Interspecific Distances Research Articles

A Measure of the DNA Barcode Gap for Applied and Basic Research.

DNA barcoding has largely established itself as a mainstay for rapid molecular taxonomic identification in both academic and applied research. The use of DNA barcoding as a molecular identification method depends on a "DNA barcode gap"-the separation between the maximum within-species difference and the minimum between-species difference. Previous work indicates the presence of a gap hinges on sampling effort for focal taxa and their close relatives. Furthermore, both theory and empirical work indicate a gap may not occur for related pairs of biological species. Here, we present a novel evaluation approach in the form of an easily calculated set of nonparametric metrics to quantify the extent of proportional overlap in inter- and intraspecific distributions of pairwise differences among target species and their conspecifics. The metrics are based on a simple count of the number of overlapping records for a species falling within the bounds of maximum intraspecific distance and minimum interspecific distance. Our approach takes advantage of the asymmetric directionality inherent in pairwise genetic distance distributions, which has not been previously done in the DNA barcoding literature. We apply the metrics to the predatory diving beetle genus Agabus as a case study because this group poses significant identification challenges due to its morphological uniformity despite both relative sampling ease and well-established taxonomy. Results herein show that target species and their nearest neighbor species were found to be tightly clustered and therefore difficult to distinguish. Such findings demonstrate that DNA barcoding can fail to fully resolve species in certain cases. Moving forward, we suggest the implementation of the proposed metrics be integrated into a common framework to be reported in any study that uses DNA barcoding for identification. In so doing, the importance of the DNA barcode gap and its components for the success of DNA-based identification using DNA barcodes can be better appreciated.

New sand fly (Diptera, Psychodidae) records and COI DNA barcodes in the state of Maranhão, Eastern Amazon, Brazil

The sand fly fauna and the usefulness of the DNA barcoding fragment of the cytochrome c oxidase subunit I (COI) gene were accessed in a forest fragment in the municipality of Governador Newton Bello, state of Maranhão, Brazil. We performed entomological collections in three independent campaigns in May and October 2021, and January 2023. Sand flies were morphologically-identified and then DNA barcoded. Sequences were deposited and analyzed in the BOLD System Database, and various species delimitation algorithms, to assess whether DNA sequences merge into taxonomic units in accordance with nominal species. In total, 1,524 sand flies were collected, comprising 32 nominal species. Nyssomyia antunesi was the most abundant species (31.5 %), followed by Psychodopygus davisi (27 %). We reported for the first time in the state of Maranhão, the presence of Lutzomyia evangelistai, Lutzomyia sherlocki, Pressatia equatorialis, and Psathyromyia barrettoi. We amplified and analyzed 67 COI barcodes of 23 species, which were merged with conspecific sequences extracted from GenBank. The maximum intraspecific p distances ranged from 0.0 % to 14.74 %, while the distances to the nearest neighbor varied from 1.67 % to 13.64 %. The phylogenetic gene tree and species delimitation tools clustered sequences into well-supported clades/clusters for each nominal species, except for Pressatia choti/Pr. equatorialis, which have the lowest interspecific genetic distance (1.67 %). We sequenced for the first time COI barcodes of Brumptomyia brumpti, Evandromyia monstruosa, Micropygomyia rorotaensis, Micropygomyia pilosa, Pintomyia christenseni, Pintomyia pacae, Pr. equatorialis, Pa. barrettoi, and Psathyromyia hermanlenti, which will be useful for further molecular identification and classification proposals of Neotropical species. This study updated the current list of the sand fly fauna for the state of Maranhão to 97, and demonstrated that COI barcodes are useful for specific identification.

Underestimated diversity: Integrative taxonomy of Mesenchytraeus (Enchytraeidae, Clitellata) from Changbai Mountain, China

