DNA Barcode Data Research Articles

A new species of Autocrates (Coleoptera: Trictenotomidae) revealed by DNA barcoding and morphological evidence

Autocrates soni sp. nov. is described from Vietnam based on morphological and molecular evidence. This beetle is characterised by reddish-brown pubescence on its ventral side and much shorter setae on its femora compared to the closely related species Autocrates vitalisi Vuillet, 1912. The validity of this new species is strongly supported by DNA barcode data from all known species of the genus Autocrates.

Shedding light on some dark branches: The under‐appreciated diversity of gymnosome pteropods and their coiled thecosome prey in the Neotropics

AbstractHoloplanktonic organisms are thought to have enormous populations sizes, expansive geographic ranges, and low species diversity. Previous work suggests that pelagic gastropods are no exception to this generalization. However, most of these data are derived from temperate species, whereas the bulk of pteropod diversity occurs in the tropics. Here, we present DNA barcode data for COI focused on the under‐studied limacinoid, gymnosome, and pseudothecosome pteropods collected from the coastal waters of Panama. We applied four molecular species delimitation approaches to determine the number of operational taxonomic units (OTUs) identifiable from our sequences and all data available from Barcode of Life Database (BOLD) and GenBank and compared these to the Barcode Index Number generated by the BOLD. Assemble Species by Automatic Partitioning (ASAP), Poisson Tree Processes (PTP), Bayesian Poisson Tree Processes (bPTP), and Generalized Mixed Yule Coalescent (GMYC) methods generally gave congruent results and suggest that pteropod diversity in the tropics is significantly underrepresented both in DNA sequence databases and in the number of named OTUs. Of 42 specimens collected and sequenced, we recovered 16 OTUs, only one of which belonged to an OTU already represented in sequence databases. Neighbor‐joining trees including the previously published sequences show that in all three groups morphospecies can include genetically divergent lineages. Many very divergent (>15% from nearest neighbor) taxa are also still represented by only a single sequence, suggesting that there is a large amount of cryptic or pseudo‐cryptic diversity still to be described. To aid this future endeavor, we include some preliminary 16S data derived from new pteropod‐specific primers.

Massive citizen science sampling and integrated taxonomic approach unravel Danish cryptogam-dwelling tardigrade fauna

Tardigrade diversity and distribution are enigmatic in most parts of the globe, and only some European countries can boast of a relatively well-studied water bear fauna. However, even these suffer from the lack of genetic data, which would substantiate faunistic data and make biogeographic comparisons easier. Denmark has never been intensively and systematically researched in this regard, thus a citizen science sampling of cryptogams (mosses, liverworts, and lichens) was launched in spring 2023, aiming at a comprehensive biodiversity survey across this insular country. Nearly 700 samples were selected out of 8.000 sent to NHMD, based on the quality of samples, representativeness of various regions of Denmark, and the type of substrate to allow unravelling of potential ecological associations between tardigrades and cryptogams. Importantly, a large fraction of morphological identifications was backed up by DNA barcode data based on ITS-2 (1001 sequences), and in some cases also on COI (93 sequences) and ITS-1 (22 sequences) molecular markers, which are recognised DNA fragments used in species delimitation. We quadruple the number of known Danish limno-terrestrial tardigrade species (55 spp. reported in this paper vs. 14 spp. reported in literature so far, most of which were contentious due to the insufficient knowledge on tardigrade taxonomy), demonstrating the power of integrative taxonomy. No fewer than nine spp. are new to science. This is the first case where tardigrade fauna of an entire country is examined both from morphological and DNA barcoding data perspective.

On the identity of the Afrotropical species of Mallota Meigen (Diptera: Syrphidae)

The Afrotropical hoverflies belonging to the genus Mallota Meigen, 1822 (Diptera: Syrphidae) are revised. Ten species are recognized, of which four are new to science: Mallota glabra sp. nov., M. hircus sp. nov., M. wyatti sp. nov. and M. stipulata sp. nov. Merodon edentulus Macquart, 1855 is considered a junior synonym of Eristalis dasyops Wiedemann, 1819. Lectotypes are designated for Eristalis dasyops, Merodon edentulus, Mallota pachymera Bezzi, 1915 and Helophilus extremus Loew, 1858. The taxonomic history of the placement and identity of the different species of Mallota is presented. Their relationships, as well as affiliations with Afrotropical representatives of the genera Eristalis Latreille, 1804 and Myathropa Rondani, 1845, is briefly discussed based on morphological and DNA barcode data.

