Species delimitation of the Afrotropical and Palaearctic Calliphora Robineau-Desvoidy and discovery of two new species in Afrotropics.

Orthoptera, among the oldest and most numerous insect lineages, is an excellent model for evolutionary studies but has numerous taxonomic problems. To mitigate these issues, the cytochrome c oxidase subunit I ( COI), standardized with the DNA barcode for Metazoa, is increasingly used for specimen identification and species delimitation. We tested the performance of COI as a DNA barcode in Orthoptera, using two analyses based on intra- and inter-specific distances, barcode gap, and Probability of Correct Identification (PCI); and estimated species richness through Automatic Barcode Gap Discovery (ABGD) and Assemble Species by Automatic Partitioning (ASAP). We filtered all sequences of Orthoptera available in Barcode of Life Data System (BOLD) and used 11 605 COI sequences, covering 1 132 species, 226 genera, and 18 families. The overall average PCI was 73.86%. For 82.2% of genera, the barcode gap boxplots were classified as “good” or “intermediate”, indicating that COI can be effective as a DNA barcode in Orthoptera, although with varying efficiency depending on the need for more information. ABGD and ASAP inferred species richness similar to labels informed by BOLD for the suborders Caelifera and Ensifera. The representation of Orthoptera in the BOLD database and the results of these analyses are discussed.

Integrative taxonomy is crucial for discovery, recognition, and species delimitation, especially in underestimated species complex or cryptic species, by incorporating different sources of evidence to construct rigorous species hypotheses. The spider genus Physocyclus Simon, 1893 (Pholcidae, Arteminae) is composed of 37 species, mainly from North America. In this study, traditional morphology was compared with three DNA barcoding markers regarding their utility in species delimitation within the genus: 1) Cytochrome c Oxidase subunit 1 (CO1), 2) Internal Transcribed Spacer 2 (ITS2), and 3) Ribosomal large subunit (28S). The molecular species delimitation analyses were carried out using four methods under the corrected p-distances Neighbor-Joining (NJ) criteria: 1) Automatic Barcode Gap Discovery (ABGD), 2) Assemble Species by Automatic Partitioning (ASAP), 3) General Mixed Yule Coalescent model (GMYC), and 4) Bayesian Poisson Tree Processes (bPTP). The analyses incorporated 75 terminals from 22 putative species of Physocyclus. The average intraspecific genetic distance (p-distance) was found to be < 2%, whereas the average interspecific genetic distance was 20.6%. The ABGD, ASAP, and GMYC methods were the most congruent, delimiting 26 or 27 species, while the bPTP method delimited 33 species. The use of traditional morphology for species delimitation was congruent with most molecular methods, with the male palp, male chelicerae, and female genitalia shown to be robust characters that support species-level identification. The barcoding with CO1 and 28S had better resolution for species delimitation in comparison with ITS2. The concatenated matrix and traditional morphology were found to be more robust and informative for species delimitation within Physocyclus.

The DNA barcode data of venomous cobra species (Naja naja and Naja kaouthia) are limited in the global database, especially from India and Bangladesh. Owing to the rapid success of DNA barcoding for discriminating a variety of species around the world, the present study aimed to generate the mitochondrial cytochrome c oxidase subunit I (COI) gene information of two morphologically identified deadly elapid species from the Mizoram state in northeast India and Rangpur in northern Bangladesh. The multiple species delimitation methods: Automatic Barcode Gap Discovery (ABGD), General Mixed Yule-Coalescent (GMYC), and Poisson-Tree-Processes (bPTP) revealed 14, 16, and 18 molecular operational taxonomic units (MOTUs) for 12 Naja species. The binocellate cobra, N. naja showed monophyletic clustering in both maximum-likelihood (ML) and Bayesian (BA) trees, single MOTU in ABGD and GMYC, and negligible intra-species genetic distance (0.2%) with two haplotypes. However, the monocellate cobra, N. kaouthia showed paraphyletic and polytomy in ML and BA phylogenies respectively; more than one MOTUs in ABGD, GMYC, and bPTP analyses; and sufficient intra-species genetic distances (0.6–2.3%) with five haplotypes related to the diverse geographical locations in Bangladesh, China, India, and Thailand. By superimposing the executed species delimitation criteria, the present molecular-based investigation concludes the presence of cryptic diversity of N. kaouthia in Indo-Bangladesh, China, and Thailand.