AbstractThe holarctic genus Mesenchytraeus is one of the species‐rich genera in the family Enchytraeidae. Changbai Mountain supports high diversity of Mesenchytraeus species, making it an ideal area to explore species boundaries. We explored species boundaries of Mesenchytraeus using an integrative approach. Morphological taxonomy recognized 10 species in this region, five of them new to science. In contrast, molecular species delimitation analyses showed that there are at least 16 species, 11 of them new to science. Clear genetic gaps were observed among species with high interspecific distances (10%–21.2%) and low intraspecific distances (0.2%–6.7%) based on uncorrected p‐distance of the COI gene. Morphological species complex M. spermatoglomeratus consists of three species, M. spermatoglomeratus Zhang, Lu & Xie, 2018 sensu stricto, M. rijina sp. n., and M. manchu sp. n. The M. duodiverticulus complex consists of M. duodiverticulus sp. n. and M. similiduodiverticulus sp. n. The M. monodiverticulus complex consists of M. monodiverticulus Shen, Chen & Xie, 2012 sensu stricto, M. ngulen sp. n., M. zhenggulen sp. n., and M. fokulen sp. n. Further new species, distinguishable with morphological as well as molecular methods, are M. similigigachaetus sp. n., M. parvidiverticulus sp. n., M. digitalisdiverticulus sp. n., and M. infradiverticulus sp. n. Finally, we inferred the morphogenetic processes of spermathecae and sperm bundles, and filtrated some morphological characters which are useful to identify species. As the first attempt in this genus, our study provides an opportunity to discuss the currently used taxonomic criteria and acquire new ideas for the taxonomy of enchytraeids.

Molecular and morphometric study of Brazilian populations of Psychodopygus davisi.

In this study, we analysed the molecular and morphometric differences of several populations of the putative sand fly vector Psychodopygus davisi (Root, 1934) (Diptera, Psychodidae, Phlebotominae) in Brazil. We amplified the 658 base pair fragments of the DNA barcoding region-cytochrome c oxidase subunit 1 (COI) gene-for 57 specimens of P. davisi and three specimens of Psychodopygus claustrei (Abonnenc, Léger & Fauran, 1979). We merged our data with public sequences of the same species available from GenBank. Then, the combined dataset-87 sequences and 20 localities-was analysed using population structure analysis and different species delimitation approaches. Geometric morphometry of wings was performed for 155 specimens of P. davisi populations from the North, Midwest and Southeast Brazilian regions, analysing the differences in centroid sizes and canonical variates. Molecular analysis indicated high intraspecific genetic distance values for P. davisi (maximum p distance = 5.52%). All algorithms identified P. davisi and P. claustrei as distinct molecular taxonomic units, despite the low interspecific distance (p distance to the nearest neighbour = 4.79%). P. davisi sequences were split into four genetic clusters by population structure analysis and at least five genetic lineages using intermediate scenarios of the species delimitation algorithms. The species validation analysis of BPP strongly supported the five-species model in our dataset. We found high genetic diversity in this taxon, which is in agreement with its wide geographic distribution in Brazil. Furthermore, the wing analysis showed that specimens from the Southeast Region of Brazil are different from those in the North and the Midwest. The evolutionary patterns of P. davisi populations in Brazil suggest the presence of candidate species, which need to be validated in future studies using a more comprehensive approach with both genomic data and morphological characters.

Assessing the Impact of Anthropic Pressures on Aquatic Macroinvertebrates: A Functional Trait Approach in the Irtysh River Watershed.

Little is known about how changes in the biodiversity and functional traits of macroinvertebrates in rivers respond to the responses of anthropic pressures and their driving factors. Macroinvertebrates were sampled at 17 sites in the Irtysh River Basin and classified macroinvertebrates into 10 traits and 38 categories between May and August 2022. Then, we performed R-mode linked to Q-mode (RLQ) analysis and calculated functional richness, evenness, divergence, and Rao's quadratic entropy (RaoQ) for each site and community-weighted means for each trait category. Our results indicated that there were pronounced alterations in species variability in the urban region. Functional divergence indicated fierce competition among species and considerable niche overlap in the urban region. Functional evenness indicated that species abundance distribution and interspecific functional distance were not uniform in the urban region. Functional richness indicated that the urban region was the strongest region in terms of niche occupation, resource utilization, and buffering capacity for environmental fluctuations. Rao's quadratic entropy showed that the trait difference of macroinvertebrates was the largest in all regions, which was caused by the gradient environmental difference. Research has revealed that urbanization significantly influences the evolutionary trajectory of macroinvertebrate fauna, culminating in an upsurge in pollution-tolerant species and a convergence of functional traits. We recommend strengthening the control of urban and industrial pollution and wise planning and management of land and water resources to mitigate the impact of anthropogenic destruction on habitat fragmentation in the Irtysh River Basin.