Charting a future for entomological taxonomy in New Zealand

ABSTRACT There is an increasing need for the identification and monitoring of insects. However, funding for entomological taxonomy, which traditionally provides the knowledge and tools for insect identification is in decline. Here, I review the history of entomological taxonomy in New Zealand and argue that the funding decline is a consequence of two broad shifts in biology. First, taxonomists test hypotheses in a qualitative manner unlike much of biology which has become increasingly quantitative. Second, there has been a shift in biology away from studying patterns and towards understanding processes and mechanisms. These scientific transformations have left the practice of taxonomy intellectually isolated from much of modern biology and led to its devaluing. I advocate for broadening taxonomy beyond the Linnaean system and to include genetic and genomic methods for identifying evolutionary lineages. This New Zealand Tree of Life would be comprised of ‘preliminary species hypotheses’ that can later be robustly tested with diverse character data and incorporated into the Linnean classification if resources and capability become available. Preliminary species hypotheses as well as associated genomic, DNA barcode, and specimen image data will support portable technologies that allow anyone to identify insects for environmental monitoring and biosecurity. This democratisation of insect identification has the potential to help build support for collections and taxonomic research. A concerted effort to accelerate the digitisation of collection data, especially genomic, DNA barcode, and specimen image data, will be necessary for implementation.

Heterogeneous microbiomes associate with shell-boring Polydora hoplura (Polychaeta, Spionidae) affecting the flat oyster Ostrea edulis

Shell-boring polychaetes belonging to the genus Polydora (Spionidae) infect commercially harvested bivalve molluscs worldwide, reducing their economic value. Due to their limited intraspecific variability, taxonomic identification becomes challenging without molecular approaches. In this study, we identified Polydora hoplura worms infecting flat oysters (Ostrea edulis) in the Gulf of Naples (Italy), providing the first DNA barcode data on the cytochrome c oxidase subunit 1 mitochondrial gene (COI) from its type locality. Three haplotypes with up to ten mutational step differences were detected, suggesting recent introductions from distant populations, including South Africa. The characterization of Polydora associated prokaryotic communities revealed a diverse and fluctuating microbiome linked to these worms, deviating from the usual species-specific pattern observed in other invertebrates. Core and some non-core bacteria could be playing symbiotic roles in nutrient provision, removal of waste products, and antioxidant and detoxifying processes to allow endolithic lifestyle within the borrowed valve chambers. The majority of these microbes though, seem to be acquired horizontally, from an interconnection between the environment and the flat oyster cavity. Indeed, there was a notable detection of opportunistic and potentially pathogenic taxa clusters, including Mycobacterium, Vibrio, Aliiroseovarius and Halarcobacter spp., which in concomitance with the prevalent polydorid infections may prompt implications for the health of the flat oyster host, and for its commercial market, including human consumption. Our study is novel in characterizing bacterial communities associated with shell-boring polychaetes, and sets the bases to propose these parasitic worms as vectors of alterable microbial exchange between oyster hosts and the environment.

Complementary studies of the Perlidae (Insecta: Plecoptera) fauna from the Paranapiacaba Mountains using DNA barcode data

The Paranapiacaba Mountains are a region of Brazil where the stonefly fauna is relatively well known. Despite this, there are still gaps in knowledge that need to be filled. In this study, through field sampling and molecular analyses, we have updated the taxonomic knowledge of Perlidae species in this region. Our study employed DNA barcoding methods to complement traditional morphological approaches, facilitating the association and description of the nymphs of Anacroneuria itajaimirim Bispo & Froehlich 2004. Additionally, we generated DNA barcode sequences for Anacroneuria iporanga Bispo & Froehlich 2004. Our results contributed to knowledge about Perlidae from the Paranapiacaba Mountains, highlighting the importance of DNA barcode data in the association of immature stages and species delimitations. Although the stoneflies of Paranapiacaba Mountains have been relatively well-studied, our results reveal that we still have deficits in knowledge of the fauna of this region.

