New Color-Patterned Species of Microtendipes Kieffer, 1913 (Diptera: Chironomidae) and a Deep Intraspecific Divergence of Species by DNA Barcodes †

SIMPLE SUMMARY: Non-biting midges are the most widely distributed, and frequently the most abundant, insect family in freshwater environments. Species delimitation concerning color patterns and the shape or distribution of thorax pigmentation, wing spots, abdomen pigmentation, and leg pigmentation are disputable and unstable in the family. This research focuses on a genus that shares the general appearance of the hypopygium, but with variations in coloration of the antennae, thorax, wings, and legs. In this study, we analyzed collected species along with public sequences, resulting in a preliminary DNA library including 21 morphospecies. DNA barcodes can successfully delimit Microtendipes species and showed deep intraspecific divergence in some species. We also confirmed that color patterns can be important diagnostic characters. As a result of this analysis, five species new to science are identified and described, and an updated key to male adults of known Microtendipes species from China is provided. ABSTRACT: The genus Microtendipes Kieffer (Diptera: Chironomidae) has a nearly worldwide distribution, comprising more than 60 species, which are further divided into two species groups based on larval stage. However, species delimitation and identification among the adults of this genus are controversial and uncertain. For instance, previous studies have provided many synonymies based on conspecific color pattern variations in Microtendipes species. Here, we used DNA barcode data to address Microtendipes species delimitation as well as to test whether color pattern variations can be diagnostic characters for interspecific identification. The 151 DNA barcodes used, 51 of which were contributed by our laboratory, represent 21 morphospecies. Species with specific color patterns could be accurately separated based on DNA barcodes. Consequently, the color patterns of adult males could be important diagnostic characters. The average intraspecific and interspecific sequence divergences were 2.8% and 12.5%, respectively, and several species exhibited deep intraspecific divergences higher than 5%. Molecular operational taxonomic units (OTUs) ranged from 21 to 73, based on methods including phylogenetic trees, the assemble species by automatic partitioning method, the Poisson tree process (PTP), and the general mixed Yule-coalescent (GMYC) method. As a result of these analyses, five new species were recognized (M. baishanzuensis sp. nov., M. bimaculatus sp. nov., M. nigrithorax sp. nov., M. robustus sp. nov., and M. wuyiensis sp. nov.).