Future of DNA-based insect monitoring.

Insects are crucial for ecosystem health but climate change and pesticide use are driving massive insect decline. To mitigate this loss, we need new and effective monitoring techniques. Over the past decade there has been a shift to DNA-based techniques. We describe key emerging techniques for sample collection. We suggest that the selection of tools should be broadened, and that DNA-based insect monitoring data need to be integrated more rapidly into policymaking. We argue that there are four key areas for advancement, including the generation of more complete DNA barcode databases to interpret molecular data, standardisation of molecular methods, scaling up of monitoring efforts, and integrating molecular tools with other technologies that allow continuous, passive monitoring based on images and/or laser imaging, detection, and ranging (LIDAR).

A new molecular phylogeny for the Tanypodinae (Diptera: Chironomidae) places the Australian diversity in a global context.

The non-biting midge subfamily Tanypodinae (Diptera: Chironomidae) is species-rich, ecologically diverse, and near-globally distributed. Within the subfamily, aspects of generic and species-level taxonomy remain poorly understood, in particular the validity of assignment of Australian and New Zealand taxa to genera erected for northern hemisphere (Holarctic) fauna. Here, we place the austral diversity within this global context by extensive geographical and taxonomic sampling in concert with a multilocus phylogenetic approach. We incorporated sequence data for mitochondrial COI, and nuclear 28S and CAD, and conducted Bayesian and maximum likelihood phylogenetic inferences and Bayesian divergence time estimation. The resolved phylogeny supported many associations of Australian taxa with their proposed Holarctic congeners, with the exception of Apsectrotanypus Fittkau, and validates several taxa as endemic. Three of four New Zealand sampled taxa had their sister groups in Australia; New Zealand Monopelopia Fittkau was sister to a German congener. This included the first record of Procladius Kieffer from New Zealand. Most nodes connecting austral and Holarctic taxa clustered around the Cretaceous-Tertiary boundary (60-80 mya), whereas New Zealand-Australia nodes were generally slightly younger (53-57 mya). Together, these data contribute substantially to our understanding of the taxonomy, systematics and biogeography of the Australian Tanypodinae and more broadly to knowledge of Australia's aquatic insect biodiversity.

Multi-marker DNA metabarcoding reflects tardigrade diversity in different habitats.

Like meiofauna in general, tardigrades are often neglected in ecological and environmental surveys. Tardigrades occur in all parts of the world, from deep marine sediments to alpine environments, and are present in most ecosystems. They are therefore potentially good candidates for biomonitoring programs. However, sampling of these minute animals is both tedious and time-consuming, impeding their inclusion in large-scale ecological surveys. In this study we argue that using a multi-marker metabarcoding approach on environmental DNA (eDNA) partly can overcome this barrier. Samples of moss, lichens, and leaf litter were investigated both by morphology-based methods and DNA metabarcoding, and the results were compared in terms of tardigrade diversity and community composition of the sampled microhabitats. DNA metabarcoding using three markers detected more species of tardigrades than identification by morphology in most samples. Also, metabarcoding detected the same community differences and microhabitat distribution patterns as morphology-based methods. In general, metabarcoding of litter samples was unreliable, with only one out of three markers consistently amplifying and detecting tardigrades. The low availability of tardigrade reference sequences in public databases restricts the taxonomic resolution in eDNA surveys, but this impediment is partly circumvented by utilizing multiple markers.

DNA barcode data reveal biogeographic trends in Arctic non-biting midges.

Chironomid flies (non-biting midges) are among the most abundant and diverse animals in Arctic regions, but detailed analyses of species distributions and biogeographical patterns are hampered by challenging taxonomy and reliance on morphology for species-level identification. Here we take advantage of available DNA barcode data of Arctic Chironomidae in BOLD to analyse similarities in species distributions across a northern Nearctic - West Palearctic gradient. Using more than 260 000 barcodes representing 4666 BINs (Barcode Index Numbers) and 826 named species (some with interim names) from a combination of public and novel data, we show that the Greenland chironomid fauna shows affinities to both the Nearctic and the West Palearctic regions. While raw taxon counts indicate a strong Greenland - North American affinity, comparisons using Chao's dissimilarity metric support a slightly higher similarity between Greenland and West Palearctic chironomid communities. Results were relatively consistent across different definitions of species taxonomic units, including morphologically determined species, BINs, and superBINs based on a ∼4.5% threshold. While most taxa found in Greenland are shared with at least one other region, reflecting circum-Arctic dispersal, our results also reveal that Greenland harbours a small endemic biodiversity. Our exploratory study showcases how DNA barcoding efforts using standardized gene regions contribute to an understanding of broad-scale patterns in biogeography by enabling joint analysis of public DNA sequence data derived from diverse prior studies.