MycoAI: Fast and accurate taxonomic classification for fungal ITS sequences.

Efficient and accurate classification of DNA barcode data is crucial for large-scale fungal biodiversity studies. However, existing methods are either computationally expensive or lack accuracy. Previous research has demonstrated the potential of deep learning in this domain, successfully training neural networks for biological sequence classification. We introduce the MycoAI Python package, featuring various deep learning models such as BERT and CNN tailored for fungal Internal Transcribed Spacer (ITS) sequences. We explore different neural architecture designs and encoding methods to identify optimal models. By employing a multi-head output architecture and multi-level hierarchical label smoothing, MycoAI effectively generalizes across the taxonomic hierarchy. Using over 5 million labelled sequences from the UNITE database, we develop two models: MycoAI-BERT and MycoAI-CNN. While we emphasize the necessity of verifying classification results by AI models due to insufficient reference data, MycoAI still exhibits substantial potential. When benchmarked against existing classifiers such as DNABarcoder and RDP on two independent test sets with labels present in the training dataset, MycoAI models demonstrate high accuracy at the genus and higher taxonomic levels, with MycoAI-CNN being the fastest and most accurate. In terms of efficiency, MycoAI models can classify over 300,000 sequences within 5 min. We publicly release the MycoAI models, enabling mycologists to classify their ITS barcode data efficiently. Additionally, MycoAI serves as a platform for developing further deep learning-based classification methods. The source code for MycoAI is available under the MIT Licence at https://github.com/MycoAI/MycoAI.

Larval Taxonomy of the Caddisfly Cernotina truncona Ross, 1947 (Trichoptera: Polycentropodidae)

The genus Cernotina Ross, 1938 is represented in the southeastern United States by three nominal species: Cernotina calcea Ross, 1938, Cernotina spicata Ross, 1938, and Cernotina truncona Ross, 1947. Of all Cernotina species, only the larva of C. spicata has been described to date. The goal of this paper is to describe, illustrate, and diagnose the larva of C. truncona using ecologically associated specimens. In addition, we used publicly available mitochondrial DNA barcoding data to evaluate the genetic relationships of these species. The larvae of Cernotina truncona can be distinguished from those of C. spicata by differences in setal placement and number on the meso- and metanota, mandibular morphology, head width, and distal setation of the tarsi. The ultrastructure of the anal claw is figured, highlighting the novel finding of small spines on the concave margin of a larva of Cernotina. With this new description, just the second described larva from this genus, only C. calcea remains unknown in the southeastern United States. The information provided herein enables the in-depth study of the ecology and life history of this diminutive caddisfly.

First record of the sap beetle Phenolia picta invading Eastern honey bee (Apis cerana) (Hymenoptera, Apidae) colonies

In Asia, the sap beetle Phenolia (Lasiodites) picta (Macleay) (Coleoptera, Nitidulidae) (hereafter P. picta) is endemic to the southeastern Palaearctic and northeastern Oriental regions. It is commonly found in orchards and plantations, where it feeds on ripe or rotten fruits. Here, P. picta was recorded for the first time to invade the hive of the Eastern honey bee (Apis cerana). Both DNA barcode and morphological data were used to identify the beetle, with the DNA barcoding comparison to the P. picta sequence in the BOLD library reaching 100% similarity. Additionally, the body sizes were measured, with the males averaging 7.87 ± 0.21 mm in length and the females averaging 8.27 ± 0.15 mm. The beetle’s body colour is primarily brownish with yellowish to pale orange spots on the elytra. P. picta invades the honey bee hive, feeds on honey and bee pupae, and tunnels through the comb, which causes the honey bees to abandon their hive. Such invasions could have severe impacts on honey bee colonies. This report provides essential information for beekeepers to use when identifying P. picta, which could be useful for preventing and controlling parasitic beetles.

