Intraspecific Distances Research Articles

Identification of Apiaceae using ITS, ITS2 and psba-trnH barcodes.

Apiaceae plants are used as medicinal herbs, pesticides, spices, and vegetables; thus, accurately identifying Apiaceae species is important. The grassland ecosystem of Heilongjiang Province in northern China has huge reserves of wild Apiaceae plants, but few reports have systematically documented their diversity. In this study, 275 Apiaceae plants of 23 species in 18 genera were collected from this area. We identified Apiaceae species by using nuclear internal transcribed spacer (ITS/ITS2) and psbA-trnH (chloroplast non-coding region) sequences based on experimental data. The identification efficiency of ITS, ITS2 and psbA-trnH sequences was determined and evaluated by sequence alignment and analysis, intraspecific and interspecific genetic distance analyses, and phylogenetic tree construction. ITS, ITS2 could distinguish 21 species from 17 genera of Apiaceae with good identification effect. When identifying species in the Apiaceae family, ITS2 can be used as the core barcode and psbA-trnH can be used as the supplementary barcode. These results can enrich the reference Apiaceae DNA barcode database.

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.

Multigene phylogeny of the Indo–West Pacific genus Enosteoides (Crustacea, Decapoda, Porcellanidae) with description of a new species from Australia

The porcellanid genus Enosteoides Johnson, 1970, currently containing six species, was raised in the 1970s to contain aberrant Indo–West Pacific forms of the diverse and cosmopolitan genus Porcellana Lamarck, 1801. Here, we describe the most aberrant form as Enosteoides spinosussp. nov., from the northeast and northwest coasts of Australia and present results on phylogenetic reconstructions of the genus, based on an 1,870 bp alignment of concatenated DNA sequences of three mitochondrial and one nuclear gene. The new species is peculiarly spiny and has a higher morphological affinity to the type species of the genus, E. ornatus (Stimpson, 1858), than to the other congeneric species. Our molecular results indicate that Enosteoides is not monophyletic. The new species and E. ornatus are encompassed in a clade, which does not share immediate common ancestry with the clade containing the other species of Enosteoides. This clade is more closely related to species of Porcellana and Pisidia. Relatively large interspecific genetic distances between and within the two clades, as compared to distances estimated in American pairs of species on each side of the Panama Isthmus, suggest ancient divergence, probably followed by extinction events or low speciation rate. Relatively large intraspecific distances between Australian populations of the new species of Enosteoides from geographically distant locations suggest some level of phylogeographic structure.

First contribution to Labiobaetis Novikova & Kluge in Cambodia (Ephemeroptera, Baetidae), with description of two new species.

Material collected in 2018 in Cambodia gives us first insights into the diversity of Labiobaetis Novikova & Kluge, 1987 in this country. No species has been reported so far. We identified two new species using a combination of morphology and genetic distance (COI, Kimura 2-parameter). They are described and illustrated based on their larvae. A key to all Labiobaetis species of continental Southeast Asia is provided. The interspecific K2P distance between the two new species is 20-21%, the intraspecific distance of one of them is 1%. The total number of Labiobaetis species worldwide is augmented to 156.

Geometric morphometric and molecular techniques for discriminating among three cryptic species of the Anopheles barbirostris complex (Diptera: Culicidae) in Thailand

Anopheles members of the Barbirostris complex are important vectors of malaria in Thailand. However, they are morphologically indistinguishable because they are closely related species. In this study, wing geometric morphometrics (GM) and DNA barcoding based on the cytochrome c oxidase subunit 1 (COI) gene were applied to differentiate cryptic species of the Barbirostris complex in Thailand. Three cryptic species of the Barbirostris complex, Anopheles dissidens (19.44%), Anopheles saeungae (24.54%), and Anopheles wejchoochotei (56.02%) were initially identified using the multiplex polymerase chain reaction assay. DNA barcoding analyses showed low intraspecific distances (range, 0.27%–0.63%) and high interspecific distances (range, 1.92%–3.68%), consistent with the phylogenetic analyses that showed clear species groups. While wing size and shape analyses based on landmark-based GM indicated differences between three species (p < 0.05). The cross-validated reclassification revealed that the overall efficacy of wing size analysis for species identification of the Barbirostris complex was less than the wing shape analysis (56.43% vs. 74.29% total performance). Therefore, this study's results are guidelines for applying modern techniques to identify members within the Barbirostris complex, which are still difficult to distinguish by morphology-based identification and contribute to further appropriate malaria control.