A taxonomic revision of genus Labrundinia Fittkau, 1962 (Diptera: Chironomidae: Tanypodinae).

The species of the genus Labrundinia (Diptera: Chironomidae: Tanypodinae: Pentaneurini) are revised, described and figured. Keys to known adult males, pupae and larvae are provided. Fourteen previously known species are redescribed, and 25 species from Neotropical region are diagnosed and described as new to science. These species treated are L. amandae sp. n., L. amazonica sp. n., L. becki Beck et Beck, L. carolae sp. n., L. costaricae sp. n., L. fera Roback, L. fiorelinii sp. n., L. fittkaui sp. n., L. fosteri Roback, L. hirsuta Roback, L. gregi sp. n., L. jasoni sp. n., L. johannseni Beck et Beck, L. kogilamae sp. n., L. longipalpis Goetghebuer, L. mayaca sp. n., L. meta Roback, L. multidentata sp. n., L. nathani sp. n., L. neopilosella Beck et Beck, L. nicaraguensis sp. n., L. opela Roback, L. panamensis sp. n., L. parabecki Roback, L. parafittkaui sp. n., L. parareniformis sp. n., L. paravirescens sp. n., L. paulae sp. n., L. pilosella Loew, L. reniformis sp. n., L. robacki sp. n., L. semicurvata sp. n., L. setosa sp. n., L. separata Edwards, L. sofiae sp. n., L. tenata Roback, L. trilucida sp. n., L. unicolor sp. n., L. virescens Beck et Beck. A diagnosis of the genus is presented and morphological differences to closely related genera are discussed.

Global arthropod beta-diversity is spatially and temporally structured by latitude.

Global biodiversity gradients are generally expected to reflect greater species replacement closer to the equator. However, empirical validation of global biodiversity gradients largely relies on vertebrates, plants, and other less diverse taxa. Here we assess the temporal and spatial dynamics of global arthropod biodiversity dynamics using a beta-diversity framework. Sampling includes 129 sampling sites whereby malaise traps are deployed to monitor temporal changes in arthropod communities. Overall, we encountered more than 150,000 unique barcode index numbers (BINs) (i.e. species proxies). We assess between site differences in community diversity using beta-diversity and the partitioned components of species replacement and richness difference. Global total beta-diversity (dissimilarity) increases with decreasing latitude, greater spatial distance and greater temporal distance. Species replacement and richness difference patterns vary across biogeographic regions. Our findings support long-standing, general expectations of global biodiversity patterns. However, we also show that the underlying processes driving patterns may be regionally linked.

DNA barcodes provide insights into the diversity and biogeography of the non-biting midge Polypedilum (Diptera, Chironomidae) in South America.