A Preliminary mitochondrial cytochrome c oxidase-I-based phylogeographic and phylogenetic analysis of Eurasian Acanthocinus griseus (Coleoptera, Cerambycidae)

Acanthocinus griseus (Fabricius, 1792) (Coleoptera: Cerambycidae, Lamiinae, Acanthocinini) has long been known for its role in the decay process of the wood in the forest ecosystem, and two critical features of the species, inhabiting standing trees and being a vector of pine wood nematodes Bursaphelenchus spp., have been noted recently. Therefore, understanding the current relationships and possible migration scenarios has been further required to assess invasion risks. The present work provided a preliminary comprehension of the phylogenetic relationships of A. griseus based on the mitochondrial cytochrome c oxidase-I (COI) gene region (658 bp), with sequences produced in the present study, from the specimens collected from timberyards, ports and forests of Kocaeli Province, Turkey, and with available sequences in GenBank of inhabitants of Eurasia, and of intercepted specimens in ports. The intraspecific genetic distance of A. griseus was 1.37-0,3%, while the interspecific distance was 10,79-13,37%, except the closeness of an A. griseus haplotype (AGR1) to A. sachalinensis (0,3%) more than its conspecifics (4,71-5,47%). The ML and BI analyses suggested identical topologies. The statistical parsimony network drew a reticular branching diagram without grouping across countries or geographic regions, which addresses ongoing gene flow. Most haplotypes from Turkey were clustered around a central haplotype (AGR11), which may point to a bottleneck effect. A haplotype previously intercepted in USA ports was identical to a haplotype sampled in Kocaeli. The present study suggests that the relationship between A. griseus and A. sachalinensis should be reconsidered from both morphological and molecular points of view. In addition, the possible ongoing intraspecific gene flow within A. griseus might be due to facilitated migration by the international wood trade.

Deepest known novel species of the genus Ophiuroglypha Hertz, 1927 (Echinodermata: Ophiuroidea) from the central rift zone, Philippine Sea

A new species of brittle star was collected in 2021 by the manned submersible “Fendouzhe” from the central rift zone deep waters, Philippine Sea, at a depth of 7729 m. It is described as Ophiuroglypha fendouzhe sp. nov., and is distinguished from its congeners based on the following features: slender arms, separated dorsal and ventral arm plates, overlapping large disc scales, and distally contiguous radial shields. We provide comprehensive descriptions of the external morphological features, including characteristics of the arm skeleton, and a phylogenetic analysis based on COI sequences. The interspecific genetic distance variation in the genus Ophiuroglypha found in this study was 3.89% to 24.25%. The new species constitutes the deepest known record for the genus Ophiuroglypha.

Molecular and morphological evidence reveals a hidden new taxon in the endemic genus Pseudocuneopsis (Bivalvia, Unionidae) from China.

A new species of freshwater mussel belonging to the genus Pseudocuneopsis, namely Pseudocuneopsiswuanasp. nov., is diagnosed and described from Guangxi Province, China. This paper provides a detailed shell morphological description, soft-body anatomical characteristics, and partial sequences of mitochondrial COI as DNA barcode data for the novel species. The new species can be distinguished from its congeners (Pseudocuneopsissichuanensis, P.yangshuoensis, and P.capitata) by shell shape, beak position, and surface sculpture. Phylogenetic analyses based on the mitochondrial COI gene reveal that Pseudocuneopsiswuanasp. nov. forms a sister group with P.yangshuoensis and exhibits an interspecific genetic distance of 5.1%. Therefore, we provide robust morphological and molecular evidence to support the validity of this new species.

A new species of Simulium (Asiosimulium) (Diptera: Simuliidae) from northeastern Thailand, with its phylogenetic relationships with related species in the subgenus Asiosimulium.

Simulium (Asiosimulium) khongchiamense sp. nov. is described based on females, males, pupae, and mature larvae collected from Khong Chiam District, Ubon Ratchathani Province, northeastern Thailand. It is characterized in the female by the medium-long sensory vesicle, scutum with 3 dark longitudinal vittae and elongate cercus; in the male by the number of upper-eye (large) facets in 17 or 18 vertical rows and 18 or 19 horizontal rows, hind basitarsus moderately enlarged and ventral plate with the posterior margin moderately concave medially; in the pupa by the head and thoracic integument sparsely covered with tubercles and gill of arborescent type with 32 or 33 filaments; and in the larva by the postgenal cleft deep, reaching the posterior margin of the hypostoma and sheath of the subesophageal ganglion dark pigmented. DNA analysis based on COI gene of all known species of the subgenus Asiosimulium, except for S. shanense and S. suchitrae, indicated that this new species can be clearly differentiated from all other related species (S. phurueaense, S. oblongum, S. saeungae, S. furvum, and S. wanchaii) with interspecific genetic distances ranging between 4.79% and 19.18%. This is the eighth species of the subgenus Asiosimulium. Taxonomic notes are given to distinguish this new species from the 7 known species members in its same subgenus. Additionally, keys to species of all members in the subgenus Asiosimulium are provided.

