DNA Barcoding Research Articles

Morphological Development and DNA Barcoding Identification of Pholis fangi Larvae and Juveniles in the Yellow Sea

Pholis fangi is a small, bottom-dwelling fish species endemic to the Yellow Sea and Bohai Sea in China. While previous research has focused on its population biology and molecular structure, little is known about the early developmental stages of this species. In this study, larval and juvenile P. fangi specimens were collected from the Sheyang Sea Area, Jiangsu Province, in 2017. Morphological features were examined using microscopy, and DNA barcoding was conducted to confirm species identification. The research documented detailed changes in yolk sac, fin development, and melanophore distribution patterns across larval and juvenile stages of P. fangi. Comparative analysis with other Pholis species revealed that melanophore distribution is a key distinguishing characteristic, allowing effective differentiation between larval and juvenile stages, as well as between Pholis species. This study provides valuable insights into the early life history of P. fangi, contributing to a better understanding of the genus Pholis. The findings demonstrate the utility of combining traditional morphological observation and molecular techniques for accurate species identification, particularly during the critical larval and juvenile developmental phases.

Integrative taxonomy sheds light on species boundaries and relationships in the inbred haplodiploid ambrosia beetle genus Xyleborus

Abstract This study investigated the relationship between genetic and morphological evidence in a species complex of haplodiploid, near-inbred ambrosia beetles, which are also vectors of an invasive tree pathogen: Xyleborus ferrugineus, Xyleborus bispinatus, and Xyleborus impressus. The unorthodox genetic system may blur species boundaries and challenges the applicability of standard species concepts. Haplo-diploid scolytine systematics is rife with contentious species delimitation, but species limits have rarely been tested empirically. To test the congruence of morphology, genetics, and geography in species identity, we analyzed 16 morphological characters and two DNA barcodes (COI mtDNA and CAD DNA sequences) in samples from across Florida. Morphological characters used to distinguish species corresponded with phylogenetic relationships in the majority of individuals. The maximum intraspecific divergence for the COI barcoding region was up to 19%, among the largest reported for animals. The phylogenetic and morphological analyses suggest that the three species are valid, but the astonishing intraspecific genetic diversity suggests either additional cryptic species or pseudogenes.

The pan-plastome of Prunus mume: insights into Prunus diversity, phylogeny, and domestication history.

Prunus mume in the Rosaceae and commonly referred to as mei or Chinese plum is widely used as a traditional ornamental flowering plant and fruit tree in China. Although some population and genetic analyses have been conducted for this species, no extensive comparisons of genetic variation from plastomes have yet been investigated. We de novo assembled a total of 322 complete P. mume plastomes in this study and did a series of comparative analyses to better resolve pan-plastomic patterns of P. mume. To determine the phylogeny and domestication history of this species, we reconstructed the phylogenetic tree of Prunus genus, and resolved the population structure of P. mume. We also examined the nucleotide variation of P. mume to find potential DNA barcodes. The assembled plastomes exhibited a typical quadripartite structure and ranged from 157,871 bp to 158,213 bp in total size with a GC content ranging from 36.73 to 36.75%. A total of 112 unique genes were identified. Single nucleotide variants (SNVs) were the most common variants found among the plastomes, followed by nucleotide insertions/deletions (InDels), and block substitutions with the intergenic spacer (IGS) regions containing the greatest number of variants. From the pan-plastome data six well-supported genetic clusters were resolved using multiple different population structure analyses. The different cultivars were unevenly distributed among multiple clades. We also reconstructed a phylogeny for multiple species of Prunus to better understand genus level diversity and history from which a complex introgressive relationship between mei and other apricots/plums was resolved. This study constructed the pan-plastome of P. mume, which indicated the domestication of P. mume involved multiple genetic origins and possible matrilineal introgression from other species. The phylogenetic analysis in Prunus and the population structure of P. mume provide an important maternal history for Prunus and the groundwork for future studies on intergenomic sequence transfers, cytonuclear incompatibility, and conservation genetics.

