Genetic differentiation of water penny beetles may be associated with the formation process of the ancient Lake Biwa.

Background: Water flow is one of several factors that determine the habitats of aquatic insects. The conversions of stagnant (lentic) and running (lotic) water are physiologically and ecologically constrained. In general, aquatic insect species only occur in one of these environments. Though Eubrianax ramicornis (Coleoptera: Psephenidae) usually resides in rivers, however, it is also known to occur near the shores of Lake Biwa in Japan. Here, we investigated the genetic differences between the two Eubrianax species of Lake Biwa and its inflowing rivers, and other regions of Japan for the first time. Lake Biwa is an ancient lake and about four million years old. Though over 60 endemic species were described from Lake Biwa, they are mostly fish and molluscs, with only two species of aquatic insects. There are few reports of endemic aquatic insects in other ancient lakes, too. Discussion of the endemism of Eubrianax and its promoting factors is expected to provide insights into the evolution of aquatic insects in ancient lakes. Methods: We surveyed and morphologically identified psephenid larvae in Lake Biwa and its inflowing rivers during the summers of 2017 and 2018 (Hayashi, 2009: Hayashi & Sota, 2009). We conducted phylogenetic analyses of two regions of COI and estimated the ages of population divergence based on fossil records of Eubrianax beetles in Lake Biwa as well as those of previously reported populations in other regions. We also analyzed two COI regions of the E. ramicornis haplotypes in Lake Biwa. Results: Four psephenid species were identified among the samples collected in Lake Biwa and its inflowing rivers. Of these, E. ramicornis was collected only at the Lake Biwa shore whereas its congeneric E. granicollis was collected only from the rivers inflowing the lake. The phylogenetic analyses showed that E. ramicornis of Lake Biwa were clearly differentiated from those in previously reported regions and this divergence was estimated to have occurred 1.40 MYA. At that time, there was no lake at the location of present-day Lake Biwa. It is thought that rivers and their dammed flood plains to the east, with gravel and sand supplied by the rising Suzuka Mountains further to the east. Such an environments is preferable for E. ramicornis and they were thought to have become isolated from the surrounding rivers during the formation of Lake Biwa. On the contrary, there was no genetic differentiation between the E. granicollis population at the Lake Biwa inflow rivers and those elsewhere. Hence, E. granicollis might have migrated to the Lake Biwa watershed later than E. ramicornis and maintained gene flow over a wide area. The genetic differentiation of E. ramicornis reported here for the first time suggests that a species with a wide distribution may rather be evolving endemic to Lake Biwa. Reports on the endemism of aquatic insects in ancient lakes are scarce. This study is expected to bring new perspectives to the study of insect evolution in ancient lakes. ### Competing Interest Statement The authors have declared no competing interest.