DNA barcoding, visual-guide resource, new localities and host associations of genus Periglischrus Oudemans, 1902 (Acari: Mesostigmata, Spinturnicidae) from Minas Gerais, Brazil

The family Spinturnicidae (Oudemans, 1902) comprises hematophagous mites found exclusively on bats. In this article, we present DNA barcodes (mitochondrial Cytochrome c Oxidase I) for 71 specimens morphologically assigned to six spinturnicid species from 34 bat specimens (11 species) obtained from 10 caves and one forest fragment in karst areas from Minas Gerais state, southeast Brazil. A visual guide with diagnostic characters for Periglischrus acutisternus Machado-Allison, 1964, P. caligus Kolenati, 1857, P. herrerai Machado-Allison, 1965, P. iheringi Oudemans, 1902, P. paravargasi Herrin and Tipton, 1975, P. torrealbai Machado-Allison, 1965 is provided. Along DNA barcode data, photo-documentation of vouchering material of each individual sequenced have been deposited at BOLD (Barcoding of Life Data System v4) database. Periglischrus herrerai, P. caligus and P. paravargasi are reported for the first time in Minas Gerais state, extending their distribution ranges.

A review of the Cochylimorpha perfusana (Guenée, 1845) species group (Lepidoptera, Tortricidae) in Europe, with the description of a new species from the Southern Carpathians (Romania)

European species of the Cochylimorpha perfusana (Guenée, 1845) species group are revised. Four species are recognized, all closely related to C. perfusana (Guenée, 1845) with which they have previously been confused. Based on morphological differences and DNA barcode data, C. callosana (Herrich-Schäffer, 1856), sp. rev. and C. dorsimaculana (Preissecker, 1908), sp. rev., are re-instated to their original species rank. Inconsistencies in their taxonomy leading to their synonymy are shown and corrected. Cochylimorpha bucegiana Z. Kovács, S. Kovács & P. Buchner, sp. nov. is described from the subalpine meadows of the Bucegi and Făgăraș Mountains (Southern Carpathians, Romania), and the other three species are redescribed. Adults and genitalia of both sexes of all four, and the habitat of three taxa are figured. The geographic distribution of each species is re-evaluated based on the examined material and a critical analysis of the literature. Literature data for which no material could be found for examination are treated as doubtful and in need of confirmation. Cochylimorpha perfusana is widely distributed in mountain areas of Europe (Alps, Carpathians and Pirin Mountains), C. callosana is widespread only in the north-western Balkan Peninsula and north-eastern Italy, and local in eastern France, Corsica and Hungary, the other two seem to be endemics, C. dorsimaculana to Wachau and Retz in Lower Austria and C. bucegianasp. nov. to the Southern Carpathians in Romania. Cochylimorpha perfusana is recorded for the first time from Bulgaria; the first confirmed records of C. callosana from Slovenia and Hungary are given; and in the north-eastern Italian fauna C. callosana replaces C. perfusana. Records of C. perfusana from Ukraine, the Czech Republic, Slovenia, and north-western Italy require confirmation.

A DNA barcoding framework for taxonomic verification in the Darwin Tree of Life Project.

Biodiversity genomics research requires reliable organismal identification, which can be difficult based on morphology alone. DNA-based identification using DNA barcoding can provide confirmation of species identity and resolve taxonomic issues but is rarely used in studies generating reference genomes. Here, we describe the development and implementation of DNA barcoding for the Darwin Tree of Life Project (DToL), which aims to sequence and assemble high quality reference genomes for all eukaryotic species in Britain and Ireland. We present a standardised framework for DNA barcode sequencing and data interpretation that is then adapted for diverse organismal groups. DNA barcoding data from over 12,000 DToL specimens has identified up to 20% of samples requiring additional verification, with 2% of seed plants and 3.5% of animal specimens subsequently having their names changed. We also make recommendations for future developments using new sequencing approaches and streamlined bioinformatic approaches.

New evidence on the identity of the European Helorus species (Hymenoptera, Proctotrupoidea, Heloridae).