INTERSPECIFIC GENETIC DISTANCES IN THE GENUS YPONOMEUTA LATR. (LEPIDOPTERA: YPONOMEUTIDAE) ON COI AND COII FRAGMENTS AND THE USE OF THE RESULTS OF THEIR COMPARATIVE ANALYSIS FOR SOLVING TAXONOMIC TASKS

Comparative genetic analysis of the nucleotide sequences of COI and COII mitochondrial fragments showed that the degree of divergence of ermine moths of the genus Yponomeuta on both fragments is comparable. In the studied taxa, interspecific genetic distances are within 0.2–9.9% for the COI fragment and 0.5–9.6% for the COII fragment. Based on the results of the nucleotide sequences analysis of the COI fragment, the conspecificity of Y. refrigeratus and Y. evonymella, the interval of genetic distances between samples of which is 0.2–0.5%, was proved, and the validity of the synonymization of these species was confirmed. Analysis of nucleotide sequences by COI and COII fragments in samples collected on the Sakhalin Is., as well as the rooting of their clusters in groups with conspecific species, made it possible to identify them and confirm the habitat of Y. polystictus and Y. spodocrossus on Sakhalin Is. In general, the molecular genetic method, based on a comparative analysis of interspecific genetic distances by mitochondrial fragments of COI and COII, is effective for determining the status of a taxon, specimen identification in morphologically weakly divergent and variable species, or in species described by one sex, with an unknown other sex, as well as for delimitation of the species in heterogeneous series.

Molecular and morphometric differentiation of secondary filariasis vector Coquillettidia mosquitoes (Diptera: Culicidae) in Thailand.

Coquillettidia mosquitoes are important nuisance-biting pests and a vector of brugian filariasis in Thailand. However, comprehensive information about these mosquitoes remains unavailable such as molecular and morphometric differences among species. The lack of vector knowledge on Coquillettidia species could affect future disease control. This study aims to investigate differences in molecular variations based on mitochondrial cytochrome oxidase subunit I (COI) gene and wing geometric traits of three Coquillettidia species, namely Cq. crassipes, Cq. nigrosignata, and Cq. ochracea in Thailand. The results of molecular analyses revealed the differences among three Coquillettidia species. The genetic difference measure based on the Kimura two-parameter model among three Coquillettidia species showed low intraspecific distances (0%-3.05%) and large interspecific distances (10.10%-12.41%). The values of intra- and inter-genetic differences of three Coquillettidia species did not overlap which showed the existence of a barcoding gap indicating the efficiency of the identification based on the COI gene. As with molecular analysis, the landmark-based geometric morphometrics approach based on wing shape analysis indicated three distinct species groups which were supported by the high total performance score of cross-validated classification (97.16%). These results provide the first evidence of taxonomic signal based on molecular and wing geometric differences to support species identification and biological variations of Coquillettidia mosquitoes in Thailand for understanding these rare vector mosquitoes in depth and leading to effective further mosquito control.

Does One Size Fit All? Variations in the DNA Barcode Gaps of Macrofungal Genera.

The nuclear ribosomal internal transcribed spacer (nrITS) region has been widely used in fungal diversity studies. Environmental metabarcoding has increased the importance of the fungal DNA barcode in documenting fungal diversity and distribution. The DNA barcode gap is seen as the difference between intra- and inter-specific pairwise distances in a DNA barcode. The current understanding of the barcode gap in macrofungi is limited, inhibiting the development of best practices in applying the nrITS region toward research on fungal diversity. This study examined the barcode gap using 5146 sequences representing 717 species of macrofungi from eleven genera, eight orders and two phyla in datasets assembled by taxonomic experts. Intra- and inter-specific pairwise distances were measured from sequence and phylogenetic data. The results demonstrate that barcode gaps are influenced by differences in intra- and inter-specific variance in pairwise distances. In terms of DNA barcode behavior, variance is greater in the ITS1 than ITS2, and variance is greater in both relative to the combined nrITS region. Due to the difference in variance, the barcode gaps in the ITS2 region are greater than in the ITS1. Additionally, the taxonomic approach of "splitting" taxa into numerous taxonomic units produces greater barcode gaps when compared to "lumping". The results show variability in the barcode gaps between fungal taxa, demonstrating a need to understand the accuracy of DNA barcoding in quantifying species richness. For taxonomic studies, variability in nrITS sequence data supports the application of multiple molecular markers to corroborate the taxonomic and systematic delineation of species.

