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Population genetic structure of the insular Ryukyu flying fox Pteropus dasymallus


Chen, Shiang-Fan et al. (2020), Population genetic structure of the insular Ryukyu flying fox Pteropus dasymallus, Dryad, Dataset,


Small isolated populations are vulnerable to both stochastic events and the negative consequences of genetic drift. For threatened species, the genetic management of such populations has therefore become a crucial aspect of conservation. Flying foxes (Pteropus spp, Chiroptera) are keystone species with essential roles in pollination and seed dispersal in tropical and subtropical ecosystems. However, many flying fox species are also threatened, having experienced dramatic population declines driven by habitat loss and hunting. The insular Ryukyu flying fox (Pteropus dasymallus) ranges from the Ryukyu Archipelago of Japan through Taiwan to the northern Philippines and has undergone precipitous population crashes on several islands in recent decades. To assess the population genetic structure and diversity in P. dasymallus, and its likely causes, we analyzed mitochondrial and microsatellite DNA. Both markers showed significant genetic differentiation among most island populations, with mitochondrial haplotypes showing some mixing across the region, likely reflecting historical colonization and/or dispersal events. In contrast, microsatellite markers showed an overall pattern of isolation by distance; however, this pattern appeared to be driven by the presence of deep ocean trenches between geographically distant populations. Thus the current distribution of P. dasymallus and its subspecific diversity appears to have arisen through vicariance coupled with a long history of restricted gene flow across oceanic barriers. We conclude that isolated island subgroups should be managed separately, with efforts directed at reducing further declines in genetic diversity.


1. Sampling

We obtained samples of P. dasymallus opportunistically over a period of 10 years (2009-2019) from wild-caught individuals and carcasses found in the wild, as well as rescued and/or captive individuals. All samples originated from eight different Taiwanese and Ryukyu islands and were classified into the five subspecies based on their geographical source following Yoshiyuki (1989), Mickleburgh et al. (1992), and Kinjo & Nakamoto (2015). A total of 77 samples were analyzed for this study after removing three duplicate samples and three putative first-degree relatives, as outlined below. Sample sizes per subspecies were 36 Formosan, 10 Yaeyama, 22 Orii’s, 1 Erabu, and 8 Daito. Formosan flying fox samples were further divided into three groups denoted as TW1 (Gueishan Island), TW2 (Lyudao), and TW3 (Taiwan’s main island) according to the islands from which they originated. Yaeyama samples originated from Iriomote-jima and Ishigaki-jima, Orii’s samples from Okinawa-jima, Erabu sample from Kuchinoerabu-jima, and Daito samples from Minamidaito-jima (Figure 1, Table S1).

Samples ranged from wing membrane biopsies, blood and frozen muscle tissue to fecal samples. Wing membrane samples were collected with a 3-mm biopsy punch and placed in 99.5% ethanol, Allprotect Tissue Reagent (Qiagen), or silica beads until extraction. For blood, a volume of 0.5 cc was taken by a professional veterinarian and preserved in an EDTA anticoagulant tube. Frozen tissue obtained from specimens and fresh feces were stored in 99.5% ethanol or RNAlater (Qiagen).

2. DNA extraction and amplification

To extract genomic DNA from wing membrane, blood, and frozen muscle samples, we used DNeasy Blood and Tissue Kits (Qiagen). For fecal samples, we used the QIAamp Investigator Kit or QIAamp Fast DNA Stool Mini Kit (Qiagen).

We amplified a section of the mtDNA control region using the primers BovL 14987 (5'-CGC-ATA-TGC-AAT-CCT-ACG-A-3') and BovR 15967 (5'-GCG-GGT-TGC-TGG-TTT-CAC-3'), which we designed for this study. PCR was carried out in a total volume of 15 μl, containing 20-100 ng of template DNA, 0.375 μl of 10 μM of each primer, and 7.5 μl of Quick Taq HS DyeMix (TOYOBO). Amplification was performed with the following profile: 2 min at 94ºC followed by 35 cycles of 30 s at 94ºC, 30 s at annealing temperature (55℃), 50 s at 68ºC, and a final extension of 10 min at 68ºC. PCR products were run on an ABI 3730XL DNA Analyzer (Applied Biosystems). Chromatograms were edited and aligned in the SeqMan and MegAlign (DNASTAR) programs. We also obtained three published P. dasymallus partial control region sequences from GenBank for one Yaeyama flying fox from Irabu-jima (NC_002612) (Nikaido et al., 2000a, b) and two individuals collected from the Batanes Islands, with one from Batan Island and the other from Sabtang Island (MN477630 and MN477629, respectively), representing the Philippine population (Tsang et al., 2019a, b).

For microsatellite DNA analyses, we successfully genotyped 76 samples at 26 polymorphic loci developed for this study (S1, Table S2). PCR was carried out in a total volume of 10 μl, containing approximately 10-50 ng of template DNA, 0.5 μl of 10 μM of each primer, and 5 μl of Quick Taq HS DyeMix. Amplification was performed with the following profile: 2 min at 94°C, followed by 40 cycles of 30 s at 94°C, 30 s at annealing temperature (54°C), 1 min at 68°C, and a final extension of 10 min at 68°C. PCR products were also run on an ABI 3730XL DNA Analyzer, and allele scoring was performed using the software GeneMarker 4.2 (SoftGenetics). Identity and parentage analyses were performed using Cervus 3.0.7 (Kalinowski et al., 2007). Samples with identical genotypes across all loci were determined to be duplicates and removed. Similarly, first-degree descendant relatives were determined on the basis of no allele mismatches, and where potential parent-offspring pairs were inferred, only one individual from each pair was retained. 

Usage Notes

microsatellite genotyping data.xlsx : This is a table of microsatellite genotyping data of 26 loci for 76 individuals of Pteropus dasymallus, Chen et al. (2020) Population Genetic Structure of the Insular Ryukyu Flying Fox Pteropus dasymallus, Biotropica, accepted. The characteristics and sequences of each microsatellite locus were provided in Table S2.

mtDNA_dloop_sequences.txt : The file contains the mtDNA d-loop sequences for 77 individuals of Pteropus dasymallus used in this study.
The abbreviation for each subspecies is as below.
DA: P. d. daitoensis
ER: P. d. dasymallus
OR: P. d. inopinatus
YA: P. d. yayeyamae
TW: P. d. formosus


Council of Agriculture, Award: 107-9.1-SB-17(1)

Ministry of Science and Technology, Taiwan, Award: MOST 107-2621-B-305-001

JSPS KAKENHI, Award: JP16H06542

Council of Agriculture, Award: 108-9.1-SB-30