Interspecific Genetic 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.

Gut microbial ecology of Philippine gekkonids: ecoevolutionary effects on microbiome compositions.

Given the rapidly changing landscapes of habitats across the globe, a sound understanding of host-associated microbial communities and the ecoevolutionary forces that shape them is needed to assess general organismal adaptability. Knowledge of the symbiotic endogenous microbiomes of most reptilian species worldwide remains limited. We sampled gut microbiomes of geckos spanning nine species and four genera in the Philippines to (i) provide baseline data on gut microbiota in these host species, (ii) test for significant associations between host phylogenetic relationships and observed microbial assemblages, potentially indicative of phylosymbiosis, and (iii) identify correlations between multiple ecoevolutionary factors (e.g. species identity, habitat tendencies, range extents, and maximum body sizes) and gut microbiomes in Philippine gekkonids. We recovered no significant association between interspecific host genetic distances and observed gut microbiomes, providing limited evidence for phylosymbiosis in this group. Philippine gekkonid microbiomes were associated most heavily with host species identity, though marked variation among conspecifics at distinct sampling sites indicates that host locality influences gut microbiomes as well. Interestingly, individuals grouped as widespread and microendemic regardless of host species identity displayed significant differences in alpha and beta diversity metrics examined, likely driven by differences in rare OTU presence between groups. These results provide much needed insight in host-associated microbiomes in wild reptiles and the ecoevolutionary forces that structure such communities.

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.

The ITS analysis and identification of Actinidia eriantha and its related species.

The dried plant material of medically important plant Actinidia eriantha especially when it remains in the form of powder often look morphologically similar to its related species. The lack of efficient methods to distinguish the authentic material from other similar species leads to chances of adulteration. The molecular authentication of herbal plant materials such as the internal transcribed spacer (ITS) sequences is considered as more reliable method compared to morphological traits. In this study, we aim to evaluate the potential of identification for roots of A. eriantha and its related species by ITS sequences. The lengths of ITS regions ranged from 624 to 636 bp with GC content ranging from 50.96% to 59.55%. A total of 194 variation sites and 46 haplotypes were formed in 185 samples. Among them, the roots of A. eriantha possessed specific sites at 85bp (C), 205bp (T), 493bp (C), 542bp (G), 574bp (C), 582bp (T) and 610bp (G), while A. hemsleyana, A. callosa, A. valvata and A. polygama have their own specific sites. The inter-specific genetic distance among 8 Actinidia species in the range 2.28% to 11.00%. The phylogenetic tree constructed with ITS, ITS1 and ITS2 region showed that the ITS sequences have higher potential for identification in 8 Actinidia species. However, as to A. eriantha, A. hemsleyana and A. valvata, these three barcodes have the same identification ability. The ITS regions indicated that different samples from same species can be grouped together, except for A. arguta and A. melanandrah. In conclusion, the ITS sequences can be used as an efficient DNA barcode for the identification of A. eriantha and its related species.

First record of the order Polyzoniida from the Indian subcontinent with an integrative description of a new genus (Diplopoda, Colobognatha, Siphonotidae).

Considering the large area of the Indian subcontinent, its known millipede diversity is sparse with only ca. 270 described species in 90 genera, 25 families and 11 orders. So far, not a single polyzoniidan millipede has been described from India. The order Polyzoniida Cook, 1895 is one of the most species-poor millipede groups with less than 80 described species, and includes taxonomically problematic groups, especially in the family Siphonotidae Cook, 1895. Here we report the first representatives of the order Polyzoniida from India, and describe the new genus Theratta n. gen., with the three species Theratta mannavan n. sp., Theratta eravikulam n. sp., and Theratta shola n. sp., using scanning electron microscopy and COI genetic barcoding. Based on the morphological characters we place Theratta n. gen., and the three new species in the tribe Rhinotini Hoffman, 1977 of the family Siphonotidae. The gonopods of the new genus are similar to those of the genus Rhinotus Cook, 1896, but differ from all other Rhinotini in the modification of the podomere II of the anterior telopod, carrying a mesal process on the posterior side and a lamellar process on the anterior side. The three newly described species show a high interspecific genetic distance of 20.925.3%. The specimens were collected from the Shola forests (high altitude montane forests) of the southern Western Ghats in Kerala, India. We suggest that more extensive sampling in this area and in the Indian subcontinent in general will yield more new millipede species for science and expand our understanding of the hitherto neglected Indian millipede fauna.