Culicoides jiangchengensis, a new species of the subgenus Sinocoides (Diptera, Ceratopogonidae) based on integrative taxonomy from China.

Biting midges of the genus Culicoides are important in both medicine and veterinary medicine because their blood-feeding regime enable them to transmit a variety of pathogens. In this study, the morphological characteristics of the new species of Culicoides (Sinocoides) jiangchengensis Wang et Liu sp. nov are described and compared with the other species of female Culicoides in the subgenus Sinocoides. Three morphological characteristics of C. jiangchengensis, such as without sensory pit in 3rd palpus segment, sensilla coeloconica on flagellomeres 1,9-13, and m1 and m2 cell of the wings with pale spots, were different from the other nine species of culicoides in subgenus Sinocoides. Genetically, C. jiangchengensis are most closely related to C. malipoensis, but they were located in different branches and the minimum interspecific distance between them was 12.6%. In addition, a checklist of 10 species in the subgenus Sinocoides Chu, 1983 (Diptera: Ceratopogonidae: Culicoides) in China, including the new species C. jiangchengensis Wang et Liu sp. nov., is provided, and an updated key to species of the subgenus Sinocoides Chu, 1983 was presented.

DNA barcoding of black flies (Diptera: Simuliidae) in Indonesia

BackgroundDNA barcoding is a valuable taxonomic tool for rapid and accurate species identification and cryptic species discovery in black flies. Indonesia has 143 nominal species of black flies, but information on their biological aspects, including vectorial capacity and biting habits, remains underreported, in part because of identification problems. The current study represents the first comprehensive DNA barcoding of Indonesian black flies using mitochondrial cytochrome c oxidase subunit I (COI) gene sequences.MethodsGenomic DNA of Indonesian black fly samples were extracted and sequenced, producing 86 COI sequences in total. Two hundred four COI sequences, including 118 GenBank sequences, were analysed. Maximum likelihood (ML) and Bayesian inference (BI) trees were constructed and species delimitation analyses, including ASAP, GMYC and single PTP, were performed to determine whether the species of Indonesian black flies could be delineated. Intra- and interspecific genetic distances were also calculated and the efficacy of COI sequences for species identification was tested.ResultsThe DNA barcodes successfully distinguished most morphologically distinct species (> 80% of sampled taxa). Nonetheless, high maximum intraspecific distances (3.32–13.94%) in 11 species suggested cryptic diversity. Notably, populations of the common taxa Simulium (Gomphostilbia) cheongi, S. (Gomphostilbia) sheilae, S. (Nevermannia) feuerborni and S. (Simulium) tani in the islands of Indonesia were genetically distinct from those on the Southeast Asian mainland (Malaysia and Thailand). Integrated morphological, cytogenetic and nuclear DNA studies are warranted to clarify the taxonomic status of these more complex taxa.ConclusionsThe findings showed that COI barcoding is a promising taxonomic tool for Indonesian black flies. The DNA barcodes will aid in correct identification and genetic study of Indonesian black flies, which will be helpful in the control and management of potential vector species.Graphical abstract

The Case for a Nuclear Barcode: Using the CAD CPS Region for Species and Genus Level Discrimination in Beetles

DNA barcoding has revolutionized how we discover, identify, and detect species. A substantial foundation has been established with millions of mitochondrial cytochrome c oxidase I sequences freely available for eukaryotes. However, issues with COI ranging from uniparental inheritance and small genetic population sizes to nuclear and asymmetric introgression can impede its use. We propose using CAD as the “nuclear barcode” to complement the COI barcode and ameliorate these concerns. We focused on beetles from taxonomically diverse species-level studies that used COI and CAD. An ambiguous barcode gap was present between intra- and interspecific genetic distances in CAD and COI; this led to difficulty with automated gap detection methods. We found pseudogenes, problematic population structure, introgression, and incomplete lineage sorting represented in the COI data. A CAD gene tree illuminated these cryptic problems. Placement tests of species and outgroups using distance-based tree building were largely successful for CAD, demonstrating its phylogenetic signal at the species and genus levels. Species placement issues were typically unique to one locus, allowing for recognition of misdiagnosis. We conclude that a CAD barcode is a valuable tool for beetle diagnostics, metabarcoding, and faunistic surveys.