The bee genus Borgatomelissa Patiny, 2000 (Anthophila: Andrenidae: Panurginae) with the description of a new species from northern Oman, and a key to species

ABSTRACT The bee genus Borgatomelissa Patiny, 2000 (Anthophila: Andrenidae: Panurginae) is found across North Africa, the Sahel, and throughout the Arabian Peninsula. Using both morphology and DNA barcodes, a new species, B. samailensis sp. n., is described from Oman, increasing the known number of species to four. Due to the holotype of B. brevipennis Walker, 1871 being lost, we designate a neotype from a specimen collected from Oman. Additionally, new country records are documented herein for B. flavimaura Ortiz-Sánchez and Patiny, 2019 and B. brevipennis, based on specimens housed in the collections of the Natural History Museum, London and Biologiezentrum Linz, Austria. A key to the species in the genus is given. http://www.zoobank.org/urn:lsid:zoobank.org:pub:6C01C20B-AA12-4021-83FC-A91634EB492C

DNA barcoding of terrestrial invasive plant species in Southwest Michigan.

Because of the detrimental effects of terrestrial invasive plant species (TIPS) on native species, ecosystems, public health, and the economy, many countries have been actively looking for strategies to prevent the introduction and minimize the spread of TIPS. Fast and accurate detection of TIPS is essential to achieving these goals. Conventionally, invasive species monitoring has relied on morphological attributes. Recently, DNA-based species identification (i.e., DNA barcoding) has become more attractive. To investigate whether DNA barcoding can aid in the detection and management of TIPS, we visited multiple nature areas in Southwest Michigan and collected a small piece of leaf tissue from 91 representative terrestrial plant species, most of which are invasive. We extracted DNA from the leaf samples, amplified four genomic loci (ITS, rbcL, matK, and trnH-psbA) with PCR, and then purified and sequenced the PCR products. After careful examination of the sequencing data, we were able to identify reliable DNA barcode regions for most species and had an average PCR-and-sequencing success rate of 87.9%. We found that the species discrimination rate of a DNA barcode region is inversely related to the ease of PCR amplification and sequencing. Compared with rbcL and matK, ITS and trnH-psbA have better species discrimination rates (80.6% and 63.2%, respectively). When ITS and trnH-psbA are simultaneously used, the species discrimination rate increases to 97.1%. The high species/genus/family discrimination rates of DNA barcoding indicate that DNA barcoding can be successfully employed in TIPS identification. Further increases in the number of DNA barcode regions show little or no additional increases in the species discrimination rate, suggesting that dual-barcode approaches (e.g., ITS + trnH-psbA) might be the efficient and cost-effective method in DNA-based TIPS identification. Close inspection of nucleotide sequences at the four DNA barcode regions among related species demonstrates that DNA barcoding is especially useful in identifying TIPS that are morphologically similar to other species.

Skmer approach improves species discrimination in taxonomically problematic genus Schima (Theaceae)

Genome skimming has dramatically extended DNA barcoding from short DNA fragments to next generation barcodes in plants. However, conserved DNA barcoding markers, including complete plastid genome and nuclear ribosomal DNA (nrDNA) sequences, are inadequate for accurate species identification. Skmer, a recently proposed approach that estimates genetic distances among species based on unassembled genome skims, has been proposed to effectively improve species discrimination rate. In this study, we used Skmer to identify species based on genomic skims of 47 individuals representing 10 out of 13 species of Schima (Theaceae) from China. The unassembled reads identified six species, with a species identification rate of 60%, twice as high as previous efforts that used plastid genomes (27.27%). In addition, Skmer was able to identify Schima species with only 0.5× sequencing depth, as six species were well-supported with unassembled data sizes as small as 0.5 Gb. These findings demonstrate the potential for the Skmer approach in species identification, where nuclear genomic data plays a crucial role. For taxonomically difficult taxa such as Schima, which have diverged recently and have low levels of genetic variation, Skmer is a promising alternative to next generation barcodes.