Variation in genetics, morphology, and recruitment of the invasive barnacle Amphibalanus eburneus (Gould, 1841) in the southern Korean peninsula.

The ivory barnacle Amphibalanus eburneus is a marine crustacean, which presents near-cosmopolitan distribution due to extensive introduction and exhibits a wide spectrum of phenotypic variation. To elucidate geographical differentiation among populations through invasion, we investigated variation in genetic structure, shell morphology, and recruitment pattern for A. eburneus, from the southern Korean Peninsula where it has been established since the late 1980s. We selected samples from four populations in corresponding ecologically-relevant regions representing all surrounding South Korean waters. From these we amplified the mitochondrial genetic marker cytochrome oxidase subunit I (COI) from 57 individuals and performed a populational genetic analyses with 11 additional GenBank sequences to evaluate population structure. To examine morphological variation, we applied two-dimensional landmark-based geometric morphometrics to the scutum and tergum for 148 and 151 individuals, respectively. Furthermore, we estimated the density of year-old individuals in the field to compare recruitment responses among localities. We detected 33 haplotypes among the four locations belonging to three distinct clades based on moderate intraspecific pairwise genetic distance (≥3.5%). The haplotypes in these clades were not locality-specific in their distribution. In contrast, we did detect interpopulation variation in opercular shape and morphospace structure, and one population could be separated from the rest based on its distinct tergum morphotype alone. This morphologically distinct population was also differentiated by displaying the lowest mean recruitment density. Our results indicate that although there is no relationship between molecular variation in the COI gene and geographic regions in South Korea, association with locality for operculum morphology, and recruitment response suggest ecological adaptation by this barnacle in a new habitat.

Net-Winged Midge Genus Blepharicera Macquart (Diptera: Blephariceridae) in China: The First DNA Barcode Database with Descriptions of Four New Species and Notes on Distribution.

Simple SummaryBlephariceridae is a small but highly specialized family in Diptera with extreme morphological adaptations for living in fast-moving waters. Among the seven blepharicerid genera in China, Blepharicera Macquart, 1843 is the species richest genus with 12 known species. There is a serious gap in knowledge concerning larval stages and connecting them to adult morphology. At present, DNA barcoding using mitochondrial (mt) cytochrome c oxidase subunit 1 gene (COI) has proven to be successful for identification in many animal groups. There are about 60 species of the genus Blepharicera known in the world. However, only one identified Blepharicera species from Europe is currently available in DNA sequence repositories, and a DNA barcode database is still lacking for Chinese Blepharicera. To fill this gap, we started a project with the objectives to collect specimens of Blepharicera across China, identify the species by morphological characters, and build an mt COI barcode database for this important insect group. Molecular and morphological markers show that four morphospecies examined represent members of the genus Blepharicera and constitute four new, distinct species. This initial attempt indicates the validity of mt COI barcodes for Blepharicera in delimiting species and contributes to the growing library of DNA barcodes of net-winged midges of the world.Mitochondrial (mt) cytochrome c oxidase 1 (COI) gene is more and more widely used for DNA barcoding, which provides a rapid and timely identification as this technique is not limited by polymorphism, sex, and life stages and fundamentally complements traditional evolutionary taxonomy. The present study generated 33 mt COI sequences of seven Chinese Blepharicera Macquart, 1843 species with an average of 594 bp, which represent the first DNA barcode database for Chinese Blepharicera. Genetic distance analysis reveals that intraspecific distances in the genus are generally less than 1.7%, and interspecific distances range from 5.4% to 20.3%. Phylogenetic analysis shows that each species recovered in our analyses is separated from all neighboring species. Based on molecular and morphological data, four Blepharicera species from China, B. beishanica sp. nov., B. dushanzica sp. nov., B. nigra sp. nov. and B. xinjiangica sp. nov., are described and illustrated as new to science. Identification keys for adults and larvae of Chinese Blepharicera are also presented. Geographical analysis shows that Southwest China is the species’ richest region. Our results will be useful in tackling taxonomic problems, understanding species distribution, and resolving nomenclature conflicts associated with Blepharicera species.