New Insights into the Taxonomy of Myotis Bats in China Based on Morphology and Multilocus Phylogeny

The genus Myotis is one of the most diverse and widely distributed mammals, providing a good model for studies of speciation and diversification across large geographic scales. However, the classification within this genus has long been chaotic. Taxonomic revisions based on multiple data sources are essential and urgent. In this study, morphometrics and genetic markers with different modes of inheritance were used to clarify the taxonomy of Myotis distributed in China. Based on 173 mitochondrial Cytb sequences and five morphological characteristics, 114 specimens collected nationwide over the past 20 years were assigned to 11 Myotis species. All Chinese samples classified into M. davidii and M. longipes were revised to M. alticraniatus and M. laniger. Then, two nuclear fragments (Rag2 and Chd1) and Cytb sequences from representative individuals of Chinese Myotis were used for multilocus phylogeny reconstruction and genetic divergence evaluation. The phylogenetic relationships were clearly demonstrated in the species tree: M. alticraniatus and M. laniger; M. fimbriatus, M. pilosus, M. macrodactylus, and M. petax; and M. pequinius, M. chinensis, and M. blythii formed three strongly supported monophyletic clades. Mitochondrial divergence was almost 10 times that of nuclear divergence, with interspecific K2P distances ranging from 8% to 20% for Cytb and 0.3% to 2.3% for concatenated nuclear genes. Low levels of genetic divergence were observed between M. alticraniatus and M. laniger, as well as M. fimbriatus and M. pilosus. These results provide new insights into the taxonomy and phylogeny of Myotis bats in China and are important for the future research and conservation of Chinese Myotis.

DNA Barcoding and Phylogenetics Relationship of Pangasiid Catfishes in Peninsular Malaysia Revealed the Impacts of Aquaculture on the Native Species Conservation

Pangasiids are an economically significant group of catfish, and many pangasiids are threatened in the wild from anthropogenic pressures, including increases in fishing pressure, habitat degradation, and improperly managed aquaculture practices. This study demonstrates the usage of DNA barcoding of the Cytochrome Oxidase subunit I (COI) gene as an identification tool in detecting potentially threatening invasive pangasiid species by establishing the diversity and phylogenetic relationship of Pangasiidae catfishes in Peninsular Malaysia. A neighbour-joining (NJ) dendrogram (Kimura-2-parameter model) generated five clades to represent distinct genera. Pangasius was further subdivided into two clades (Clade A: Pangasius bocourti-P. djambal and Clade B: P. nasutus-P. conchophilus). Given the marginal genetic divergence, indigenous and non-native species should be treated cautiously in allopatrically distributed species. The analysis used Automatic Barcode Gap Discovery (ABGD) and revealed barcode gaps between the intraspecific and interspecific distances. The sequences were partitioned into five groupings, corresponding with the species delineation based on the distribution of pairwise differences, which could not be differentiated using the NJ dendrogram. ABGD allows the recognition of one or two additional species using the recursive approach, but other taxonomic methods should be considered for a solid conclusion. DNA barcoding demonstrates the identification of closely related species, thus justifying its application towards the conservation of these fish.

Improving the COI DNA barcoding library for Neotropical phlebotomine sand flies (Diptera: Psychodidae)

