Genetic divergences and evolutionary relationship of the genus Platycerus (Coleoptera, Lucanidae) and its yeast symbionts in East Asia

概要

Fluctuations in climate during the Pleistocene had a profound impact on the geographical distribution of organisms in temperate zones. The distribution of cool temperate zone species towards the warmer areas depends on the existence of high mountains, which may serve as refuges and corridors for the species. During the cycle of expansion and contraction of these habitats, many animals and plants produced repeating episodes of population subdivision and secondary contact.
　The genus Platycerus (Coleoptera, Lucanidae) is a group of small stag beetles that is widely distributed in the northern hemisphere and includes more than 50 described species. This genus is generally adapted to cool temperate broad-leaved forests in East Asia, where are known10 species in the Japanese archipelago, one species in the Korean Peninsula, and more than 20 species in China. Although they have quite different geographical backgrounds in the East Asian area (Japan, Korea and China), the differentiation of the genus Platycerus in the East Asian area have been little studied except for in Japan (e.g., Kubota et al. 2011).
　In 2010, the presence of xylose-fermenting Scheffersomyces yeasts within the microbe-storage organ (mycangia) in stag beetles was firstly reported. For Japanese Platycerus species, beetle species and their yeast symbionts are known to have co-evolved but incompletely.
　The aim of this study was to understand the diversification patterns of East Asian Platycerus and whether divergences correspond to major biogeographic events in this region and to clarify the evolutionary relationship between the genus Platycerus and its yeast symbionts in East Asia, which seems to contribute the understanding of the formation process of temperate forest insect fauna in East Asia ultimately.

Phylogeographic history of Platycerus hongwonpyoi in South Korea
　Platycerus hongwonpyoi is the only species belonging to the genus Platycerus in Korea. Based on 130 samples from 16 sites in seven mountain areas, South Korean P. hongwonpyoi exhibited remarkable geographic differentiation in the mitochondrial COI gene, although there was no variation in nuclear Wg or ITS sequences. The divergence in COI sequences was greater among sites or among areas than within sites, so that COI sequences appear to have undergone significant geographical divergence. However, some of the major clade included individuals from multiple mountain areas, suggesting that secondary gene flow between populations from different mountain areas occurred. This may have resulted in the relatively high local genetic diversities. And the population size of South Korean P. hongwonpyoi seems to have decreased over the past several tens of thousands of years.
　The Korean Peninsula has more simple topography than Japan, with mountains lower than 2000 m in elevation and a single continuous mountain range running longitudinally. This topography may facilitate the northward range shift by P. hongwonpyoi after the last glacial period, in which the longitudinal mountain range acted as a corridor. As a result, the genus Platycerus followed quite different divergence patterns in South Korea compared with Japan, which may be due to the topographical differences between these geographical areas.

