Mitogenomes have been widely used for phylogenetic reconstruction of various Dipteran groups, but specifically for chironomid, they have not been carried out to resolve the relationships. Diamesinae (Diptera: Chironomidae) are important bioindicators for freshwater ecosystem monitoring, but its evolutionary history remains uncertain for lack of information. Here, coupled with one previously published and 30 new mitogenomes of Diamesinae, we carried out comparative mitogenomic analysis and phylogenetic analysis. Mitogenomes of Diamesinae were conserved in structure, and all genes arranged in the same order as the ancestral insect mitogenome. All protein‐coding genes in Diamesinae were under stronger purifying selection than those of other nonbiting midge species, which may exhibit signs of adaptation to life at cold living conditions. Phylogenetic analyses strongly supported the monophyly of Diamesinae, with Boreheptagyiini deeply nested within Diamesini. In addition, phylogenetic relationship of selected six genera was resolved, except Sympotthastia remained unstable. Our study revealed that the mitogenomes of Diamesinae are highly conserved, and they are practically useful for phylogenetic inference.

Over the past decade, molecular approaches to species delimitation have seen rapid development. However, species delimitation based on a single locus, for example, DNA barcodes, can lead to inaccurate results in cases of recent speciation and incomplete lineage sorting. Here, we compare the performance of Automatic Barcode Gap Discovery (ABGD), Bayesian Poisson tree processes (PTP), networks, generalized mixed Yule coalescent (GMYC) and Bayesian phylogenetics and phylogeography (BPP) models to delineate cryptic species previously detected by DNA barcodes within Tanytarsus (Diptera: Chironomidae) non‐biting midges. We compare the results from analyses of one mitochondrial (cytochrome c oxidase subunit I [COI]) and three nuclear (alanyl‐tRNA synthetase 1 [AATS1], carbamoyl phosphate synthetase 1 [CAD1] and 6‐phosphogluconate dehydrogenase [PGD]) protein‐coding genes. Our results show that species delimitation based on multiple nuclear DNA markers is largely concordant with morphological variation and delimitations using a single locus, for example, the COI barcode. However, ABGD, GMYC, PTP and network models led to conflicting results based on a single locus and delineate species differently than morphology. Results from BPP analyses on multiple loci correspond best with current morphological species concept. In total, 10 lineages of the Tanytarsus curticornis species complex were uncovered. Excluding a Norwegian population of Tanytarsus brundini which might have undergone recent hybridization, this suggests six hitherto unrecognized species new to science. Five distinct species are well supported in the Tanytarsus heusdensis species complex, including two species new to science.

Background: The mitochondrial genome (mitogenome) has been extensively used for phylogenetic and evolutionary analysis in Diptera, but the study of mitogenome is still scarce in the family Chironomidae. Methods: Here, the first complete mitochondrial genomes of four Chironomid species representing Diamesinae, Orthocladiinae, Prodiamesinae and Tanypodinae are presented. Coupled with published mitogenomes of two, a comparative mitochondrial genomic analysis between six subfamilies of Chironomidae was carried out. Results: Mitogenomes of Chironomidae are conserved in structure, each contains 37 typical genes and a control region, and all genes arrange the same gene order as the ancestral insect mitogenome. Nucleotide composition is highly biased, the control region displayed the highest A + T content. All protein coding genes are under purifying selection, and the ATP8 evolves at the fastest rate. In addition, the phylogenetic analysis covering six subfamilies within Chironomidae was conducted. The monophyly of Chironomidae is strongly supported. However, the topology of six subfamilies based on mitogenomes in this study is inconsistent with previous morphological and molecular studies. This may be due to the high mutation rate of the mitochondrial genetic markers within Chironomidae. Our results indicate that mitogenomes showed poor signals in phylogenetic reconstructions at the subfamily level of Chironomidae.

Tanytarsus van der Wulp, with 355 currently known valid species, is a comparatively large genus of nonbiting midges (Diptera: Chironomidae). Relationships among Tanytarsus and associated genera and among species in the genus have been exceptionally difficult to resolve using morphology or single gene genealogies. Here, the phylogeny of Tanytarsus sensu lato is reconstructed based on the combined analysis of five nuclear markers, including both ribosomal (18S) and protein‐coding (AATS1, CAD, PGD and TPI) genes. Our results indicate that Tanytarsus is paraphyletic with Caladomyia Säwedal placed among South American Tanytarsus , Virgatanytarsus Pinder as part of a Gondwanan clade, and Corynocera Zetterstedt within the Tanytarsus norvegicus (Kieffer) species group. Based on these results, we synonymize Caladomyia and Virgatanytarsus with Tanytarsus . We propose retaining the older name Corynocera until an eventual International Code of Zoological Nomenclature ruling on precedence can be made regarding the more frequent usage of the younger name Tanytarsus . As expected, the previously synonymized Nimbocera Reiss is confirmed to be a junior synonym of Tanytarsus with T . rhabdomantis (Trivinho‐Strixino & Strixino) grouping with other Neotropical Tanytarsus . The genus Sublettea Roback remains valid outside of Tanytarsus + Cladotanytarsus Kieffer. The monophyly of some established species groups is well supported, while other groups are refuted or remain uncertain. Based on a calibrated molecular divergence time analysis, Tanytarsus diverged from its sister group Cladotanytarsus during the Late Cretaceous to Early Paleogene (61–79 Ma). The genus most likely originated in the Oriental and Palearctic regions.