Molecular identification of ornamental loaches (Cypriniformes, Cobitoidei) of North East India using mitochondrial genes

North East (NE) India, referred as Biodiversity Hot Spot, is the natural habitat of a large variety of ornamental fishes. Among all the ornamental fishes available in NE India, Loaches are of great importance in the ornamental fish trade. In this study, 38 specimens from ten loaches species of North-eastern India are characterized using three mitochondrial genes i.e. cytochrome c oxidase subunit I (COI), 16S rRNA and cytochrome b (Cyt b). The average genetic distance values for all the three mitochondrial genes have increased gradually when moving up from species to genus to family levels. Among the genes, Cyt b gene showed relatively higher genetic distance values than mitochondrial 16S rRNA and COI genes. Some of the specimens of Botia and Lepidocephalichthys showed higher intraspecific genetic distance values compared to other specimens under analysis.

DNA Barcoding of genus Culicoides biting midges (Diptera: Ceratopogonidae) in the Brazilian Amazon

Culicoides biting midges are capable to transmit Oropouche virus, Bluetongue virus and Mansonella spp. This study aimed to assess the utility of DNA barcode as an alternative method in the Culicoides species identification. The study was conducted in Jamari National Forest. Biting midges were collected using HP light traps during four months, February, April, August and October 2018. Insects were morphologically identified to the species level, and rest of the body were subjected to DNA extraction and PCR targeting a fragment of the cytochrome c oxidase subunit I (COI) gene, which were analyzed and deposited in GenBank. A phylogenetic gene tree was reconstructed using RAxML software, and the sequences were assigned at Molecular Operational Taxonomic Unit (MOTU) level by species delimitation algorithms. According to morphological approach, 18 species of 2 subgenera and 7 species groups were identified. A total of 191 new COI barcodes from 18 species were generated. Of these, fifteen species have been deposited for the first time in all datasets in the world. These sequences allowed the correct identification of 188 and 187 specimens according to the BM and BCM criteria, respectively. The intraspecific genetic distances ranged from 0 to 16.5%, while the interspecific ones ranged from 2.1 to 27.1%. The nominal species Culicoides glabellus and C. tetrathyris splitted into three and two MOTUs, respectively, except for mPTP, indicating a cryptic diversity in these species. Also, sequences of C. pseudodiabolicus formed two MOTUs using all algorithms, except for PTP and ABGD, suggesting the existence of two potential species. In contrast, some barcodes of C. quasiparaensis and C. paraensis merged into a single MOTU, which can be explained by the complex characteristics of the paraensis group, since these species have similar morphological characters. Here, we provided the first COI barcodes for biting midges in Rondônia and Brazil, and demonstrates that these are sufficient to discriminate between some species.

Molecular characterization and genetic divergence of seven Culex mosquito (Diptera: Culicidae) species using Mt COI gene from Odisha State, India

BackgroundCulex mosquitoes are involved in the transmission of arboviral diseases worldwide. Bio-ecology and identification of mosquitoes are of paramount importance to develop species-specific vector control strategies. Genetic-based species-specific approaches that reduce the burden of vector-borne diseases are made possible by molecular techniques.ResultsIn the present study, the mitochondrial COI gene of Culex mosquitoes was used for molecular identification in addition to morpho-taxonomy. Our findings suggested the presence of important Culex mosquito vectors viz, Culex vishnui, Culex tritaeniorhynchus, Culex bitaeniorhynchus, Culex quinquefasciatus, Culex gelidus, Culex fuscocephala, and Culex fuscanus in the southern part of Odisha state, India. We examined the phylogeny and genetic diversity of the above seven different Culex populations from different geographical locations. An average intra-specific K2P distance of the COI gene was found to be 0.9%. Further, to measure the diversity of the Culex population among different geographical strains, haplotype diversity and nucleotide diversity were compared. Culex fuscanus showed high polymorphisms and mutations with high nucleotide diversity (0.013) and the Culex quinquefasciatus showed the lowest variation in P(i), 0.0013 in the intra-population polymorphism analysis of COI sequences. Similarly, the Haplotype diversity (Hd) found in Culex gelidus and Culex fuscocephala with the value of 0.972 and Culex quinquefasciatus (0.583) showed the lowest value of haplotype diversity. A haplotype network was constructed to establish the genealogical relationship between haplotypes. The phylogenetic tree was constructed that produces distinctive conspecific clusters in different Culex species. Population genetic study has illustrated the occurrence of genetic differentiation within the population.ConclusionsThe findings of this study contribute to greater evidence that DNA barcode sequences can be used to monitor mosquito species diversity. This study also adds valuable information about the systematics and molecular biology of seven public health important mosquito species acting as a significant vector for Japanese encephalitis in various Asian continents. This information is further used for the effective implementation of region-specific vector control strategies.