Species of Helorus Latreille 1802 are rarely collected endoparasitoids of Chrysopidae larvae (Neuroptera). Previous work on the limits between the European species of this species-poor genus, based on morphology only, has left some uncertainties. Here, we approach these cases and revisit previous taxonomic decisions using freshly collected and museum material. We generated the first large-scale Heloridae DNA barcode dataset, combined these with morphological data in an integrative taxonomic approach, and added information from studying all relevant type material. We found five species, Helorusanomalipes (Panzer, 1798), H.coruscus Haliday, 1857 stat. rev., H.nigripes Förster, 1856, H.ruficornis Förster, 1856, and H.striolatus Cameron, 1906, for which we provide an updated identification key. DNA barcode data are added to publicly available DNA barcode reference databases, for all species, except H.nigripes.

The obligate fig-pollinator family Agaonidae in Germany (Hymenoptera, Chalcidoidea)

All native and many cultivated fig plants are pollinated by representatives of the family Agaonidae (fig wasps), which are specialised, secondarily phytophagous relatives of parasitoid wasps that evolved an obligate mutualism with fig trees. So far, distribution of Agaonidae in Europe has been limited to southern, mostly Mediterranean areas, for example, in Greece, Croatia, Hungary, Italy, France, Spain and Portugal. Here, we report the first four records of the family for Germany, all in the form of the widespread species Blastophaga psenes (Linnaeus, 1758). New verified records are from three States in western and south-western Germany, Baden-Wuerttemberg (Radolfzell at Lake Constance and Sasbach am Kaiserstuhl near Freiburg), Saarland (Saarbrücken) and Northrhine-Westalia (Bochum) and all are based on citizen-scientist observations and collections. The new records are considerably more northern than previously recorded localities and, strikingly, geographically distant from these. All records can be attributed to the presence of large male caprifig trees (Ficus carica L. var. caprificus), whose three generations of fruits host the development stages of Blastophaga psenes. We generated DNA barcode data of specimens from three localities and added them to the national GBOL (German Barcode of Life) database and the international Barcode of Life database (BOLD). The somewhat surprising occurrence of the species/family in Germany might be attributable to increasing temperatures as a result of global warming, but this needs further investigation. Additionally, the presence of fig wasps, assuming it stabilises, could offer new opportunities for fig farming in Germany.

Cryptic or underworked? Taxonomic revision of the Antistrophus rufus species complex (Cynipoidea, Aulacideini)

Cryptic species present challenges across many subdisciplines of biology. Not all “cryptic” species, however, are truly cryptic; many are simply underexplored morphologically. We examined this idea for the Antistrophus rufus species complex, which previously contained three species thought to be morphologically cryptic. To determine whether the A. rufus complex are truly cryptic species, we assessed species boundaries of members of the A. rufus species complex using morphological, ecological, and DNA barcode data, and tested whether a set of 50 morphological characters could adequately diagnose these species. We revealed that this complex includes five species, and that there are useful phenotypic diagnostic characters for all members of this species complex. This enabled redescription of four species and the description of Antistrophus laurenae Nastasi, sp. nov., which induces externally inconspicuous galls in stems of Silphium integrifolium Michx., a host not associated with other members of the complex. We use these new diagnostic characters to construct a key to the five species of the rufus complex. We conclude that the A. rufus complex was not a true case of cryptic species. Our Bayesian analysis of DNA barcode data suggests possible cospeciation of members of the rufus complex and their Silphium host plants, but further study is necessary to better understand the evolution of host use in the lineage.

Characterising the north-western European species of Phaenoglyphis Förster, 1869 (Hymenoptera: Figitidae: Charipinae) with novel insights from DNA barcode data.

The taxonomy of the hymenopteran parasitoid subfamily Charipinae (Hymenoptera: Cynipoidea: Figitidae) has, until recently, been in a state of chaos. While this situation has improved significantly in recent years, most of the efforts were focused on morphological data of typically old specimens. Here, we present the first integrative approach to describe the diversity of the genus Phaenoglyphis Förster, 1869 from north-western Europe. For seven (of a total of 17) species, we provide DNA barcode data. Phaenoglyphisbelizini Pujade-Villar, 2018 and Phaenoglyphisevenhuisi Pujade-Villar & Paretas-Martínez, 2006 are recorded for the first time from Germany. All DNA barcodes and specimen data were added to the publicly available GBOL and BOLD reference database. The presence of a 6 bp long deletion in the CO1 barcode region that is characteristic to the genus and unique amongst Figitidae supports the monophyly of Phaenoglyphis.