A study on the intertidal animal diversity of Xia-Sanheng Island by using morphological and DNA barcoding identification

Intertidal animals are an essential part of the ecosystem, and species diversity can reflect the state of the local ecological environment. However, traditional morphological identification is prone to corresponding classification errors or research limitations. With the development of molecular biology, many techniques and bioinformatics classification methods have been applied to identify species efficiently in recent years. This research aimed to examine the feasibility of DNA barcoding within the identification of intertidal animal species collected from the Xia-Sanheng Island. A total of 41 cox1 gene sequences were obtained by experimentalization or downloaded from the National Center for Biotechnology Information (NCBI) database. Then morphological classification, molecular identification, phylogenetic tree analysis, and Automatic Barcode Gap Discovery (ABGD) method were used to analyze the animal samples. We found that although the molecular classification of molluscs was not accurate enough, the collected specimens could be divided into 15 species, 12 families, and 4 phyla. Tetraclita japonica, Thais luteostoma, and Mytilus coruscus were the dominant species on the Xia-Sanheng Island. Additionally, there may be a new species, Platynereis sp., which needs further confirmation. We suggest that further identification of marine biodiversity should be carried out by combining morphological and molecular biological methods.

One Step beyond Species Description: Unveiling a Fine-Scale Diversity within the Genus Dzhanokmenia Kostjukov (Hymenoptera: Eulophidae).

Although Chalcidoidea is one of the megadiverse superfamilies in Hymenoptera, numerous species are still being discovered and described. However, the difficulties in delimiting intra- and interspecific variation hinder this process. In this study, DNA barcoding methods using the COI gene were employed to investigate the morphological variation within Dzhanokmenia Kostjukov, 1977. The nuclear locus, 28S D2, was used to infer a phylogeny to gain an understanding of the relationship of Dzhanokmenia with other potentially close genera. Through a preliminary DNA barcode library established here, including eight species, we calibrated the intraspecific variation in certain diagnostic characters for the new species described here, D. brevifunis Ganbaatar & Cao sp. nov. Maximum likelihood results show that Dzhanokmenia is clustered with the genera associated with Tetrastichus, such as Chaenotetrastichus Graham, 1987, Baryscapus Förster, 1856, Tetrastichus Haliday, 1844, and Oomyzus Rondani, 1870 involved in this study. Our results indicate that the species diversity of Dzhanokmenia is understudied and tentatively confirm that Dzhanokmenia has a potential close relationship with Baryscapus. Along with the DNA barcode library, the referenced phylogeny datasets improve the understanding of the systematic position of Dzhanokmenia within the subfamily Tetrastichinae and the definition of this genus in terms of morphology, thereby facilitating species delimitation, discovery, and description within Dzhanokmenia.

Genetic Identification and Phylogeny of Clerodendrum Phlomidis Growing in Egypt Using Some DNA Barcodes

Genetic Identification and Phylogeny of Clerodendrum Phlomidis Growing in Egypt Using Some DNA Barcodes

Morphological and genetic identification of the gill monogenean parasite (Diclidophora merlangi) that infects Twobar Seabream Fish (Acanthopagrus bifasciatus) in the Arabian Gulf, Saudi Arabia.

Ectoparasites, particularly monogeneans, negatively affect fish health and growth. This study identified monogenean parasites in the twobar seabream, Acanthopagrus bifasciatus (Sparidae), inhabited the Arabian Gulf (Saudi Arabia). Following that, forty A. bifasciatus fish samples were visually examined for monogeneans. Parasite species were collected from the gills and then analyzed morphometrically, morphologically, and molecularly using the partial regions of the large subunit of ribosomal RNA (28S rRNA) and mitochondrial cytochrome C oxidase subunit I (COI) genes. Fish species were also identified using a DNA barcoding approach based on the COI gene. The monogenean species of Diclidophora merlangi (Diclidophoridae) were found in 45% of the fish species studied. The generic features of the Diclidophora genus distinguish this species. This species discriminated itself from congeners by having a muscular bulb with 17 grooved and recurved hooks, 218±10 (184-267) post-ovarian testes, and four pairs of pedunculated clamps of relative sizes. Partial 28S rRNA sequencing from monogeneans revealed that they grouped with members of the genus Diclidophora, forming a monophyletic group that supported the morphological descriptions. Molecular identification revealed that D. merlangi has a unique barcode made up of a COI sequence. The host identity was established as A. bifasciatus based on the COI gene sequences. Furthermore, a molecular phylogenetic study was performed to determine the phylogenetic affinity of parasite species and fish hosts. This study on Diclidophora species is considered the first record of this genus in the examined area.