The Application of DNA Barcoding in Crustacean Larvae Identification from the Zhongsha Islands, South China Sea

Marine crustaceans are known as a group with high morphological diversity and great economic value. Most species have planktonic larval stages that are difficult to identify to species level using traditional approaches because of insufficient morphological diagnostic characters or taxonomic descriptions. We used DNA barcoding and molecular species identification to investigate the species diversity and distribution of crustacean larvae in the Zhongsha Islands waters, South China Sea. In total, 108 sequences were obtained from crustacean larvae collected in the Zhongsha Islands waters in 2019 using vertical hauls between the depths of 5 and 200 m. The molecular classification approach confirmed that 108 sequences represented crustaceans typical to the South China Sea, with 70 species identified, representing 43 genera, 23 families, and 4 orders. However, the cytochrome c oxidase subunit I gene sequences of only 27 species identified from the larval samples matched with available sequences taken from adults in GenBank. The comparison of K2P distances yielded a notable gap of 3.5–10.7% between intraspecific and interspecific distances across the sequence dataset. More than 80% of the crustacean larvae species belonged to the order Decapoda, and they displayed marked differences in their distributionsin the Zhongshan Islands waters. The orders Calanoida and Amphipoda were represented by the fewest species, which were recorded only at the edge of the Zhongsha Atoll.

INTRASPECIFIC AND INTERSPECIFIC GENETIC DISTANCES IN ERMINE MOTHS OF THE GENUS YPONOMEUTA LATR. (LEPIDOPTERA: YPONOMEUTIDAE)

An analysis of intraspecific variability based on the barcoding fragment mtCOI showed that species of Y. padella–complex with larger ranges are characterized by relatively larger intervals of genetic distances between representatives of different populations compared to species distributed in a limited area. Thus, for the Transpalaearctic species Y. evonymella, the intraspecific distance lies within 0–1%, while the average indices of intraspecific distances were revealed in the Euro-American Y. malinella and the European Y. irrorella (0–0,5%), and the minimal range of distances were recorded in the Euro-American Y cagnagella and European Y. rorrella (0–0,2%). For the East Asian Y. orientalis, in which samples were studied only from the Russian Far East, intraspecific variability was not revealed. The interspecific distance intervals within the Y. padella–complex turned out to be very low and with small range, not exceeding 2,6% (Y. padella – Y. orientalis, Y. padella – Y. irrorella) and 2,8% (Y. malinella – Y. irrorella, Y. malinella – Y. orientalis), which confirmed the weak divergence of the species group not only in morphological markers, but also in the barcoding fragment mtCOI. In a number of studied species, the intervals of intraspecific and interspecific distances have borderline indicators or slightly overlapping ones – 0,2% (Y. cagnagella), 0,2–0,3% (Y. padella), 0,5–0,7% (Y. malinella), which point out the unreliability of fragment COI usage to identify such weakly divergent species as the species of the Y. padella– complex. Summarizing the above, it should be concluded that identification of a number of taxa, especially relatively young and weakly divergent ones, is difficult based on the nucleotide sequences of the barcoding fragment mtCOI without the use of a complex of other markers.