DNA Barcode Gap Analysis for Multiple Marker Genes for Phytoplankton Species Biodiversity in Mediterranean Aquatic Ecosystems

Simple SummaryEnvironmental DNA metabarcoding (eDNA) has strong potential in the assessment of biodiversity in aquatic ecosystems. The incompleteness of DNA barcode reference libraries represents a current limit to unveiling the whole biodiversity of an aquatic ecosystem. Therefore, barcode gap analyses at species level are of great significance, in particular at local/regional level, for the advancement of eDNA metabarcoding application to aquatic ecosystems surveillance and future biodiversity assessment.The implementation of DNA metabarcoding and environmental DNA (eDNA) to the biodiversity assessment and biomonitoring of aquatic ecosystems has great potential worldwide. However, DNA metabarcoding and eDNA are highly reliant on the coverage of the DNA barcode reference libraries that are currently hindered by the substantial lack of reference sequences. The main objective of this study was to analyze the current coverage of DNA barcode reference libraries for phytoplankton species of the aquatic Mediterranean ecoregion in the southeast of Italy (Apulia Region) in order to assess the applicability of DNA metabarcoding and eDNA in this area. To do so, we investigated three main DNA barcode reference libraries, BOLD Systems, GenBank and SILVA, for the availability of DNA barcodes of the examined phytoplankton species. The gap analysis was conducted for three molecular gene markers, 18S, 16S and COI. The results showed a considerable lack of barcodes for all three markers. However, among the three markers, 18S had a greater coverage in the reference libraries. For the 18S gene marker, the barcode coverage gap across the three types of ecosystems examined was 32.21–39.68%, 60.12–65.19% for the 16S marker gene, and 72.44–80.61 for the COI marker gene. Afterwards, the interspecific genetic distance examined on the most represented molecular marker, 18S, was able to distinguish 80% of the species mined for lakes and 70% for both marine and transitional waters. Conclusively, this work highlights the importance of filling the gaps in the reference libraries, and constitutes the basis towards the advancement of DNA metabarcoding and eDNA application for biodiversity assessment and biomonitoring.

Testing the effectiveness of rbcLa DNA-barcoding for species discrimination in tropical montane cloud forest vascular plants (Oaxaca, Mexico) using BLAST, genetic distance, and tree-based methods.