Island mosquitoes of Thailand: an update on species diversity and DNA barcoding.

Mosquitoes (Diptera: Culicidae) are among the most medically significant insects, with several species acting as vectors for human pathogens. Although there are frequent reports of mosquito-borne diseases in the border island areas of Thailand, comprehensive data on the diversity and DNA barcoding of these mosquito species remain limited. This study investigated mosquito diversity in two main archipelagos in Thailand-the Trat archipelago (comprising Chang Island and Kood Island) and the Ranong archipelago (comprising Chang Island and Phayam Island)-and generated DNA barcode data from the mosquitoes found there. The survey across these islands discovered a total of 41 species, highlighting the presence of several species known to be vectors for human diseases. Thirty-seven mosquito species from the island areas were documented to provide reference DNA barcode sequences for mosquitoes in Thailand's island regions. Two species, Aedes fumidus and Finlaya flavipennis, have been added as new COI sequence records in the database. DNA barcoding was highly effective in classifying almost all species by identifying barcoding gaps, except for Anopheles baimaii and Anopheles dirus, which could not be distinguished. Additionally, the study noted that geographical variations might influence certain mosquito species, such as Anopheles barbirostris A3 and Mansonia dives, causing them to be split into two distinct subgroups. The findings of this study are crucial, as they aid in classifying mosquito species using molecular techniques and expand our knowledge of disease vectors in these biodiverse regions.

Detailed DNA barcoding of mayflies in a small European country proved how far we are from having comprehensive barcode reference libraries.

Mayflies (Ephemeroptera) are among the crucial water and habitat quality bioindicators. However, despite their intensive long-term use in various studies, more reliable mayfly DNA barcode data have been produced in a negligible number of countries, and only ~40% of European species had been barcoded with less than 50% of families covered. Despite being carried out in a small area, our study presents the second-most species-rich DNA reference library of mayflies from Europe and the first comprehensive view from an important biodiversity hotspot such as the Western Carpathians. Within 1153 sequences, 76 morphologically determined species were recorded and added to the Barcode of Life Data System (BOLD) database. All obtained sequences were assigned to 97 BINs, 11 of which were unique and three represented species never barcoded before. Sequences of 16 species with high intraspecific variability were divided into 40 BINs, confirming the presence of cryptic lineages. Due to the low interspecific divergence and the non-existing barcoding gap, sequences of six species were assigned to three shared BINs. Delimitation analyses resulted in 79 and 107 putative species respectively. Bayesian and maximum-likelihood phylogenies confirmed the monophyly of almost all species and complexes of cryptic taxa and proved that DNA barcoding distinguishes almost all studied mayfly species. We have shown that it is still sufficient to thoroughly investigate the fauna of a small but geographically important area to enrich global databases greatly. In particular, the insights gained here transcend the local context and may have broader implications for advancing barcoding efforts.

Revised description of the blueberry bud mite, Acalitus vaccinii (Acari: Trombidiformes: Eriophyidae), and a key to all Eriophyoidea on Vaccinium

In 2014 the blueberry bud mite, Acalitus vaccinii (Acari: Trombidformes: Eriophyidae), was detected in the Mpumalanga province of South Africa for the first time causing significant damage to cultivated blueberries. This was the first detection of this pest outside of North America, to which it is native. However, its taxonomic description at that time lacked critical detail and omitted characters and life stages important for easy and accurate identification. Using an integrative taxonomic approach, we combined phase contrast light microscopy with low-temperature SEM and DNA barcoding data to revise the description of A. vaccinii using South African specimens. Additional characters not included in previous descriptions but reported here are the h1 (accessory) setae, leg I & II u’ (mesal) setae, and leg II bv (femoral) setae. Detailed descriptions and measurements of all life stages are included, along with a discussion of morphological variation and biology. Two DNA sequences of common barcode regions (nuclear and mitochondrial) are provided to further aid in identification. In addition, a key to all known species of eriophyoid mites present on Vaccinium is provided.