BackgroundThe subfamily Phlebotominae comprises 1028 species of sand fly, of which only 90 are recognized as vectors of pathogenic agents such as Trypanosoma, Leishmania, and Bartonella. In Thailand, leishmaniasis—a sand fly-borne disease—is currently endemic, with 36 documented sand fly species. However, many cryptic species likely remain unidentified. To improve our understanding of the distribution, habitat preferences, and role in disease transmission of these sand flies, further research is necessary.MethodsSand flies were collected using CDC light traps from 13 locations across four provinces in Thailand between October 2022 and October 2023. Initially, species identification was based on morphological characteristics, employing identification keys, and subsequently confirmed through mitochondrial cytochrome oxidase c subunit I (COI) and cytochrome b (Cytb) sequencing. Species identities were verified using BLASTN and BOLD searches. Species delimitation was conducted using Automatic Barcode Gap Discovery (ABGD) and Assemble Species by Automatic Partitioning (ASAP) with three substitution models. Additionally, intraspecific and interspecific genetic variation, neutrality tests (including Tajima’s and Fu and Li’s D* tests), phylogenetic analyses, and TCS haplotype network analysis were performed using the obtained sequences.ResultsA total of 3693 phlebotomine sand flies were collected, with 2261 (61.22%) identified as female. Integrative analyses combining morphological data, BLASTN searches, phylogenetic assessments, and species delimitation confirmed the identification of four genera: Sergentomyia, Grassomyia, Phlebotomus, and Idiophlebotomus, encompassing 12 species: Sergentomyia anodontis, Se. sylvatica, Se. perturbans, Se. barraudi, Se. hivernus, Se. khawi, Se. siamensis, Grassomyia indica, Phlebotomus barguesae, Ph. stantoni, Idiophlebotomus asperulus, and Id. longiforceps. Furthermore, molecular analysis revealed cryptic and complex species, including two putatively novel species, Se. sp. 1 and Se. sp. 2, as well as a unique haplotype.ConclusionsThis study, which integrated genetic and morphological identification techniques, identified 12 sand fly species and unveiled cryptic and complex species, including two putatively novel species (Se. sp. 1 and Se. sp. 2) and a unique haplotype. The findings underscore the utility of mitochondrial genes, combined with species delimitation methodologies, as reliable approaches for identifying diverse sand fly species.Graphical

The genetic and morphological variation within Rhyacodrilus falciformis Bretscher, 1901 (Clitellata: Naididae) in Europe was explored using an integrative approach, with three unlinked genetic markers [the mitochondrial cytochrome c oxidase subunit I (COI), the nuclear histone 3 (H3) and internal transcribed spacer region (ITS)] combined with morphology, to investigate whether this taxon constitutes a single or several species. Using Automatic Barcode Gap Discovery on the COI data set, the specimens were divided into seven clusters, used as hypothetical species that were further tested with the other data sources. Single‐gene trees were estimated for all three markers, using coalescence analysis and they were in many parts incongruent with each other. Only one of the clusters was supported by all trees; it was also morphologically differentiated from the other clusters by the shape of its modified penial chaetae. This group consists of two specimens from the Crotot Cave in south‐eastern France, and morphologically they fit a previously described but invalid variety, ‘pigueti’, which is here described as a new species, Rhyacodrilus pigueti Achurra &amp; Martinsson sp. n. The study highlights the fact that a single data source (e.g. COI barcodes) seldom provides a sufficient basis for taxonomic decisions such as species delimitation.