Scalable design of orthogonal DNA barcode libraries

Orthogonal DNA barcode library design is an essential task in bioengineering. Here we present seqwalk, an efficient method for designing barcode libraries that satisfy a sequence symmetry minimization (SSM) heuristic for orthogonality, with theoretical guarantees of maximal or near-maximal library size under certain design constraints. Seqwalk encodes SSM constraints in a de Bruijn graph representation of sequence space, enabling the application of recent advances in discrete mathematics1 to the problem of orthogonal sequence design. We demonstrate the scalability of seqwalk by designing a library of >106 SSM-satisfying barcode sequences in less than 20 s on a standard laptop.

The Problems of DNA-Barcoding the Shads of Genus Alosa (Alosidae) of the Ponto-Caspian Basin

—Numerous studies show that species identification of representatives of the genus Alosa using various genetic markers is often difficult and the search for more specific biomarkers is required. For the first time we analyzed polymorphism of COI gene fragment of mitochondrial DNA of two representatives of this genus (A. tanaica and A. kessleri), supplemented with new data on A. immaculata, from the waters of the Ponto-Caspian basin in comparative aspect with other representatives of the herring (Clupeoidea) genera Alosa, Clupea, Clupeonella, Sprattus, and Sardinops. The main result was the conclusion that within the genus Alosa, it is not possible to identify species using the marker used. On the one hand, specimens collected from morphologically distinct individuals and identified as different species have the same haplotypes. On the other hand, samples belonging to different species differ from each other by an insignificant number of nucleotide substitutions and do not form independent clades on the phylogram and haplotype network. This indicates the absence of genetic differentiation between the studied samples of herrings of genus Alosa into separate species and species groups when using DNA barcoding based on the COI gene. The reasons for such a phenomenon may be the following: (1) incorrect identification of species in catches, since shads (Alosidae) have high morphological flexibility and in many species, the main external morphological characters often overlap; (2) recent time of speciation by the standards of biological evolution for shads of genus Alosa; (3) difference in proportion of interspecific hybrids, which can vary significantly between populations of the same species.

Even one tardigrade is enough. Molecular methods revealed presence of pantropical species <em>Echiniscus lineatus</em> (Heterotardigrada, Echiniscidae) in the fauna of Seychelles

A brief report on the tardigrade species Echiniscus lineatus discovered in a moss cushion collected in Seychelles is given. The species represents a new pantropical element in the tardigrade fauna of Seychelles. With this record the number of echiniscid tardigrade species in Seychelles has reached five. Morphological identification of the species is supported with the analysis of obtained sequences of nuclear and mitochondrial DNA barcode genes (ITS-1, ITS-2 and COI). Analysis of the genetic data using the method of TCS haplotype networks revealed separated position of the Seychellian population of E. lineatus and supported the hypothesis of independent colonisation of Seychelles.

A review of Coelopoetinae (Lepidoptera, Gelechioidea, Pterolonchidae), a moth subfamily confined to western North America, with descriptions of seven new species

Systematics and taxonomy of the gelechioid subfamily Coelopoetinae are reviewed. Following the current classification, this group is considered to form its own monotypic subfamily in Pterolonchidae with one recognized genus, Coelopoeta, after a convoluted and, in part, arguably conjectural, historical systematic treatment. On morphological basis (appearance, male genitalia) and with support from DNA barcodes, the genus is divided into two discrete units probably meriting recognition as separate genera. The species groups are informally treated as the nominate C. glutinosi species group, and the C. fissurina species group. In the absence of knowledge of females or the biologies of any of the species of the C. fissurina group, species of both groups are here provisionally included in Coelopoeta. In total, 10 species are recognized, seven of which are here described as new: C. glutinosi species group: C. alboflava Kaila, sp. nov., C. aprica Kaila, sp. nov., C. aurora Kaila, sp. nov., C. fulminea Kaila, sp. nov. and C. sariae Kaila, sp. nov.; C. fissurina species group: C. fissurina Kaila, sp. nov. and C. valalbui Kaila, sp. nov. The three previously known species, C. glutinosi Walsingham, 1907, C. maiadella Kaila, 1995 and C. phaceliae Kaila, 1995 are redescribed. All three of these species belong to the glutinosi species group. A lectotype is designated for C. glutinosi Walsingham, 1907. Some southwestern Coelopoeta species are potentially under threat of decline or even extinction due to the apparently increasingly intense and frequent forest fires. This threat is significant as the species with known life histories spend their entire life cycles above ground in low vegetation.