DNA-barcoding is a species identification tool that uses a short section of the genome that provides a genetic signature of the species. The main advantage of this novel technique is that it requires a small sample of tissue from the tested organism. In most animal groups, this technique is very effective. However, in plants, the recommended standard markers, such as rbcLa, may not always work, and their efficacy remains to be tested in many plant groups, particularly from the Neotropical region. We examined the discriminating power of rbcLa in 55 tropical cloud forest vascular plant species from 38 families (Oaxaca, Mexico). We followed the CBOL criteria using BLASTn, genetic distance, and monophyly tree-based analyses (neighbor-joining, NJ, maximum likelihood, ML, and Bayesian inference, BI). rbcLa universal primers amplified 69.0% of the samples and yielded 91.3% bi-directional sequences. Sixty-three new rbcLa sequences were established. BLAST discriminates 80.8% of the genus but only 15.4% of the species. There was nil minimum interspecific genetic distances in Quercus, Oreopanax, and Daphnopsis. Contrastingly, Ericaceae (5.6%), Euphorbiaceae (4.6%), and Asteraceae (3.3%) species displayed the highest within-family genetic distances. According to the most recent angiosperm classification, NJ and ML trees successfully resolved (100%) monophyletic species. ML trees showed the highest mean branch support value (87.3%). Only NJ and ML trees could successfully discriminate Quercus species belonging to different subsections: Quercus martinezii (white oaks) from Q. callophylla and Q. laurina (red oaks). The ML topology could distinguish species in the Solanaceae clade with similar BLAST matches. Also, the BI topology showed a polytomy in this clade, and the NJ tree displayed low-support values. We do not recommend genetic-distance approaches for species discrimination. Severe shortages of rbcLa sequences in public databases of neotropical species hindered effective BLAST comparisons. Instead, ML tree-based analysis displays the highest species discrimination among the tree-based analyses. With the ML topology in selected genera, rbcLa helped distinguish infra-generic taxonomic categories, such as subsections, grouping affine species within the same genus, and discriminating species. Since the ML phylogenetic tree could discriminate 48 species out of our 55 studied species, we recommend this approach to resolve tropical montane cloud forest species using rbcLa, as an initial step and improve DNA amplification methods.

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>C>A>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.

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.

Review of Ophioplinthaca Verrill, 1899 (Echinodermata, Ophiuroidea, Ophiacanthidae), description of new species in Ophioplinthaca and Ophiophthalmus, and new records from the Northwest Pacific and the South China Sea.

The ophiuroid genus Ophioplinthaca is well characterized by the deep incisions in the disc. Prior to this study, it contained 32 accepted species, but species limits and geographic distributions were not well understood. The manned submersible vehicle 'Shenhaiyongshi' was used to collect ophiuroid specimens from the deep-sea seamounts and cold seeps in the South China Sea and Northwest Pacific at 602-3600 m depth, during 2018 to 2020. The genus Ophioplinthaca was reviewed using both morphological data and a phylogenetic analysis, based on COI sequences. The taxonomic status of the genus Ophiophthalmus Matsumoto, 1917, a junior homonym of Ophiophthalmus Fitzinger, 1843 (a reptile) was clarified by proving prevailing usage of the ophiuroid name. A total of eight species were identified, including two new species, described asOphioplinthacabrachispina sp. nov. and Ophiophthalmusserratus sp. nov., and two new records. The new species are characterized by unique features of the arm skeletons. Tabular keys to all Ophioplinthaca and Ophiophthalmus species are provided. Interspecific and intraspecific genetic distance of Ophioplinthaca species ranged from 2.32% to 19.72%, and from 0.26% to 0.90%, respectively. The data suggest that species of the genus Ophioplinthaca are more widely spread around the Northwest Pacific region deep-sea seamounts than previously known.

Exploring Large-Scale Patterns of Genetic Variation in the COI Gene among Insecta: Implications for DNA Barcoding and Threshold-Based Species Delimitation Studies.

Simple SummaryInsecta is the most diverse group in the kingdom Animalia, and it accounts for approximately two-thirds of all animals. The most commonly used gene for species delimitation in animals is cytochrome c oxidase subunit 1 (COI). We calculated the genetic distance of 64,414 insect species, downloaded from BOLD, and found that approximately one-quarter of the species of Insecta showed high intraspecific genetic variation (>3%). Owing to the high intraspecific genetic variation in insects, false positives may easily occur in threshold-based species delimitation based on the COI gene. Compared to the fixed thresholds, the thresholds that were calculated from the “threshOpt” and “localMinima” algorithms that are embedded in the Spider package are recommended in threshold-based species delimitation studies.The genetic variation in the COI gene has had a great effect on the final results of species delimitation studies. However, little research has comprehensively investigated the genetic divergence in COI among Insecta. The fast-growing COI data in BOLD provide an opportunity for the comprehensive appraisal of the genetic variation in COI among Insecta. We calculated the K2P distance of 64,414 insect species downloaded from BOLD. The match ratios of the clustering analysis, based on different thresholds, were also compared among 4288 genera (35,068 species). The results indicate that approximately one-quarter of the species of Insecta showed high intraspecific genetic variation (>3%), and a conservative estimate of this proportion ranges from 12.05% to 22.58%. The application of empirical thresholds (e.g., 2% and 3%) in the clustering analysis may result in the overestimation of the species diversity. If the minimum interspecific genetic distance of the congeneric species is greater than or equal to 2%, it is possible to avoid overestimating the species diversity on the basis of the empirical thresholds. In comparison to the fixed thresholds, the “threshOpt” and “localMinima” algorithms are recommended for the provision of a reference threshold for threshold-based species delimitation studies.