South America, particularly within its tropical belt, is renowned for its unparalleled high levels of species richness, surpassing other major biomes. Certain neotropical areas harbor fragmented knowledge of insect diversity and face imminent threats from biodiversity loss and climate change. Hence, there is an urgent need for rapid estimation methods to complement slower traditional taxonomic approaches. A variety of algorithms for delimiting species through single-locus DNA barcodes have been developed and applied for rapid species diversity estimates across diverse taxa. However, tree-based and distance-based methods may yield different group assignments, leading to potential overestimation or underestimation of putative species. Here, we investigate the performance of different DNA-based species delimitation approaches to rapidly estimate the diversity of Polypedilum (Chironomidae, Diptera) in South America. Additionally, we test the hypothesis that significant differences exist in the community structure of Polypedilum fauna between South America and its neighboring regions, particularly the Nearctic. Our analysis encompasses a dataset of 1492 specimens from 598 locations worldwide, with a specific focus on South America. Within this region, we analyzed a subset of 247 specimens reported from 37 locations. Using various methods including the Barcode Index Number (BIN), Bayesian Poisson tree processes (bPTP), multi-rate Poisson tree processes (mPTP), single-rate Poisson tree processes (sPTP), and generalized mixed Yule coalescent (sGMYC), we identify molecular operational taxonomic units (MOTUs) ranging from 267 to 520. Our results indicate that the sGMYC method is the most suitable for estimating putative species in our dataset, resulting in the identification of 75 species in the Neotropical region, particularly in South America. Notably, this region exhibited higher species richness in comparison to the Palearctic and Oriental realms. Additionally, our findings suggest potential differences in species composition of Polypedilum fauna between the Neotropical and the adjacent Nearctic realms, highlighting high levels of endemism and species richness in the first. These results support our hypothesis that there are substantial differences exist in species composition between the Polypedilum fauna in South America and the neighboring regions.

BAGS: An automated Barcode, Audit & Grade System for DNA barcode reference libraries.

Biodiversity studies greatly benefit from molecular tools, such as DNA metabarcoding, which provides an effective identification tool in biomonitoring and conservation programmes. The accuracy of species-level assignment, and consequent taxonomic coverage, relies on comprehensive DNA barcode reference libraries. The role of these libraries is to support species identification, but accidental errors in the generation of the barcodes may compromise their accuracy. Here, we present an R-based application, Barcode, Audit & Grade System (BAGS) (https://github.com/tadeu95/BAGS), that performs automated auditing and annotation of cytochrome c oxidase subunit I (COI) sequences libraries, for a given taxonomic group of animals, available in the Barcode of Life Data System (BOLD). This is followed by implementing a qualitative ranking system that assigns one of five grades (A to E) to each species in the reference library, according to the attributes of the data and congruency of species names with sequences clustered in barcode index numbers (BINs). Our goal is to allow researchers to obtain the most useful and reliable data, highlighting and segregating records according to their congruency. Different tests were performed to perceive its usefulness and limitations. BAGS fulfils a significant gap in the current landscape of DNA barcoding research tools by quickly screening reference libraries to gauge the congruence status of data and facilitate the triage of ambiguous data for posterior review. Thereby, BAGS has the potential to become a valuable addition in forthcoming DNA metabarcoding studies, in the long term contributing to globally improve the quality and reliability of the public reference libraries.

Coverage and quality of DNA barcode references for Central and Northern European Odonata.

BACKGROUND: Dragonflies and damselflies (Odonata) are important components in biomonitoring due to their amphibiotic lifecycle and specific habitat requirements. They are charismatic and popular insects, but can be challenging to identify despite large size and often distinct coloration, especially the immature stages. DNA-based assessment tools rely on validated DNA barcode reference libraries evaluated in a supraregional context to minimize taxonomic incongruence and identification mismatches. METHODS: This study reports on findings from the analysis of the most comprehensive DNA barcode dataset for Central European Odonata to date, with 103 out of 145 recorded European species included and publicly deposited in the Barcode of Life Data System (BOLD). The complete dataset includes 697 specimens (548 adults, 108 larvae) from 274 localities in 16 countries with a geographic emphasis on Central Europe. We used BOLD to generate sequence divergence metrics and to examine the taxonomic composition of the DNA barcode clusters within the dataset and in comparison with all data on BOLD. RESULTS: Over 88% of the species included can be readily identified using their DNA barcodes and the reference dataset provided. Considering the complete European dataset, unambiguous identification is hampered in 12 species due to weak mitochondrial differentiation and partial haplotype sharing. However, considering the known species distributions only two groups of five species possibly co-occur, leading to an unambiguous identification of more than 95% of the analysed Odonata via DNA barcoding in real applications. The cases of small interspecific genetic distances and the observed deep intraspecific variation in Cordulia aenea (Linnaeus, 1758) are discussed in detail and the corresponding taxa in the public reference database are highlighted. They should be considered in future applications of DNA barcoding and metabarcoding and represent interesting evolutionary biological questions, which call for in depth analyses of the involved taxa throughout their distribution ranges.