To date, five species of reddish-brown Neotriplax have been described, but their highly similar body color and other phenotypic traits make accurate taxonomy challenging. To clarify species-level taxonomy and validate potential new species, the cytochrome oxidase subunit I (COI) was used for phylogenetic analysis and the geometric morphometrics of elytron, pronotum, and hind wing were employed to distinguish all reddish-brown Neotriplax species. Phylogenetic results using maximum likelihood and Bayesian analyses of COI sequences aligned well with the current taxonomy of the Neotriplax species group. Significant K2P divergences, with no overlap between intra- and interspecific genetic distances, were obtained in Neotriplax species. The automatic barcode gap discovery (ABGD), assemble species by automatic partitioning (ASAP), and generalized mixed Yule coalescent (GMYC) approaches concurred, dividing the similar species into eight molecular operational taxonomic units (MOTUs). Geometric morphometric analysis using pronotum, elytron, hind wing shape and wing vein patterns also validated the classification of all eight species. By integrating these analytical approaches with morphological evidence, we successfully delineated the reddish-brown species of Neotriplax into eight species with three new species: N. qinghaiensis sp. nov., N. maoershanensis sp. nov., and N. guangxiensis sp. nov. Furthermore, we documented the first record of N. lewisii in China. This study underscores the utility of an integrative taxonomy approach in species delimitation within Neotriplax and serves as a reference for the taxonomic revision of other morphologically challenging beetles through integrative taxonomy.

The soft scales (Hemiptera: Coccoidea: Coccidae) are a group of sap-sucking plant parasites, many of which are notorious agricultural pests. The quarantine and economic importance of soft scales necessitates rapid and reliable identification of these taxa. Nucleotide sequences of the mitochondrial cytochrome c oxidase subunit I (COI) gene (barcoding region) and 28S rDNA were generated from 340 individuals of 36 common soft scales in China. Distance-based [(best match, Automated Barcode Gap Discovery (ABGD)], tree-based (neighbor-joining, Bayesian inference), Klee diagrams, and general mixed Yule coalescent (GMYC) models were used to evaluate barcoding success rates in the data set. Best match showed that COI and 28S sequences could provide 100 and 95.52% correct identification, respectively. The average interspecific divergences were 19.81% for COI data and 20.38% for 28S data, and mean intraspecific divergences were 0.56 and 0.07%, respectively. For COI data, multiple methods (ABGD, Klee, and tree-based methods) resulted in general congruence with morphological identifications. However, GMYC analysis tended to provide more molecular operational taxonomic units (MOTUs). Twelve MOTUs derived from five morphospecies (Rhodococcus sariuoni, Pulvinaria vitis, Pulvinaria aurantii, Parasaissetia nigra, and Ceroplastes rubens) were observed using the GMYC approach. In addition, tree-based methods showed that 28S sequences could be used for species-level identification (except for Ceroplastes ceriferus - Ceroplastes pseudoceriferus), even with low genetic variation (<1%). This report demonstrates the robustness of DNA barcoding for species discrimination of soft scales with two molecular markers (COI and 28S) and provides a reliable barcode library and rapid diagnostic tool for common soft scales in China.

DNA barcoding methods are potentially useful for the detection and identification of species difficult to identify using morphology-based taxonomy, such as the larvae of marine taxa. Combining barcoding methods with massively parallel sequencing and low-cost DNA library preparation improves the feasibility of processing several samples but limits the taxonomic resolution of resulting sequences. In light of these limitations, we looked for suitable barcode regions for echinoderm larvae, first by examining the cytochrome c oxidase subunit I (COI) region, the most commonly used barcode for echinoderms. Using a dataset containing over 4000 COI sequences representing nearly 400 echinoderm species across all five extant classes, the COI gene was found to lack a sufficiently conserved region for barcoding. Taxonomic resolution was also found lacking, with no clear barcode gap across classes and the results of Automatic Barcode Gap Discovery (ABGD) failing to reflect current taxonomic assignments. While sequences may be mislabeled, the aggregation of multiple species in one ABGD partition suggests that course-grained taxonomic resolution may still result from using COI alone. The search for short alternative mitochondrial regions across 110 genomes of distinct species using ecoPrimers software revealed that class-specific primers targeting 12S and 16S ribosomal RNA (rRNA) regions are prime candidates for barcoding echinoderm sequences. The results of in silico PCR across the 110 mitochondrial genomes indicate that compared to existing COI primers, the candidate 12S and 16S primers yield similar taxonomic coverage, with slightly lower resolution. Taking into consideration the previous analysis of COI, we suggest the use of multiple markers (COI, 12S, 16S) to adequately capture the understudied diversity of echinoderm larvae. In vitro PCR of the 12S and 16S primers is also needed to validate the results of the in silico analyses.

