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

The genus 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 species. Here, we used DNA barcode data to address 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 ( sp. nov., sp. nov., sp. nov., sp. nov., and sp. nov.).