Phylogenetic utility of five genes for dipteran phylogeny: a test case in the Chironomidae leads to generic synonymies.

In this study we examine the utility of three mitochondrial (COI, COII, 16S) and two nuclear (CAD and EF-1a) markers for estimating lower-level phylogenetic relationships within the dipteran family Chironomidae. As a test case we use species of the genus Micropsectra and the putatively closely related genera Krenopsectra, Parapsectra and Paratanytarsus. We also examine the phylogenetic evidence for the currently accepted species groups within the genus Micropsectra. In our results, highly variable EF-1a sequences within some species indicate the first find of paralogous gene copies in nematocerous Diptera. Among the other genes, COI is found to have the weakest while CAD contains the strongest phylogenetic signal. The resulting phylogeny displays a well-supported, but paraphyletic Micropsectra with regard to Krenopsectra acuta and five Parapsectra species, indicating taxonomic synonymy of these genera with 100% posterior probability. The genus Parapsectra is polyphyletic within Micropsectra while Paratanytarsus remains monophyletic although with low posterior probability. Micropsectra acuta, M. bumasta, M. fallax, M. nohedensis, M. mendli, M. uliginosa, M. chionophila, M. nana, M. styriaca and M. wagneri will all be new combinations as a consequence of the synonymy.

The Chironomidae (Diptera) of Svalbard and Jan Mayen.

Non-biting midges of the fly family Chironomidae are extremely abundant and diverse in Arctic regions and are essential components of Arctic ecosystems. Modern identification tools based on documented records of Arctic chironomid species are therefore important for ecological research and environmental monitoring in the region. Here, we provide an updated review of the chironomid fauna of the Svalbard archipelago and the island of Jan Mayen, Norway. Our results show that a total of 73 species distributed across 24 genera in four subfamilies are known from these areas. Our review treats 109 taxa, including nomina dubia and misidentifications. It includes morphological identification keys to all known species as well as photographs of most taxa and DNA barcodes of 66 species. Taxonomic remarks are given for selected taxa, including previous misidentifications and erroneous records. Chironomus islandicus, Tvetenia bavarica, Limnophyes schnelli, Metriocnemus brusti and Metriocnemus fuscipes as well as the genera Allocladius, Corynoneura and Bryophaenocladius are reported from Svalbard for the first time, while Procladius (Holotanypus) frigidus, Stictochironomus psilopterus, Chaetocladius incertus, Orthocladius (Orthocladius) mixtus and Smittia longicosta, previously considered as junior synonyms or nomina dubia, are revived as valid species based on examination of type material or literature. Twenty species within eleven genera are introduced with interim names. Metriocnemus similis is regarded as a junior synonym of Metriocnemus ursinus, and Smittia incerta, Smittia flexinervis and Smittia spitzbergensis are regarded as nomina dubia. Valid taxa no longer considered as part of the Svalbard fauna are Parochlus kiefferi, Arctopelopia barbitarsis, Procladius (Holotanypus) crassinervis, Diamesa lindrothi, Diamesa incallida, Diamesa lundstromi, Chironomus hyperboreus, Sergentia coracina, Camptocladius stercorarius, Chaetocladius dissipatus, Chaetocladius dentiforceps, Chaetocladius laminatus, Chaetocladius perennis, Cricotopus (Cricotopus) humeralis, Cricotopus (Cricotopus) polaris, Hydrosmittia ruttneri, Limnophyes edwardsi, Metriocnemus picipes, Metriocnemus tristellus, Orthocladius (Eudactylocladius) gelidus, Orthocladius (Euorthocladius) thienemanni, Orthocladius (Orthocladius) obumbratus, Orthocladius (Orthocladius) rhyacobius, Paralimnophyes, Paraphaenocladius impensus, Psectrocladius (Monopsectrocladius) calcaratus, Psectrocladius (Psectrocladius) psilopterus, Psectrocladius (Psectrocladius) ventricosus, Smittia lasiophthalma, Smittia lasiops and Zalutschia tatrica.