A new species of Janiralata Menzies, 1951 (Isopoda: Asellota, Janiridae) from Korea

A new isopod species, Janiralata kwangsooi sp. nov., from Dokdo Island located in the East Sea off the Korean Peninsula is described here. This species is distinguished from its congeners by the number of antennular articles, setation of mouthparts and pereopods, length and width ratio of appendages, and shape of male pleopods I and II. To aid species identification, a taxonomic key to species of Janiralata Menzies, 1951 in the Japanese and Korean waters is provided. A partial sequence of mitochondrial cytochrome-c-oxidase (COI) of this new species is also provided for DNA barcoding and compared with those of congeners publicly available in GenBank.

Assembly and Comparative Analyses of the Chloroplast Genomes of the Threatened Plant Rosa anemoniflora

Due to insufficient molecular biology and genetic research on Rosa anemoniflora, this endangered plant has not yet received effective protection. Therefore, the complete chloroplast genome sequence of R. anemoniflora, along with comparative analysis of the chloroplast genomes of related species, is necessary and crucial for reconstructing phylogenetic relationships and developing genetic markers to conserve these species. A series of analyses, including genome structure, GC content, gene number, selection pressure, and nucleotide diversity, were performed by comparing the chloroplast genomes of R. anemoniflora and its relatives. The results indicate that the chloroplast genomes of R. anemoniflora and its close relatives are highly conserved in all genome characteristics, and all protein-coding genes in R. anemoniflora have not experienced significant positive or negative selection pressures. Comparative analysis revealed several variation hotspots, such as the atpH-atpI region, which can serve as a DNA barcode for distinguishing R. anemoniflora from its close relatives. Finally, the results confirmed that R. anemoniflora belongs to Rosa section Synstylae and that R. anemoniflora and its close relatives likely originated from the Fujian and Taiwan regions of China and diverged approximately 3.24 million years ago. This study provides crucial information for future biodiversity conservation and genetic resource management.

Unveiling South African insect diversity: DNA barcoding’s contribution to biodiversity data

Insects are one of the most species-rich groups on Earth. They comprise much of animal diversity and play vital roles in ecosystems, including pollination, pest control, and decomposition. However, only a fraction of this diversity has been formally described. South Africa is recognised as one of the most biologically diverse countries globally, with an estimated 44 000 insect species. Many crops rely on insect pollinators, including canola, apples, oranges, and sunflowers. A shortage of wild pollinators currently threatens crop yields, yet our knowledge of insect diversity within South Africa is sparse. There are few taxonomic specialists relative to South Africa’s biodiversity, and the methods used for insect identification can be time-consuming and expensive. DNA barcoding provides an important research tool to accelerate insect biodiversity research. In this review, we queried the public DNA barcoding BOLD (Barcode of Life Data System) database for records of “Insecta” within South Africa, and 416 211 published records assigned to 28 239 unique BINs (Barcode Index Numbers) were returned. We identified five taxonomic orders with more BINs than known species in southern Africa (Hymenoptera, Diptera, Thysanoptera, Plecoptera, and Strepsiptera). Most of the barcoded records were derived from Malaise trap sampling in Gauteng, Mpumalanga and Limpopo, while the rest of South Africa remains poorly sampled. We suggest that there is a need for a comprehensive national sampling effort alongside increased investment in taxonomic expertise to generate critical baseline data on insect biodiversity before species are lost to extinction.

Assessment of fish diversity in the East China Sea hairtail national aquatic germplasm resources conservation zone using DNA barcoding