DNA barcode analysis of the endangered green turtle (Chelonia mydas) in the breeding grounds of Badagry, Lagos, Nigeria

The Nigerian coastline is long and heavily populated. It is also the nesting site of a huge numbers of species that are dependent on that ecosystem for their reproduction cycle. Artisanal fishermen and the local populace are dependent on these species as a cheap and alternative source of protein. The Sea Turtles having been classified as threatened by the IUCN are a major part of that ecosystem. Currently, there is however a dearth of research works on the species. The usefulness of the DNA barcoding region (CO1) of the mitochondrial genome is used to analyze populations of Chelonia mydas along Badagry beach in Lagos, Nigeria. Seven samples were obtained and CO1 sequences were generated from them. Twenty seven more sequences were harvested from the GenBank of which only five were used in the final analysis either because there were errors (insertions, deletions or stop codons) or because of redundancies. A Six hundred and Eighteen (618bp) index free sequences was obtained. The intraspecific divergence in the Nigerian samples was very low, being between 0.00 and 0.002, while the intraspecific distance when the other sequences were added increased a bit ranging from 0.002 to 0.54. Neighbor- joining phylogenetic tree produced two distinct clade, with the Nigerian samples being clearly mapped into one with bootstrap value of 100% while others mapped into another. The best substitution model is TN93+G. The Tajima index was also very low indicating a population with very low genetic divergence. The average codon usage also followed the pattern A (29.3) > C (28.7) > T (27.3)> and a G (14.8). An expansion of the sampling points and increase number of samples is recommended for a more robust ideal of the relatedness within and without.

Molecular identification and phylogenetic analysis of spider mites (Prostigmata: Tetranychidae) of Turkey.

The family Tetranychidae includes many agriculturally important species known as spider mites. Their morphological identification is quite difficult due to the tiny size of their taxonomic characters and the requirement for high-level expertise. This may lead to pest misidentification and thus failure in pest management. DNA-based species identification seems to offer an alternative solution to overcome these issues. In the present study, two common molecular markers-Cytochrome oxidase subunit I (COI) and Internal transcribed spacer 2 (ITS2)-were used to identify 10 spider mite species from Turkey. Furthermore, genetic distances for several of them were assessed. Panonychus ulmi and Bryobia kissophila had the lowest (1.1%) and highest (4.5%) intra-specific genetic distances, respectively. In addition, integrative taxonomy allowed to identify Eotetranychus quercicola in Turkey as a new record. The sequences herein obtained will allow rapid species identification using molecular techniques and will contribute to resolve the phylogenetic history of spider mites.

Genetic diversity of Insect farm populations of Protaetia brevitarsis seulensis (Kolbe, 1886) (Coleoptera, Scarabaeidae) in South Korea inferred from COI gene sequences

A Korean indigenous species, Protaetia brevitarsis seulensis (PBS), is popularly reared at many insect farms as valuable industrial insect resources in South Korea. However, there is no comprehensive study on the genetic diversity within the Korean PBS population as well as between morphologically closed species. Employing the rapidly evolved genetic markers such as mitochondrial DNA is important in the detection of haplotype diversity and rapid species identification of larval stage and/or its processed goods. We here examined how many genetically different haplotypes distribute in the reared PBS collected from 14 insect farms and the interspecific genetic difference between congeners using mitochondrial COI gene sequences (DNA barcoding region). As a results, the PBS was clearly separated from two related species with large genetic distances (7.98%~8.86%). In the PBS, 57 haplotypes were detected from 223 specimens with low intraspecific genetic distance (~1.3%). Among them, the most dominant haplotypes are subsequently detected in H4 (25.6%), H38 (15.7%) and H26 (10.3%). From the haplotype network and distribution analyses, we consider that many haplotypes have been mixed by artificial collections from various local places and exchanged between insect farms within South Korea. We expect that this data will be helpful in rapid species identification for its processed goods, and understanding the exact population diversity and detection of the original habitats for each haplotype of the reared PBS throughout comparing with natural populations in the further study.&#x0D;

Identification, genetic diversity, and comparative evolution of the striped snakehead Channa striata (Bloch, 1793) in Wallacea, Indonesia