Sand fly species are traditionally identified using morphological traits, though this method is hampered by the presence of cryptic species. DNA barcoding is a widely used tool in the case of insects of medical importance, where it is necessary to know quickly which species are present in a transmission area. Here, we assess the usefulness of mitochondrial cytochrome c oxidase subunit I (COI) DNA barcoding as a practical tool for species identification, correct assignment of isomorphic females, and to evaluate the detection of cryptic diversity that occurs in the same species. A fragment of the COI gene was used to generate 156 new barcode sequences for sand flies from different countries of the Neotropical region, mainly Colombia, which had been identified morphologically as 43 species. The sequencing of the COI gene allowed the detection of cryptic diversity within species and correctly associated isomorphic females with males identified by morphology. The maximum intraspecific genetic distances ranged from 0 to 8.32% and 0 to 8.92% using uncorrected p distances and the Kimura 2-parameter (K2P) model, respectively. The minimum interspecific distance (nearest neighbor) for each species ranged from 1.5 to 14.14% and 1.51 to 15.7% using p and K2P distances, respectively. Three species had more than 3% maximum intraspecific distance: Psychodopygus panamensis, Micropygomyia cayennensis cayennensis, and Pintomyia evansi. They also were split into at least two molecular operational taxonomic units (MOTUs) each, using different species delimitation algorithms. Regarding interspecific genetic distances, the species of the genera Nyssomyia and Trichophoromyia generated values lower than 3% (except Nyssomyia ylephiletor and Ny. trapidoi). However, the maximum intraspecific distances did not exceed these values, indicating the presence of a barcode gap despite their proximity. Also, nine sand fly species were DNA barcoded for the first time: Evandromyia georgii, Lutzomyia sherlocki, Ny. ylephiletor, Ny. yuilli pajoti, Psathyromyia punctigeniculata, Sciopemyia preclara, Trichopygomyia triramula, Trichophoromyia howardi, and Th. velezbernali. The COI DNA barcode analysis enabled the correct delimitation of several Neotropical sand fly species from South and Central America and raised questions about the presence of cryptic species for some taxa, which should be further assessed.Graphical

Authentication of Averrhoa carambola L. Using DNA Barcoding: Detection of SNPs and Evaluation of Phylogenetic Relationship

Abstract: Background: Averrhoa carambola L. (star fruit) is an underutilized plant in the family Oxalidaceae. The plant has various pharmacological activities and has fleshy juicy fruit with high nutritional value and is rich in vitamins. Objectives: This study aims to authenticate and examine the evolutionary relationships between various Averrhoa taxa. Materials and Methods: The species identification was verified using the well-established DNA barcoding method. For this, a few predetermined loci including rpoB, matK, atpF-atpH, and rbcL were employed. Results: Amplicons were observed with a molecular weight of 450bp, 950bp, 800bp, and 650bp respectively as reported earlier. The sequences of these loci were submitted to NCBI. The sequences were approved with accession numbers MN447319, MN733917, MT195537, and MT195538 respectively. The amplified loci of the species and related taxa of the genus were compared using bioinformatics tools. 204 numbers of SNPs/indels were identified out of which some are unique for the species. K2P analysis showed that the maximum intraspecific distance (1.3%) was lower than the minimum interspecific distance (2.6%). Conclusion: Among the different universal barcoding loci, matK was found to be most informative in terms of the occurrence of SNPs and genetic distance. So, the information can be utilized for species authentication in the family Oxalidaceae. Keywords: Averrhoa carambola, DNA Barcoding, atpF–atpH, matK, rpoB.

Revealing cryptic diversity and population divergence in subtropical aphids throughDNAbarcoding

AbstractAphids are worldwide distributed pests, which feed on plant sap and cause serious losses to agricultural and forestry industries. Relatively few comprehensive studies have been conducted on aphid diversity in subtropics, although these regions may harbour hidden aphid diversity due to high plant diversity. In this study, we conducted DNA barcoding analysis for aphids in subtropical regions based on a comprehensive DNA barcode reference library including 5821 sequences and related ecological information (e.g. geography, host plant) of 379 morphospecies. In the reference library, 2140 sequences and related data of 151 morphospecies belonging to 77 genera and 11 subfamilies were newly produced by the current study. The average Kimura 2‐parameter distances within species, genera and subfamilies were 0.92%, 3.55% and 8.3%, respectively. The minimum interspecific distances were greater than the maximum intraspecific distances in 79.8% species, suggesting that barcode gaps existed in most aphid species. Automatic barcode gap discovery, Barcode Index Number, Bayesian Poisson tree processes and generalized mixed Yule‐coalescent revealed 419, 401, 462 and 455 single species‐representing clusters, respectively. A total of 32 morphospecies were assigned to more than one subclade respectively, suggesting geography or host plant‐mediated divergences and the existence of cryptic diversity. This study demonstrates that subtropical regions should have a higher species diversity of aphids, and cryptic species in certain morphospecies indicated in this study need to be investigated based on integrative taxonomic practices in the future. The DNA barcode reference library constructed herein provides a robust baseline data set to support future research in taxonomy, phylogenetics, ecology and evolution of aphids.