Accurate species delimitation is important, especially for endangered species. As one of the most conspicuous bivalve taxa, giant clams are threatened throughout their geographic range. Many phylogeographic studies have revealed strong population structure among giant clams in the Indo-Pacific, suggesting cryptic diversity within these species. However, less attention has been paid to their identification and delimitation. In this study, we assembled a comprehensive dataset of mitochondrial cytochrome c oxidase subunit I (COI) sequences for Tridacna species, focusing on new sequences from Hainan Island in the South China Sea and previously published ones from Japan, Taiwan, Singapore, the Philippines, Indonesia, Australia, the Solomon Islands and the Red Sea. Three nominal species, Tridacna crocea, T. squamosa and T. noae, were recognized at Hainan Island on the basis of distance-based DNA barcoding, with mean interspecific K2P distances of 10.6–24.7% for seven Tridacna species (T. crocea, T. squamosa, T. noae, T. maxima, T. mbalavuana, T. derasa and T. gigas). The most abundant species, T. noae, represents the first record of this species from Hainan Island. Using a combination of phylogenetic and DNA-based species delimitation analyses (automatic barcode gap discovery, generalized mixed Yule coalescent and Bayesian Poisson tree processes), we found strong support for a total of 13 operational taxonomic units (OTUs) for the seven nominal Tridacna species. These results, coupled with the fact that each OTU occupies different regions in the Indo-Pacific, strongly suggest multiple cryptic species of giant clams. Our findings point to the need for taxonomic revisionary work on giant clams throughout the Indo-Pacific; such work will have important conservation implications.

The subfamily Phlebotominae comprises important insects for public health. The use of complementary tools such as molecular taxonomy is necessary for interspecific delimitation and/or discovery of cryptic species. Here, we evaluated the DNA barcoding tool to identify different species in the southwestern Brazilian Amazon. For this, we collected sand flies in forest fragments along the highway BR-317, in the municipality of Brasiléia, state of Acre, Brazil. The specimens were DNA-barcoded using a fragment of the cytochrome c oxidase subunit I (COI) gene. The sequences were analyzed to generate K2P pairwise genetic distances and a Neighbour-joining tree. The sand fly barcodes were also clustered into Molecular Operation Taxonomic Units (MOTU) using Automatic Barcode Gap Discovery (ABGD) approach. A total of 59 COI sequences comprising 22 nominal species and ten genera were generated. Of these, 11 species had not been sequenced before, thus being new COI sequences to science. Intraspecific genetic distances ranged between 0 and 4.9%, with Pintomyia serrana presenting the highest values of genetic distance, in addition to having been partitioned into three MOTUs. Regarding the distances to the nearest neighbour, all species present higher values in relation to the maximum intraspecific distance, in addition to forming well supported clusters in the neighbour-joining analysis. The DNA barcoding approach is useful for the molecular identification of sand flies from Brasiléia, state of Acre, and was efficient in detecting cryptic diversity of five species which can be confirmed in future studies using an integrative approach. We also generated new COI barcodes for Trichophoromyia auraensis, Nyssomyia shawi, and Psychodopygus paraensis, which may play a role in the transmission of Leishmania spp. in the Brazilian Amazon.