Abstract. Irmawati, Meimulya, Tassakka ACMAR, Nadiarti, Rukminasari N, Kadriah IAK, Nasrullah H, Alimuddin. 2022. Identification, genetic diversity, and comparative evolution of the striped snakehead Channa striata (Bloch, 1793) in Wallacea, Indonesia. Biodiversitas 23: 3327-3337. Striped snakehead, Channa striata populations have declined in some areas of Wallacea. This study aimed to identify the species, analyze the genetic diversity, and trace the evolutionary relationships of snakeheads in the Wallacea region with snakeheads in other geographic areas. This study was identifying all specimens in this study as C. striata. The Wallacean C. striata sequences comprised three haplotypes with five polymorphic sites (single nucleotide polymorphisms, SNPs). No mutations were detected in the COI nucleotides of C. striata in the Tempe Lake complex, Patampanua River, and Bojo River; therefore, they belonged to the same haplotype. The C. striata from the Tangkoli tertiary drainage had unique haplotypes that differed from other C. striata haplotypes. The intraspecific genetic distance within C. striata was low while the interspecific genetic distance within the genus Channa was quite large, indicating that mutation rates are quite high in the genus Channa. Haplotype evolution showed that C. striata in Wallacea shared a common ancestral pathway with C. striata in Kalimantan, Java, and Bali and had a different ancestral pathway with C. striata in Lampung (Sumatra). The nucleotide composition of the C. striata COI gene followed the general pattern of the Teleostei and the Pisces superclass (T&gt;C&gt;A&gt;G) with the G+C base percentage lower than the A+T percentage. The data from this study will be useful as a basis for designing germplasm conservation strategies and designing strategies to maintain C. striata populations and develop strains to meet future needs.

DNA barcoding suggests new species for the Mansonia subgenus (Mansonia, Mansoniini, Culicidae, Diptera) in the area surrounding the Jirau hydroelectric dam, Porto Velho municipality, Rondônia state, Brazil

Previous studies have linked the construction of hydroelectric dams with increases in the density of mosquitoes, especially Mansonia. In Brazil, Mansonia mosquitoes are still poorly studied at the taxonomic, biological, ecological and epidemiological levels, and nothing is known about the genetic diversity and the cryptic speciation of the group. The current study analyzed the molecular taxonomy of Mansonia species captured in the area surrounding the Jirau hydroelectric dam, Rondônia state, Brazil. Samples were collected from fifteen locations between 2018 and 2019. Genomic DNA of the specimens was extracted, and the DNA barcode region of the Cytochrome Oxidase, subunit I gene was amplified with PCR and both DNA strands were sequenced. The dataset was analyzed using MEGA, Mr. Bayes and DnaSP software. The results provided COI sequences for 100 specimens collected in the area surrounding from Jirau hydroelectric dam. These belonged to five species of the Mansonia subgenus, identified morphologically as Mansonia humeralis, Mansonia amazonensis, Mansonia titillans, Mansonia dyari and Mansonia indubitans. Findings showed that the COI gene is an effective and accessible DNA barcode that provides a high-resolution tool for delimiting species within the subgenus Mansonia, with the tree construction (Bayesian Inference) well supported and non-overlapping intraspecific and interspecific (K2-P) genetic distance values. These findings also indicate the occurrence of cryptic speciation within M. dyari and near of M. titillans. This is the first study to apply molecular tools to the taxonomy of Mansonia species from Brazil.

Phylogenetic analysis of black piper (Piper spp.) population collected in different locations of Viet Nam based on the ITSU1-4 gene region