Based on the fishery resource survey data from 2019 to 2022, the species composition and taxonomic diversity of fish in the East China Sea hairtail national aquatic germplasm resources conservation zone (ECSHZ) were studied through the analysis of 185 COI sequences. The results showed that the 185 fish samples collected belonged to 105 species, 21 orders, 46 families, and 89 genera in terms of their taxonomic status. There are discrepancies between morphological identification and genetic identification results for four fish species. The intraspecific genetic distance ranged from 0 % to 1.0 %, with an average value of 0.3 %. Meanwhile, the interspecies within genus distance ranged from 5.3 % to 22.7 %, with an average of 14.5 %. The outcomes of the division using Automatic Barcoding Gap Discovery (ABGD) and Bayesian phylogenetics and phylogeography (BPP) were concordant, identifying a total of 105 species groups. ABGD and BPP have shown that there was no overlap between intra-species variation and inter-species variation, supporting the existence of a barcoding gap. The results demonstrate the superiority of DNA barcoding in species identification. A phylogenetic tree was constructed using the maximum likelihood method based on the COI gene, which revealed certain limitations of COI in representing phylogenetic relationships at the genus and higher taxonomic levels. Another phylogenetic tree was constructed using 13 mitochondrial protein-coding genes with more genetic information, allowing for a better study of evolutionary relationships of ECSHZ fish species. The tree supported that Carangidae, Polynemidae, Paralichthyidae, Pleuronectidae, Sphyraenidae, and Cynoglossidae were clustered in an independent clade, with Priacanthidae and Lobotidae separating from Perciformes. This study offers DNA barcodes for ECSHZ fish species, providing crucial data support for conserving fish diversity.

DNA barcoding of common freshwater eleotrids (Gobiiformes: Eleotridae) in the Philippines, with a global comparison and notes on their conservation and management

Eleotrids, also known as gudgeons or sleeper gobies, are found mostly in freshwater and mangrove habitats of tropical and subtropical regions. Some eleotrids exhibit minimal morphological variation at the species level. In this study, DNA barcoding was used to assess the molecular diversity of common freshwater eleotrids in the Philippines in relation to other eleotrid species from other countries with available DNA sequences in public databases. A total of 422 mitochondrial DNA cytochrome c oxidase I (COI) gene sequences were analyzed: 107 of which were generated from this study, while the remaining 315 sequences were mined from Barcode of Life Data System and GenBank. Both the neighbor-joining and maximum likelihood trees constructed based on the COI sequences clustered most of the species in concordance with their taxonomic classification. The mean Kimura 2-parameter genetic distances within species and genera were 1.31% and 11.60%, respectively. As expected, the mean interspecific distance was higher than the mean intraspecific distance except in some species. This study revealed the presence of nine distinct eleotrid species belonging to five genera in the Philippines. These species include Giuris laglaizei, G. tolsoni, Eleotris acanthopomus, E. fusca, E. melanosoma, Belobranchus sp., Belobranchus belobranchus, Butis butis, and Ophiocara ophicephalus. This study also revealed the presence of possibly undescribed Belobranchus species in the Philippines. This undescribed Belobranchus species differs by a high percentage of genetic divergence from the other species of Belobranchus (3.00% from B. segura and 13.40% from B. belobranchus). Furthermore, high genetic distance (2.62%) was also observed between G. laglaizei species of Laguna de Bay and Mindanao. This study strongly recommends a taxonomic review of the Laguna de Bay G. laglaizei and the Philippine Belobranchus spp., as these species may harbor species complexes that require closer examination so that proper conservation and management measures can be implemented for each species.

Species Delimitation of Argonauta Paralarvae Reveals an Extensive Morphological Variability in the Northern Humboldt Current System

Argonauts or paper nautiluses are pelagic octopod cephalopods with a cosmopolitan distribution in tropical and subtropical waters around the world. Unlike other species of octopus, these are characterized by the fact that the female has a shell that serves as the breeding chamber for the eggs. Over time, this structure has been used as a taxonomic diagnostic character, causing problems in the systematics of this genus, with around 50 synonymies reported. Only two species, Argonauta argo and A. nouryi, have been reported in the Northern Humboldt Current System; however, there is taxonomic uncertainty regarding these species, which is reflected in the paralarvae (the first stage of life after hatching). In the paralarvae, the chromatophore patterns are considered to be conservative and reliable taxonomic characteristics. The objective of this study is to demonstrate the extensive variability in the chromatophore arrangement of Argonauta paralarvae in the Northern Humboldt Current using DNA barcoding and five species delimitation models. Our results include up to 11 different paralarvae morphotypes according to the pattern of chromatophores (number and arrangement) and 2 shell morphotypes. Species delimitation methods divided the 13 Argonauta morphotypes into two consensus molecular taxonomic units (MOTUs), A. argo and A. nouryi. Additionally, the results revealed an extensive morphological variability in the paralarvae and female shells of A. nouryi, demonstrating the importance of molecular data in studies involving species with different life stages, especially when this extensive morphological variability obscures conventional analyses.