A New Species in Notobitiella Hsiao in China Confirmed by Morphological and Molecular Data (Hemiptera: Heteroptera: Coreidae).

Simple SummaryPlenty of new species are published every year, but what if a new species could be erected as a new genus by the morphological characteristics of genitalia or a new species of an existing genus by the other morphological characteristics? Recently, we collected a new species of Coreidae facing this problem. The characteristics of genitalia between the new and existing species in Notobitiella (monotypic genus) were too different to compare with the difference among interspecies in tribe Cloresmini. It was reasonable to erect a new genus for this new species. However, considering the similarity between other characteristics, we decided to refer to the results of molecular data. Results showed that the genetic distance within the interspecific interval of the genus and the sister group relationship also supported that the new species should be included in the genus Notobitiella. Finally, a new species was confirmed, named Notobitiella bispina sp. nov. Our research highlighted how molecular data can be used to assist in species delimitation when encountering similar problems.To date, only one species of genus Notobitiella, N. elegans Hsiao, was found in Yunnan, China. Recently, we confirmed a new species, Notobitiella bispina sp. nov., by morphological and molecular data based on new specimens collected from Yunnan, China. The new species is morphologically similar to N. elegans except for the male genitalia and the sternum of the seventh abdominal segment of the female. The extraordinary difference of the genitalia between these two species inspired us to erect a new genus for this new species. However, considering their sister group relationship to other genera in tribe Cloresmini and the 12.56~12.64% genetic distance (meeting the interspecific genetic distance within genus of tribe Cloresmini) by a complete COI gene, this species was more reasonable as a new species of the genus Notobitiella, and the diagnosis of the genus was revised. The photos of the body and critical morphological characteristics for both male and female were provided for accurate identification.

Lack of Statistical Rigor in DNA Barcoding Likely Invalidates the Presence of a True Species' Barcode Gap

DNA barcoding has been largely successful in satisfactorily exposing levels of standing genetic diversity for a wide range of taxonomic groups through the employment of only one or a few universal gene markers. However, sufficient coverage of geographically-broad intra-specific haplotype variation within genomic databases like the Barcode of Life Data Systems (BOLD) and GenBank remains relatively sparse. As reference sequence libraries continue to grow exponentially in size, there is now the need to identify novel ways of meaningfully analyzing vast amounts of available DNA barcode data. This is an important issue to address promptly for the routine tasks of specimen identification and species discovery, which have seen broad adoption in areas as diverse as regulatory forensics and resource conservation. Here, it is demonstrated that the interpretation of DNA barcoding data is lacking in statistical rigor. To highlight this, focus is set specifically on one key concept that has become a household name in the field: the DNA barcode gap. Arguments outlined herein specifically center on DNA barcoding in animal taxa and stem from three angles: (1) the improper allocation of specimen sampling effort necessary to capture adequate levels of within-species genetic variation, (2) failing to properly visualize intra-specific and interspecific genetic distances, and (3) the inconsistent, inappropriate use, or absence of statistical inferential procedures in DNA barcoding gap analyses. Furthermore, simple statistical solutions are outlined which can greatly propel the use of DNA barcoding as a tool to irrefutably match unknowns to knowns on the basis of the barcoding gap with a high degree of confidence. Proposed methods examined herein are illustrated through application to DNA barcode sequence data from Canadian Pacific fish species as a case study.