The internal transcribed spacer (ITS) of nuclear ribosomal DNA is one of the most commonly used DNA markers in plant phylogenetic and DNA barcoding analyses and it has been recommended as a core plant DNA barcode. To compare and find out the genetic diversity difference, some individuals Peper were collected in different localities in Vietnam when using the ITS of nuclear ribosomal DNA. The ITS gene region from the nuclear genomes was tested for suitability as DNA barcoding regions of thirty-nine Peper individuals. Universal primers were used and sequenced products were analyzed using the Maximum Likelihood method and Tamura-Nei model in the MEGA X program. We did not observe high variability in intraspecific distance within the ITSu1-4 gene region between individuals ranging from 0.000 to 0.155. The size of the gene region has fluctuated from 667 to 685 bp between different individuals with the percentage (G + C) contained in the ITSu1-4 gene region which ranged from 54.776% to 60.805%, mean = 60.174%. The values of Fu’s Fs, D, Fu and Li’s D* and F* were negative as well (Fs = -0.209, D = -1.824; p &lt; 0.05, D* = -1.205; not significant, p &gt; 0.10 and F* = -1.699; not significant, 0.10 &gt; p &gt; 0.05) indicating an excess of recently derived haplotypes and suggesting that either population expansion or background selection has occurred. The value of Strobeck’s S is high (S = 0.684). The results of evolutionary relationships of taxa obtained 3 groups with the highest value of Fst are shown in the pairs of groups II and III (Fst = 0.151) and the lowest is in groups II and I (Fst = 0.015). All of the new sequences have been deposited in GeneBank under the following accession numbers MZ636718 to MZ636756. This database is an important resource for researchers working on Species of Peper in Vietnam and also provides a tool to create ITSu1-4 databases for any given taxonomy.

DNA barcoding and species delimitation of the genus Oxynoemacheilus (Teleostei: Nemacheilidae) in Anatolia.

The DNA barcoding approach was used for the determination of evolutionary relationships and species delimitation of the genus Oxynoemacheilus (Teleostei: Nemacheilidae). The COI barcode region (615 bp amplicon) was used to barcode 444 individuals from 64 morphologically identified species in the genus Oxynoemacheilus and 189 haplotypes were identified. The average of the interspecific p distance (9.59%) was about 21-fold higher than the average intraspecific distance (0.44%). A general genetic threshold of 1.46% sequence divergence was defined for species delimitation. The multiple species delimitation methods (BCM, GMYC, bPTP and TCS) revealed a total of 62 molecular operational taxonomic units for 64 morphospecies with a new loach species from the BuyukMelen River. Neighbour-joining, maximum likelihood and Bayesian inference analyses indicated that all haplotypes were clustered into 62 clades, which corresponded to Oxynoemacheilus species, with strong bootstrap support (≥95%). Furthermore, all samples grouped in concurrence with the taxonomic status of the species except for species groups (O. germencicus-O. cinicus-O. mesudae and O. leontinae-O. namiri) that were showed intraspecific overlap in genetic diversity for COI-based barcodes. In conclusion, our analyses indicate that COI-based barcodes provide reliable species discrimination. Therefore, we currently recommend COI barcodes as the suitable barcode forgenus Oxynoemacheilus.

Large-scale DNA barcoding of the subfamily Culterinae (Cypriniformes: Xenocyprididae) in East Asia unveils a geographical scale effect, taxonomic warnings and cryptic diversity.

Geographical scale might be expected to impact significantly the efficiency of DNA barcoding as spatially comprehensive sampling provides opportunities to uncover intricate relationships among closely related species and to detect cryptic diversity for widespread taxa. Here, we present a DNA barcoding study on a Xencyprididae subfamily (Culterinae) involving the production of 998 newly generated DNA barcodes from East Asian drainages (80 localities). Together with 513 barcodes mined from BOLD and GenBank, a reference library consisting of 1511 DNA barcodes (116 localities) for 42 species was assembled, accounting for 66% of known Culterinae species. Intraspecific genetic distances are positively correlated to geographical scale, while a negative correlation is detected between interspecific genetic distances and geographical scale. The present study demonstrates that geographical scale influences the efficiency of DNA barcoding by narrowing the width of the barcoding gap. DNA‐based species delimitation analyses delimited 44 molecular operational taxonomic units (MOTUs). Rampant cryptic diversity is detected within eight species with multiple MOTUs, whereas 25 species present mismatch between morphological and molecular delimitations. A total of 18 species are lumped into nine MOTUs due to low interspecific divergence and/or mixed lineages. Several MOTU divergences are hypothesized to relate to known biogeographical barriers and geological events during the Pliocene and Pleistocene. This study provides new insights into the taxonomy and phylogeography of the subfamily Culterinae.