Genetic Diversity and Population Structure in the Ryukyu Flying Fox Inferred from Remote Sampling in the Yaeyama Archipelago

Abstract

There are 122 mammal species in Japan, and 37 of them are bats. Especially, the species of genus Pteropus have big body size and the ability to fly hundreds of kilometers and have important roles in pollination and seed dispersal. The Ryukyu flying fox (Pteropus dasymallus) is one of the Pteropus species in Japan, distributed in the Ryukyu archipelago, Taiwan, and possibly the Philippines, and is divided into 5 subspecies. Although they are listed as VU (vulnerable) in IUCN Red List, few genetic analyses have been conducted for their conservation. The purpose of this study is to evaluate genetic diversity and investigate the genetic structure of Yaeyama flying fox, one of the subspecies of the Ryukyu flying fox. We conducted mtDNA haplotype analysis and microsatellite analysis with the 18 markers we developed.mtDNA analysis was conducted with the samples collected in 8 islands (Miyako, Ishigaki, Kohama, Kuroshima, Hateruma, Taketomi, Iriomote, Yonaguni) We identified 39 haplotypes in 526bp of the control region of 142 samples. 14 haplotypes were shared between some islands, and haplotype network for the 8 islands did not show any clear genetic structure. However, haplotype diversity was higher in Ishigaki and Iriomote compared to other islands, and some haplotypes were only found in particular islands, so there might be some genetic structure which could not be revealed by mtDNA analysis.Therefore, we also conducted microsatellite analysis with 155 samples collected in 6 islands (Miyako, Ishigaki, Kohama, Taketomi, Iriomote, Yonaguni). As a result of genetic diversity analysis, PCoA, STRUCTURE, and calculation of Fst, Yonaguni (the west end of the distribution area) population showed clear genetic differentiation from other populations, low genetic diversity, and a high inbreeding level. Ishigaki, Kohama, Taketomi, and Iriomote (the center of the distribution area) populations had gene flow between them and high genetic diversity. Miyako (the east end of the distribution area) population showed slight genetic differentiation and had the middle level of genetic diversity. Gene flow between Ishigaki and Miyako through islands between them might be preventing inbreeding of Miyako population.We revealed genetic diversity, and genetic differentiation and gene flow between islands of Yaeyama flying fox for the first time. These results will be useful for setting

Population genetic structure of the insular Ryukyu flying fox Pteropus dasymallus

“…On the other hand, strong differentiation among island populations of flying foxes separated by 200-300 km, such as that between Orii's and Yaeyama, suggests that this distance represents an upper limit for recurrent gene flow in these bats. Supporting this, limited movements were previously reported for both Orii's based on radio tracking data (Nakamoto et al, 2009(Nakamoto et al, , 2012 and Yaeyama subspecies based on gene flow within Yaeyama Islands, Japan (Taki et al, 2020). Despite this, our structure-based clustering analyses did reveal a small number of putative migrants, with two individuals recorded in Taiwan (TW1) and one in Yaeyama assigned to the Orii's genetic cluster.…”

“…Indeed, in our study, it is notable that island groups characterized by stronger genetic differentiation were also more likely to occur on opposite sides of deep-sea trenches (Hutchison & Templeton, 1999), and we found that the trend of isolation by distance was no longer significant after taking this into account. (Nakamoto et al, 2009(Nakamoto et al, , 2012 and Yaeyama subspecies based on gene flow within Yaeyama Islands, Japan (Taki et al, 2020). Despite this, our structure-based clustering analyses did reveal a small number of putative migrants, with two individuals recorded in Taiwan (TW1) and one in Yaeyama assigned to the Orii's genetic cluster.…”

“…In 2004, an additional small population of the Formosan flying fox was recorded for the first time on Gueishan Island (Turtle Island), 9.7 km off the northeastern coast of Taiwan, and some individuals have occasionally been found on the main island of Taiwan since 2006 (Wu, 2010), although the origin of these populations is unclear. Other indirect evidence of movements across water comes from the population census of Orii's flying foxes (Nakamoto et al, 2011) and from inferred gene flow from the Yaeyama flying fox (Taki et al, 2020).…”

Genetic diversity and population structure in the Ryukyu flying fox inferred from remote sampling in the Yaeyama archipelago

“…Tissue samples, on the other hand, were obtained on Ishigaki island using the standard procedure for bats, which involved capturing P . dasymallus individuals at night using mistnets and taking one 3mm wing biopsy from the plagiopatagium or between the 4 th and 5 th finger in the distal part of the chiropatagium, depending on wing condition (see detailed protocol used in [ 29 , 30 ]).…”

“…18 microsatellite loci (Accession number: LC506191-LC506193, LC506193, LC506197-LC506198, LC506200-LC506202, LC506205-LC506209, LC506212-LC506213, LC506220, LC506223) which we developed specifically for P . dasymallus [ 30 ] were amplified through PCR using a PCR System 9700 Thermal Cycler (GeneAmp). Forward primers were synthesized with an M13 tag sequence ( ) for fluorescent labeling.…”