Phylogenetic Structure Revealed through Combining DNA Barcodes with Multi-Gene Data for Agrodiaetus Blue Butterflies (Lepidoptera, Lycaenidae)

SIMPLE SUMMARY: The species-rich subgenus Agrodiaetus Hübner, 1822 is a distinct monophyletic lineage within the diverse blue butterfly genus Polyommatus Latreille, 1804. Although the subgenus has attracted the attention of numerous researchers, a large number of unresolved taxonomic problems persist in Agrodiaetus. In our study, we analyzed the taxonomic structure of the subgenus via combining short mitochondrial DNA barcodes of several extremely rare species, for which multi-locus data were unavailable, with multi-gene mitochondrial and nuclear data for the other Agrodiaetus taxa. This approach resulted in a high phylogenetic resolution of the tree obtained, even for the clades, which were solely represented by DNA barcodes. The status and taxonomic position of the enigmatic species P. muellerae, P. afghanicus, P. frauvartianae, P. bogra and P. anticarmon from Afghanistan, Pakistan, Iran and Turkey are revealed and discussed. The complete list of species groups and species of the subgenus Agrodiaetus is presented. ABSTRACT: The need for multi-gene analysis in evolutionary and taxonomic studies is generally accepted. However, the sequencing of multiple genes is not always possible. For various reasons, short mitochondrial DNA barcodes are the only source of molecular information for some species in many genera, although multi-locus data are available for other species of the same genera. In particular, such situation exists in the species-rich butterfly subgenus Polyommatus (Agrodiaetus). Here, we analyzed the partitioning of this subgenus into species groups by using three data sets. The first data set was represented by short mitochondrial DNA barcodes for all analyzed samples. The second and third data sets were represented by a combination of short mitochondrial DNA barcodes for part of the taxa with longer mitochondrial sequences COI + tRNA-Leu + COII (data set 2) and with longer mitochondrial COI + tRNA-Leu + COII and nuclear 5.8S rDNA + ITS2 + 28S rDNA sequences (data set 3) for the remaining species. We showed that the DNA barcoding approach (data set 1) failed to reveal the phylogenetic structure, resulting in numerous polytomies in the tree obtained. Combined analysis of the mitochondrial and nuclear sequences (data sets 2 and 3) revealed the species groups and the position within these species groups, even for the taxa for which only short DNA barcodes were available.