Phylogenetic analysis provides insights into the evolution of Asian fireflies and adult bioluminescence.

Fireflies are one of the best-known examples of luminescent organisms. The limited geographic distribution and rarity of some firefly genera have hindered molecular phylogenetic analysis, resulting in uncertainty in regard to firefly phylogeny. Here, using genome skimming next-generation sequencing, we sequenced 23 Asian firefly species from 15 genera (Lampyridae: 14; Rhagophthalmidae: one) and assembled their mitochondrial genomes (mitogenomes) and nuclear ribosomal DNA (rDNA) repeat unit. The mitogenomes (including 15 mitochondrial genes: COX1-3, ATP6&8, ND1-6&4L, CYTB, 12S, and 16S) were recovered for almost all 23 species; furthermore, three regions of the nuclear rDNA repeat unit (18S, 28S, and 5.8S) were recovered for 22 out of the 23 species. The mitogenomes of 11 genera and 22 species as well as the complete rDNA from 22 species are reported here for the first time. Combined with previously published sequences of mitochondrial and rDNA coding regions, 166 species (170 populations with four overlapping in Lampyridae) were included in the current analyses. We selected different species groups and coding regions to infer phylogenies, and then employed tree certainty (TC) and internode certainty (IC) to quantify any phylogenetic incongruence. Phylogenetic analysis of 18 coding regions (15 mitochondrial genes and three regions of the nuclear rDNA repeat unit) from different species groups showed that the 144-species selection group (excluding 22 species outside Lampyridae) had relatively high TC (101.39). Further phylogenetic analysis of the 144 species using different coding regions indicated that the phylogeny of the 13 coding regions (10 mitochondrial genes: COX1-2, ATP6&8, ND1, ND4-5, CYTB, 12S and 16S; three rDNA regions: 18S, 5.8S, and 28S) demonstrated higher TC (103.02) than the phylogenies based on the 18 coding regions (TC = 101.39), conserved-regions (c-regions, i.e., 12S, 16S, COX1, 18S, and 28S) (TC = 95.11), or conserved-sites (c-sites, TC = 92.31) for the mitochondrial genes. In contrast, the c-sites strengthened the deeper nodes of the 144-species phylogeny compared to the c-regions. All of the 144-species phylogenies using different coding regions (except the c-regions) consistently recovered the monophyly of each of the three luminous families and their combination (Lampyridae, Rhagophthalmidae, and Phengodidae) with high IC support. Our phylogenetic analyses clarified the position of firefly genera Lamprigera, Vesta, Stenocladius, Pyrocoelia, Diaphanes, Abscondita, Pygoluciola, Emeia, Pristolycus, and Menghuoius. We also inferred the evolutionary pattern of adult bioluminescence in Lampyridae based on the phylogenies of 166 and 144 species. Our data suggest that the common ancestor of Lampyridae possessed adult bioluminescence, with a higher loss rate than gain rate of bioluminescence during its lineage evolution. Our results provide insight into Asian firefly phylogeny, and also enrich mitogenome and rDNA data resources for further study.