New species, redescriptions and new records of deep-sea brittle stars (Echinodermata: Ophiuroidea) from the South China Sea, an integrated morphological and molecular approach

Deep-sea ophiuroids were collected by the manned submersible ‘Shenhaiyongshi’ from the South China Sea at depths of 500–3550 m, in 2017 to 2020. A total of 18 species were identified, including three new species and eight new records, increasing the total number of species known from the South China Sea to 304. Most of the ophiuroids recorded from the South China Sea were found in shallow waters (77.9%) and a few of them occurred only in deep water (20.4%). Three new species are described as Ophiacantha aster sp. nov., Ophiomoeris petalis sp. nov. and Ophiopristis shenhaiyongshii sp. nov. We provide comprehensive descriptions of morphological features, including characteristics of the arm skeletons, and a phylogenetic analysis based on COI and 16S sequences. Overall intraspecific and interspecific genetic distance variations among the families found in this study were 0.5% to 2.47% and 1.16% to 44.16%, respectively, along the South Pacific region to the South China Sea. Our phylogenetic analysis suggested that COI partial genes resolved the interspecies complexity in the class Ophiuroidea better than 16S partial genes. The order Euryalida had low interspecies genetic distance variation within the class Ophiuroidea. The present study suggests a high probability that species of Asteroschema and Gorgonocephalus are more widely spread around the Indo-Pacific region than previously expected.

Identifying the Genetic Distance Threshold for Entiminae (Coleoptera: Curculionidae) Species Delimitation via COI Barcodes.

Simple SummaryOver 12,000 species of the subfamily Entiminae (Coleoptera: Curculionidae) have been described worldwide, but there has yet to be realization of the potential for DNA barcodes to assist in species-level identification. Here, we analyzed the variation in intra- and interspecific genetic distance of 621 public- and 27 self-determined sequences, to determine parameters for species identification of Entiminae. Our study found no universal barcoding gap at the subfamily level, although a genetic distance threshold of 9.18% can delimit more than 88% of Entiminae species. We also inferred additional empirical threshold values for 14 genera (species > 2) that can delimit congeneric species.The subfamily Entiminae is the largest group in the family Curculionidae, and it has long represented a challenge in traditional and molecular classification. Here, we analyzed intra- and interspecific genetic distances of 621 public COI barcode sequences (658bp) from 39 genera and 110 species of Entiminae, to determine parameters most congruent in retaining established species. We found that the mean intraspecific genetic distance (3.07%) was much smaller than the mean interspecific one (21.96%), but there is a wide range of overlap between intra- and interspecific genetic distances (0.77–18.01%), indicating that there is no consistent, universal barcoding gap. Specifically, DNA barcoding gap analysis for morphospecies revealed that 102 of 110 morphospecies had barcoding gaps, and 9.18% was the optimum threshold of genetic distances for 97 species delimitation. We further confirmed this threshold with barcodes from 27 morphologically identified specimens (including 21 newly reported barcodes) sequenced from five genera and seven species. We also identified thresholds to delimit congeneric species within 14 selected genera (species > 2), which varied from 7.42% (Trichalophus) to 13.48% (Barypeithes). We herein present optimal parameters for species identification in the Entiminae. Our study suggests that despite no universal genetic distance threshold value in subfamily Entiminae, 9.18% is optimal for most species. We recommend a wider sampling of geographic populations to better account for intraspecific distance variation, and that genetic distance thresholds for species delimitation should be refined at the genus level.

Multi-Gene Phylogeny of the Ciliate Genus Trachelostyla (Ciliophora, Hypotrichia), With Integrative Description of Two Species, Trachelostyla multinucleata Spec. nov. and T. pediculiformis (Cohn, 1866).