South Africa harbors a rich and diverse terrestrial snail fauna. Despite their importance in ecosystem functioning and structure, their alpha taxonomic diversity is poorly understood, and most groups have not been the subject of molecular systematic scrutiny. During the present study, we used the mitochondrial cytochrome c oxidase subunit I (COI) locus to start a DNA barcode reference library, to examine intra‐ and interspecific genetic divergence, and to explore the presence of cryptic diversity among terrestrial gastropods from forested regions in the country. We generated 399 COI barcodes comprising 70 morphospecies in 26 genera and 15 families, representing nearly 10% of the described gastropod diversity in South Africa. Our results showed a substantial overlap between mean intraspecific and interspecific genetic variation and no barcoding gap. Three species delimitation methods—Poisson Tree Processes (PTP), Refined Single Linkage (RESL) algorithm in Barcode of Life Data Systems, and the Automatic Barcode Gap Discovery (ABGD)—applied to selected widespread species retrieved incongruent operational taxonomic units (OTUs). These results, coupled with high intraspecific variation, suggested cryptic diversity among several terrestrial gastropod genera in the country. We further demonstrated that DNA barcoding analysis holds the potential for highlighting cryptic lineages in snails and for revealing taxa in need of taxonomic revision. We recommend that future taxonomic studies of South African terrestrial snails incorporate multiple sources of information for species delimitation.

Cryptic diversity in supposedly species-poor genera of Enchytraeidae (Annelida: Clitellata)

Delimiting species of water mites of the genus Hydrodroma (Acari: Hydrachnidiae: Hydrodromidae) from North America and Europe: Integrative evidence of species status from COI sequences and morphology

DNA barcoding is used to identify cryptic species, survey environmental samples, and estimate phyletic and genetic diversity. Armored scale insects are phytophagous insects and are the most species-rich taxa in the Coccoidea superfamily. This study developed a DNA barcode library for armored scale insect species collected from southern China during 2021-2022. We sequenced a total of 239 specimens, recognized as 50 morphological species, representing two subfamilies and 21 genera. Sequencing analysis revealed that the average G + C content of the cytochrome oxidase subunit I (COI) gene sequence was very low (~18.06%) and that the average interspecific divergence was 10.07% while intraspecific divergence was 3.20%. The intraspecific divergence value was inflated by the high intraspecific divergence in ten taxa, which may indicate novel species overlooked by current taxonomic treatments. All the Automated Barcode Gap Discovery, Assemble Species by Automatic Partitioning, Taxon DNA analysis and Bayesian Poisson Tree Process methods yielded largely consistent results, indicating a robust and credible species delimitation. Based on these results, an intergeneric distance threshold of ≤ 5% was deemed appropriate for the differentiation of armored scale insect species in China. This study establishes a comprehensive barcode library for the identification of armored scale insects, future research, and application.

In modern systematics, different sources of evidence are commonly used for the discovery, identification, and delimitation of species, especially when morphology fails to delineate between species or in underestimated species complexes or cryptic species. In this study, morphological data and two DNA barcoding markers-cytochrome c oxidase subunit I (COI) and internal transcribed spacer 2 (ITS2)-were used to delimit species in the spider genus Loxosceles from North America. The molecular species delimitation analyses were carried out using three different methods under the corrected p-distance Neighbor-Joining (NJ) criteria: 1) Assemble Species by Automatic Partitioning (ASAP), 2) General Mixed Yule Coalescent model (GMYC), and 3) Bayesian Poisson Tree Processes (bPTP). The analyses incorporated 192 terminals corresponding to 43 putative species of Loxosceles, of which 15 are newly recognized herein, as putative new species, based on morphology and congruence between molecular methods with COI. The average intraspecific genetic distance (p-distance) was <2%, whereas the average interspecific genetic distance was 15.6%. The GMYC and bPTP molecular methods recovered 65-79 and 69 species respectively, overestimating the diversity in comparison with morphology, whereas the ASAP method delimited 60 species. The morphology of primary sexual structures (males palps and female seminal receptacles) was congruent with most of the molecular methods mainly with COI, showing that they are robust characters for identification at the species level. For species delimitation COI was more informative than ITS2. The diversity of Loxosceles species is still underestimated for North America, particularly in Mexico which holds the highest diversity of this genus worldwide.