second largest lake in the Philippines and one of the 15 ancient lakes in the world, used to contain 20 endemic cyprinid species, which had attracted the attention of evolutionary biologists in the past. Over the years, there has been a steady decline in the abundance and diversity of endemic fishes in the lake because of overfishing and introduction of non-native species. This study represents the first molecular survey of the ichthyofauna of Lake Lanao. A total of 75 specimens of 12 different species belonging to nine genera, eight families, and five orders were DNA barcoded using the mitochondrial cytochrome c oxidase subunit I (COI) gene. Average Kimura 2-parameter genetic distances were 0.24% (within species), 8.31% (between species), 9.69% (within family), and 24.86% (between families). Possible hybrids between Oreochromis species were detected. Glossogobius spp. was highlighted for further taxonomic investigation because barcoding indicated unidentified species of this genus. The partial sequence of mitochondrial COI gene was found to be a good DNA barcode for fast and accurate species identification of fishes in Lake Lanao and for tagging species that warrant further taxonomic investigation.

DNA barcoding of the family Sparidae along the coast of China and revelation of potential cryptic diversity in the Indo- West Pacific oceans based on COI and 16S rRNA genes

Sparids are of considerable economic importance in marine fishery and aquaculture in China, and the species diversity of this group is considered relatively high. However, the accurate species identification and delimitation of sparids in China remain unaddressed. In this study, we used mitochondrial cytochrome oxidase subunit І (COI) and 16S ribosomal RNA (16S) genes to conduct DNA barcoding and species delimitation in eleven sparid species from the coastal waters of China. Based on Kimura-2 parameter genetic distances, the mean intraspecific/interspecific variation for COI and 16S were calculated as 0.004/0.152 and 0.002/0.072, respectively. All the conspecific individuals formed monophyletic clusters in neighbour-joining trees of both markers. An obvious barcoding gap was detected for each species, and a common genetic threshold of 1.3% sequence divergence was defined for species delimitation in both markers. Although the sequence variation of 16S was generally lower than that of COI, the results indicated that sparid species could be effectively and accurately identified and delimited by COI as well as 16S. Thus, we propose that the COI gene serve as the standard DNA barcode for sparids, and that the 16S gene could also be an ideal candidate barcode. Moreover, each of the six sparid species ( Argyrops spinifer, Rhabdosargus sarba, Dentex hypselosomus, Acanthopagrus latus, Acanthopagrus australis and Acanthopagrus berda ) showed high intraspecific divergence (>1.3% genetic threshold) with the remarkable geographic lineages in the Indo-West Pacific oceans, which supported that potential unrecognized cryptic species were in them. The potential cryptic diversity revealed here might be primarily attributed to the allopatric divergences caused by the long-term geographic isolation between the Indian and West Pacific oceans or between the opposite sides of the Indian Ocean. The results further suggest that a revision of taxonomic status of these species is required, followed by development of a biodiversity conservation strategy.

Constructing a DNA barcode reference library for southern herbs in China: A resource for authentication of southern Chinese medicine.

Southern Chinese Medicine (SCM) is an important sect of Traditional Chinese Medicine (TCM) with its own special cultural style. Species identification is essential for TCM quality control because authentic herbs are possibly substituted with adulterants that would threaten the health of the public or even cause death. Here, we provided the first local reference DNA barcode library based on the second internal transcribed spacer (ITS2) for the molecular identification of SCM. A total of 1512 specimens of southern herbs representing 359 species were collected under the instructions and identification of taxonomic experts. Genomic DNA was extracted, and the PCR reaction proceeded according to standard procedures. After Sanger sequencing, sequence assembling and annotation, a reliable ITS2 barcode library with 1276 sequences from 309 species of Southern herbs was constructed. The PCR efficiency of the whole samples was 84.39%. Characteristics of the ITS2 barcode were analyzed, including sequence lengths and GC contents in different taxa. Neighbor-joining trees based on Kimura 2-Parameter (K2P) genetic distances showed a 67.56% successful rate of species identification with ITS2 barcode. In addition, 96.57% of species could be successfully identified at the genus level by the BLAST method. Eleven plant species were discovered to be cryptic. In addition, we found that there is an incorrect sequence existing in the public database, making a reliable local DNA barcode reference more meaningful. ITS2 barcodes exhibit advantages in TCM identification. This DNA barcode reference library could be used in Southern Chinese Medicine quality control, thus contributing to protecting public health.