DNA metabarcoding adds valuable information for management of biodiversity in roadside stormwater ponds.

ABSTRACT: Stormwater ponds are used to compensate for the adverse effects that road runoff might have on the natural environment. Depending on their design and placement, stormwater ponds can act as both refugia and traps for local biodiversity. To evaluate the impact of stormwater ponds on biodiversity, it is critical to use effective and precise methods for identification of life associated with the water body. DNA metabarcoding has recently become a promising tool for identification and assessment of freshwater biodiversity.Using both morphology and DNA metabarcoding, we analyze species richness and biological composition of samples from 12 stormwater ponds and investigate the impact of pond size and pollution levels in the sediments and water column on the macroinvertebrate community structure.DNA metabarcoding captured and identified more than twice the number of taxa compared to morphological identification. The (dis)similarity of macroinvertebrate community composition in different ponds showed that the ponds appear better separated in the results obtained by DNA metabarcoding, but that the explained variation is higher for the results obtained by morphologically identification, since it provides abundance data.The reliance on morphological methods has limited our perception of the aquatic biodiversity in response to anthropogenic stressors, thereby providing inaccurate information for appropriate design and management of stormwater ponds; these drawbacks can be overcome by DNA metabarcoding. Synthesis and applications. The results indicate that DNA metabarcoding is a useful tool in identifying species, especially Diptera, which are difficult to determine. Application of DNA metabarcoding greatly increases the number of species identified at each sampling site, thereby providing a more accurate information regarding the way the ponds function and how they are affected by management. OPEN PRACTICES: This article has earned an Open Data Badge for making publicly available the digitally-shareable data necessary to reproduce the reported results. The data is available at https://www.ebi.ac.uk/ena/data/view/PRJEB30841.

DNA barcode reference libraries for the monitoring of aquatic biota in Europe: Gap-analysis and recommendations for future work.

Effective identification of species using short DNA fragments (DNA barcoding and DNA metabarcoding) requires reliable sequence reference libraries of known taxa. Both taxonomically comprehensive coverage and content quality are important for sufficient accuracy. For aquatic ecosystems in Europe, reliable barcode reference libraries are particularly important if molecular identification tools are to be implemented in biomonitoring and reports in the context of the EU Water Framework Directive (WFD) and the Marine Strategy Framework Directive (MSFD). We analysed gaps in the two most important reference databases, Barcode of Life Data Systems (BOLD) and NCBI GenBank, with a focus on the taxa most frequently used in WFD and MSFD. Our analyses show that coverage varies strongly among taxonomic groups, and among geographic regions. In general, groups that were actively targeted in barcode projects (e.g. fish, true bugs, caddisflies and vascular plants) are well represented in the barcode libraries, while others have fewer records (e.g. marine molluscs, ascidians, and freshwater diatoms). We also found that species monitored in several countries often are represented by barcodes in reference libraries, while species monitored in a single country frequently lack sequence records. A large proportion of species (up to 50%) in several taxonomic groups are only represented by private data in BOLD. Our results have implications for the future strategy to fill existing gaps in barcode libraries, especially if DNA metabarcoding is to be used in the monitoring of European aquatic biota under the WFD and MSFD. For example, missing species relevant to monitoring in multiple countries should be prioritized for future collaborative programs. We also discuss why a strategy for quality control and quality assurance of barcode reference libraries is needed and recommend future steps to ensure full utilisation of metabarcoding in aquatic biomonitoring.

Environmental DNA filtration techniques affect recovered biodiversity.

Freshwater metazoan biodiversity assessment using environmental DNA (eDNA) captured on filters offers new opportunities for water quality management. Filtering of water in the field is a logistical advantage compared to transport of water to the nearest lab, and thus, appropriate filter preservation becomes crucial for maximum DNA recovery. Here, the effect of four different filter preservation strategies, two filter types, and pre-filtration were evaluated by measuring metazoan diversity and community composition, using eDNA collected from a river and a lake ecosystem. The filters were preserved cold on ice, in ethanol, in lysis buffer and dry in silica gel. Our results show that filters preserved either dry or in lysis buffer give the most consistent community composition. In addition, mixed cellulose ester filters yield more consistent community composition than polyethersulfone filters, while the effect of pre-filtration remained ambiguous. Our study facilitates development of guidelines for aquatic community-level eDNA biomonitoring, and we advocate filtering in the field, using mixed cellulose ester filters and preserving the filters either dry or in lysis buffer.