Genetic divergence of the genus Platycerus in China
　The sample collections were conducted in 2016-2018 from 11 localities along five mountainous areas, distributed from north to west of the Sichuan Basin. In total, 83 Platycerus samples of 14 species were collected in China. Of them, one species was described by us, as a new species, Platycerus auriceps Kubota et Zhu, 2018.
　The genetic analyses were conducted based on mitochondrial COI gene, and nuclear Wg and ITS sequences. There is no critical contradiction in the phylogenetic relationship among three genes, although nuclear genes have less information than COI gene.
　Based on the COI gene, three major clades were recognized. Platycerus hongwonpyoi in South Korea and China formed a monophyletic clade. Since the genetic and morphological divergences of P. hongwonpyoi are large, I consider P. hongwonpyoi as a species complex, The divergence was greater among species within clades than among clades. This suggests that speciation has been relatively important in genetic divergence in China. The divergence time of Platycerus in East Asia into Asian Continent and Japan was inferred to be around 10.6 Mya. If the genetic divergence rate is similar between China and Japan, Chinese Platycerus seems to have been speciated earlier than Japanese one. This suggests a hypothesis that the dispersal of Platycerus from Asian Continent to Japan. The historical effective population size for all populations was inferred to have decreased starting from several hundred thousand years ago, although that for the western clade (Clade III) seemed to increase within the past 100,000 years.
　The type of posterolateral corner of pronotum had been an important diagnosis in higher taxonomy of Platycerus, i.e., subtriangulaly produced corner: sharp (S) type; arcuately lobed corner: round (R) type. In this study, it was clarified that S type species and R type species have evolved parallelly in Asian Continental Platycerus and that the altitude and latitude of S type species collection sites were lower and higher than those of R type species, respectively. Although the function of pronotum corner and mechanism of habitat choice of Platycerus are still unknown, the results of this study suggest that the pronotum type has evolved as an ecological trait depending on the climate condition. As the results, S type seems to have evolved from R type in China and R type seems to have evolved from S type in Japan.
　China is a continental country, but with a complex topography, including the very large and steep mountains, and large rivers around Sichuan Basin, which work as barriers of dispersal for Platycerus. This topography in China is relatively similar to that in Japan compared with Korea. However, the mountain areas and river systems in China are much larger than those in Japan, which result in the large variation of climate conditions. The vertical distribution ranges of R type species in southern and high mountain areas seems to have varied more than those of S type species by climate change during Pleistocene, which contributed to the isolation and secondary contact of R type species. As the result, intensive speciation and putative introgression of COI were easier in R type species than in S type species. As the results, the genetic divergence of Chinese Platycerus showed unique process, which was relatively similar to that of Japan compared with that of South Korea.

Evolutionary relationship between the genus Platycerus and its yeast symbionts in East Asia
　Recently, it was reported that there are xylose fermenting Scheffersomyces yeasts within the microbe-storage organ (mycangia) in stag beetles (Tanahashi et al. 2010). As a taxon of stag beetles, Japanese Platycerus species and their yeast symbionts have co-evolved but incompletely (Kubota et al. in preparation).
　In this study, in total, I prepared 13 females of 11 Platycerus species from seven sites in China. I also used four females of three other lucanid species in China and one female of Prismognathus in South Korea as outgroup. I exmanined ITS and IGS sequences of yeast symbionts with them, and COI gene of their host beetles.
　Based on the ITS and IGS sequences, all the yeast symbionts with Platycerus species (China, Korea, and Japan) and Chinese and Korean Prismognathus species belong to the monophyletic lineage, although the yeasts with Japanese Prismognathus angularis belongs to a different lineage from it.
　Based on the IGS sequences, the yeasts with Platycerus species, and Chinese and Korean Prismognathus species were diverged into two clades including four subclades.
　I proposed a likely scenario of the evolutionary process of the yeast symbionts concerning Platycerus and Prismognathus in East Asia as followings (Figure). The origins of Platycerus and Primognathus of East Asia were in China. They possessed ancestral yeasts of (Clade I + Clade II + outgroup). Firstly, a part of Prismognathus moved to Japan, and its yeast symbionts evolved uniquely (to outgroup). Next, the yeasts with Platycerus and Prismognathus in China were separated into Clade I and Clade II. Clade I was used by Platycerus and Clade II was shared by two genera. A part of Platycerus with the yeasts of Clade I moved to Japan, and these yeasts evolved to Clade Ia. Finally, a part of Japanese Platycerus (P. viridicuprus) was contacted with Platycerus hongwonpyoi or Prismongnathus dauricus around Tsushima Islands near the boundary between Japan and South Korea. There, the yeasts of Clade IIa were transmitted from Korean species to Platycerus viridicuprus horizontally through the common use of host wood.

Conclusion
　In this study, it was clarified that East Asian Platycerus have diverged differently according to the topographies in China, Korea, and Japan. In China, S type species evolved from R type species repeatedly in northern and lower areas.
　The yeasts with Platycerus and Prismognathus can be delimit clearly in Japan, but they were mixed together in Asian Continent, during the genera of Platycerus and Prismognathus’ evolutionary process. This is an example that the symbiotic microorganisms can affect on the evolutionary processes of distantly related species.