Many hypotrich genera, including Trachelostyla, are taxonomically challenging and in a need of integrative revision. Using morphological data, molecular phylogenetic analyses, and internal transcribed spacer 2 (ITS2) secondary structures, we attempt to cast more light on species relationships within the genus Trachelostyla. The present multifaceted approach reveals that (1) a large-sized species with numerous macronuclear nodules, isolated from sandy littoral sediments in southern China, is new to science and is endowed here with a name, T. multinucleata spec. nov.; (2) two other Chinese populations previously identified as T. pediculiformis represent undescribed species; and (3) multigene phylogeny is more robust than single-gene trees, recovering the monophyly of the genus Trachelostyla with high bootstrap frequency. Additionally, ITS2 secondary structures and the presence of compensatory base changes in helices A and B indicate the presence of four distinct taxa within the molecularly studied members of the genus Trachelostyla. Molecular data are more suitable for delimitation of Trachelostyla species than morphological characters as interspecific pairwise genetic distances of small subunit (18S) rDNA, ITS1-5.8S-ITS2, and large subunit (28S) rDNA sequences do not overlap, whereas ranges of multiple morphometric features might transcend species boundaries.

DNA barcoding identification of Greek freshwater fishes.

Biodiversity is a key factor for the functioning and efficiency of an ecosystem. Greece, though covering a relatively small surface area, hosts a great deal of species diversity. This is especially true for freshwater fishes. In recent years, the traditional methods of species identification have been supplemented by the use of molecular markers. The present study therefore aims to extensively produce DNA barcodes for Greek freshwater fish species and investigate thoroughly if the presently accepted species classification is in agreement with molecular data. A 624-bases long fragment of the COI gene was sequenced, from 406 freshwater fish specimens belonging to 24 genera and originating from 18 lake and river sites. These sequences were used along with 596 sequences from the same genera, recovered from BOLD, for the construction of phylogenetic trees and the estimation of genetic distances between individuals. In total, 1002 sequences belonging to 72 species were analyzed. The method was found to be effective for 55 of 72 studied species. 17 closely related species with low interspecific genetic distances were observed, for which further study is proposed. It should also be noted that, in four cases, cryptic diversity was observed, where groups originally identified as one species exhibited genetic distance great enough to be separated into discrete species. Region specific haplotypes were also detected within populations of 14 species, giving the possibility to identify even the geographic origin of a species. Our findings are discussed in the light of the rich history of the Balkan peninsula and provide a significant steppingstone for the further study of Greek and European freshwater fish biodiversity.

Evaluation of partial 12S rRNA, 16S rRNA, COI and Cytb gene sequence datasets for potential single DNA barcode for hylids (Anura: Hylidae)

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

Molecular classification and comparative phylogeographic study of insectivorous bat species (Pipisitrellus coromandra) from Punjab, Pakistan.

Molecular based identification of bat fauna in Pakistan has been relatively less explored. The current study was therefore planned to report for the first time the molecular classification of insectivorous bats (Pipistrellus coromandra) based on mitochondrion gene (COI) from Punjab, Pakistan. Specimens were collected from five different locations followed by DNA extraction with subsequent gene amplification and sequencing. All samples in the study had shown close identity matches with species (Pipistrellus coromandra) from India and (Pipistrellus tenuis) from Vietnam with percentage identity score of 96.11 and 95.58 respectively except one sequence which only revealed 86.78% identity match on Basic Local Alignment Search Tool (BLAST) and could only be assigned to genus level Pipistrellus sp. The results indicated negligible intra-population genetic distance among collected samples whereas the comparison with species from other countries had shown high intraspecific (P. coromandra) and interspecific (P. tenuis) mean genetic distances. The current study hence successfully proved the efficiency of COI gene as a molecular marker for species identification and in analyzing the patterns of genetic variation with species from other countries.