Trace DNA from insect skins: a comparison of five extraction protocols and direct PCR on chironomid pupal exuviae.

Insect skins (exuviae) are of extracellular origin and shed during moulting. The skins do not contain cells or DNA themselves, but epithelial cells and other cell-based structures might accidentally attach as they are shed. This source of trace DNA can be sufficient for PCR amplification and sequencing of target genes and aid in species identification through DNA barcoding or association of unknown life stages. Species identification is essential for biomonitoring programs, as species vary in sensitivities to environmental factors. However, it requires a DNA isolation protocol that optimizes the output of target DNA. Here, we compare the relative effectiveness of five different DNA extraction protocols and direct PCR in isolation of DNA from chironomid pupal exuviae. Chironomidae (Diptera) is a species-rich group of aquatic macroinvertebrates widely distributed in freshwater environments and considered a valuable bioindicator of water quality. Genomic DNA was extracted from 61.2% of 570 sampled pupal exuviae. There were significant differences in the methods with regard to cost, handling time, DNA quantity, PCR success, sequence success and the ability to sequence target taxa. The NucleoSpin(®) Tissue XS Kit, DNeasy(®) Blood and Tissue kit, and QuickExtract(™) DNA Extraction Solution provided the best results in isolating DNA from single pupal exuviae. Direct PCR and DTAB/CTAB methods gave poor results. While the observed differences in DNA isolation methods on trace DNA will be relevant to research that focuses on aquatic macroinvertebrate ecology, taxonomy and systematics, they should also be of interest for studies using environmental barcoding and metabarcoding of aquatic environments.

Species delimitation in northern European water scavenger beetles of the genus Hydrobius (Coleoptera, Hydrophilidae).

The chiefly Holarctic Hydrobius species complex (Coleoptera, Hydrophilidae) currently consists of Hydrobius arcticus Kuwert, 1890, and three morphological variants of Hydrobius fuscipes (Linnaeus, 1758): var. fuscipes, var. rottenbergii and var. subrotundus in northern Europe. Here molecular and morphological data are used to test the species boundaries in this species complex. Three gene segments (COI, H3 and ITS2) were sequenced and analyzed with Bayesian methods to infer phylogenetic relationships. The Generalized Mixed Yule Coalescent (GMYC) model and two versions of the Bayesian species delimitation method BPP, with or without an a priori defined guide tree (v2.2 & v3.0), were used to evaluate species limits. External and male genital characters of primarily Fennoscandian specimens were measured and statistically analyzed to test for significant differences in quantitative morphological characters. The four morphotypes formed separate genetic clusters on gene trees and were delimited as separate species by GMYC and by both versions of BPP, despite specimens of Hydrobius fuscipes var. fuscipes and Hydrobius fuscipes var. subrotundus being sympatric. Hydrobius arcticus and Hydrobius fuscipes var. rottenbergii could only be separated genetically with ITS2, and were delimited statistically with GMYC on ITS2 and with BPP on the combined data. In addition, six or seven potentially cryptic species of the Hydrobius fuscipes complex from regions outside northern Europe were delimited genetically. Although some overlap was found, the mean values of six male genital characters were significantly different between the morphotypes (p < 0.001). Morphological characters previously presumed to be diagnostic were less reliable to separate Hydrobius fuscipes var. fuscipes from Hydrobius fuscipes var. subrotundus, but characters in the literature for Hydrobius arcticus and Hydrobius fuscipes var. rottenbergii were diagnostic. Overall, morphological and molecular evidence strongly suggest that Hydrobius arcticus and the three morphological variants of Hydrobius fuscipes are separate species and Hydrobius rottenbergii Gerhardt, 1872, stat. n. and Hydrobius subrotundus Stephens, 1829, stat. n. are elevated to valid species. An identification key to northern European species of Hydrobius is provided.

