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.

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.

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.

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.

Pentaneurellakatterjokki Fittkau & Murray (Chironomidae, Tanypodinae): redescription and phylogenetic position.

The monotypic genus Pentaneurella Fittkau & Murray was originally described based on larvae, pupal exuviae and pharate males. The latter prevented the observation of key features, such as wing dimensions, abdominal coloration pattern, and hypopygial apodemes (sternapodeme and phallapodeme), and the description of the adult male was considered incomplete by the authors. Herein, the adult female of Pentaneurellakatterjokki is described for the first time, and the adult male, pupa and larva are redescribed and figured based on specimens recently collected in Germany and Norway. We also discuss the phylogenetic position of Pentaneurella.

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.

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.

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.

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.

When DNA barcoding and morphology mesh: Ceratopogonidae diversity in Finnmark, Norway.

DNA barcoding in Ceratopogonidae has been restricted to interpreting the medically and veterinary important members of Culicoides Latreille. Here the technique is utilised, together with morphological study, to interpret all members of the family in a select area. Limited sampling from the county of Finnmark in northernmost Norway indicated the presence of 54 species, including 14 likely new to science, 16 new to Norway, and one new to Europe. No species were previously recorded from this county. Only 93 species were known for all of Norway before this survey, indicating how poorly studied the group is. We evaluate and discuss morphological characters commonly used in identification of biting midges and relate species diagnoses to released DNA barcode data from 223 specimens forming 58 barcode clusters in our dataset. DNA barcodes and morphology were congruent for all species, except in three morphological species where highly divergent barcode clusters indicate the possible presence of cryptic species.

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.

A comprehensive DNA sequence library is essential for identification with DNA barcodes.

In this study we examine the possibility of utilising partial cox1 gene sequences as barcodes to identify non-biting midges (Diptera: Chironomidae). We analysed DNA from 97 specimens of 47 species in the genera Cladotanytarsus, Micropsectra, Parapsectra, Paratanytarsus, Rheotanytarsus, Tanytarsus and Virgatanytarsus with a main focus on Micropsectra, Parapsectra and Paratanytarsus. Our findings show that (1) cox1 is easily amplified from extracts from different life stages with the standard barcoding primers. (2) Although K2P-distances between con-specific sequences varied up to 4.9%, con-specifics clustered together with 91-100% bootstrap support in maximum parsimony analysis. This indicates that barcodes may be excellent tools to identify species that are already in a cox1 library. (3) Both neighbour joining and maximum parsimony failed to reconstruct monophyletic genera. Thus, if a well-matching cox1 sequence is not already available in the library, the prospects of approximately identifying an unknown taxon, even to the correct genus of subtribe Tanytarsina, are not good.