DOI: 10.5061/dryad.qfttdz0fp
title: Population genetic structure of the insular Ryukyu flying fox Pteropus dasymallus

description: 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.
description: <b>1. Sampling</b> We obtained samples of <i>P. dasymallus</i> 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<i> et al. </i>(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). <b>2. DNA extraction and amplification</b> 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 <i>P. dasymallus</i> partial control region sequences from GenBank for one Yaeyama flying fox from Irabu-jima (NC_002612) (Nikaido<i> et al.</i>, 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<i> et al.</i>, 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<i> et al.</i>, 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. 
description: microsatellite genotyping data.xlsx : This is a table of
 microsatellite genotyping data of 26 loci for 76 individuals of
 <em>Pteropus dasymallus</em>, Chen <em>et al.</em>
 (2020) Population Genetic Structure of the Insular Ryukyu Flying Fox
 <em>Pteropus dasymallus</em>, 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
 <em>Pteropus dasymallus</em> used in this study.<br> The
 abbreviation for each subspecies is as below.<br> DA: <em>P.
 d. daitoensis</em><br> ER: <em>P. d.
 dasymallus</em><br> OR: <em>P. d.
 inopinatus</em><br> YA: <em>P. d.
 yayeyamae</em><br> TW: <em>P. d.
 formosus</em>