Ancient origin and maternal inheritance of blue cuckoo eggs.

Maternal inheritance via the female-specific W chromosome was long ago proposed as a potential solution to the evolutionary enigma of co-existing host-specific races (or 'gentes') in avian brood parasites. Here we report the first unambiguous evidence for maternal inheritance of egg colouration in the brood-parasitic common cuckoo Cuculus canorus. Females laying blue eggs belong to an ancient (∼2.6 Myr) maternal lineage, as evidenced by both mitochondrial and W-linked DNA, but are indistinguishable at nuclear DNA from other common cuckoos. Hence, cuckoo host races with blue eggs are distinguished only by maternally inherited components of the genome, which maintain host-specific adaptation despite interbreeding among males and females reared by different hosts. A mitochondrial phylogeny suggests that blue eggs originated in Asia and then expanded westwards as female cuckoos laying blue eggs interbred with the existing European population, introducing an adaptive trait that expanded the range of potential hosts.

A review of Norwegian Gymnometriocnemus (Diptera, Chironomidae) including the description of two new species and a new name for Gymnometriocnemusvolitans (Goetghebuer) sensu Brundin.

Examination of the syntypes of Metriocnemusvolitans Goetghebuer, 1940 revealed that these specimens belong to the genus Chaetocladius and are not con-specific with Gymnometriocnemusvolitans (Goetghebuer, 1940) sensu Brundin (1956) and Sæther (1983). A literature search showed that Gymnometriocnemuskamimegavirgus Sasa & Hirabayashi, 1993 fits well with the species figured and diagnosed by Brundin (1956) as well as with specimens of this species from Norway. We present arguments for Chaetocladiusvolitans (Goetghebuer) comb. n. and for the use of Gymnometriocnemuskamimegavirgus for Gymnometriocnemusvolitans sensu Brundin. In addition, we provide DNA barcode data that indicate the presence of at least seven Gymnometriocnemus species in Norway of which six are collected as male adults. Two of these, Gymnometriocnemus (Gymnometriocnemus) pallidussp. n. and Gymnometriocnemus (Raphidocladius) autumnalissp. n. are regarded as new to science and diagnosed based on adult male morphology and DNA barcodes. The species Gymnometriocnemus (Gymnometriocnemus) marionensis Sæther, 1969 is re-established and a key to all Holarctic species is provided.

Exploring Genetic Divergence in a Species-Rich Insect Genus Using 2790 DNA Barcodes.

DNA barcoding using a fragment of the mitochondrial cytochrome c oxidase subunit 1 gene (COI) has proven to be successful for species-level identification in many animal groups. However, most studies have been focused on relatively small datasets or on large datasets of taxonomically high-ranked groups. We explore the quality of DNA barcodes to delimit species in the diverse chironomid genus Tanytarsus (Diptera: Chironomidae) by using different analytical tools. The genus Tanytarsus is the most species-rich taxon of tribe Tanytarsini (Diptera: Chironomidae) with more than 400 species worldwide, some of which can be notoriously difficult to identify to species-level using morphology. Our dataset, based on sequences generated from own material and publicly available data in BOLD, consist of 2790 DNA barcodes with a fragment length of at least 500 base pairs. A neighbor joining tree of this dataset comprises 131 well separated clusters representing 121 morphological species of Tanytarsus: 77 named, 16 unnamed and 28 unidentified theoretical species. For our geographically widespread dataset, DNA barcodes unambiguously discriminate 94.6% of the Tanytarsus species recognized through prior morphological study. Deep intraspecific divergences exist in some species complexes, and need further taxonomic studies using appropriate nuclear markers as well as morphological and ecological data to be resolved. The DNA barcodes cluster into 120-242 molecular operational taxonomic units (OTUs) depending on whether Objective Clustering, Automatic Barcode Gap Discovery (ABGD), Generalized Mixed Yule Coalescent model (GMYC), Poisson Tree Process (PTP), subjective evaluation of the neighbor joining tree or Barcode Index Numbers (BINs) are used. We suggest that a 4-5% threshold is appropriate to delineate species of Tanytarsus non-biting midges.