Computational protocol: Increased population sampling confirms low genetic divergence among Pteropus (Chiroptera: Pteropodidae) fruit bats of Madagascar and other western Indian Ocean islands

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[…]     Ear clips were collected in the field from released individuals in the context of a research program of the Institut Pasteur de Madagascar and preserved in EDTA. Additional tissue samples associated with specimens housed in the Field Museum of Natural History and from captive animals at the Lubee Bat Conservancy were also included (Table 1). Table 1. Newly sequenced samples included in this study. Abbreviations as follows. CS: Institut Pasteur; Chauve-souris; SMG: Steven M. Goodman; ISIS: Lubee Foundation; AJ, AJL, R, H, GC, MH, MT, and V: Lubee Bat Conservancy band identification numbers.     Genomic DNA was extracted from a small tissue sample using either the Nucleospin DNA Extraction Kit or the Qiagen DNeasy Tissue Kit. The mitochondrial gene, cytochrome-b (cyt-b), was amplified using the primers L14724 and H15915 . PCR reactions were conducted in a total volume of 25 µL including 1 x Buffer (100 mM Tris-HCL, pH 8.3, 500 mM KCl), 2 mM MgCl2, 1 mM dNTP, 0.25 µM of each primer, 0.5 U Taq polymerase, and 1 µL template DNA. PCR cycles consisted of an initial denaturation of 94 °C for 2 min, 35 cycles of 94 °C denaturation for 1 min, 48 °C annealing for 1 min, and 72 °C extension for 1.5 min, and a final extension of 10 min at 72 °C. Five µL of amplified product were incubated with 0.4 µL ExoSapIT (USB) and 1.6 µL water at 37 °C for 15 minutes followed by 80 °C for 15 minutes. Cleaned PCR products were sequenced using the primers used for amplification and an internal primer when necessary (PterCytbInt1 5’ GGRGCAACAGTCATYACYAA 3’) in a total volume of 5 µL (1 x buffer, 1 µM primer, 0.2 µL BigDye v3, and 1.0 µL cleaned PCR product). Sequencing reactions were run on an ABI 3730xl DNA Analyzer capillary machine and sequences were checked by eye and assembled into contigs in Sequencher 4.8 (GeneCodes, Ann Arbor, Michigan).     We supplemented these data with sequences from GenBank (see Table S1) and used MacClade  to align sequences by hand and check for stop codons. All sequences have been deposited in GenBank (JF327207 – JF327326). We reconstructed the phylogenetic relationships among DNA sequences under maximum parsimony (MP), maximum likelihood (ML), and Bayesian inference using other Pteropodidae (Ptenochirus jagori and Cynopterus brachyotis) as outgroups to root phylogenies based on previous phylogenetic work . Maximum parsimony bootstrap analyses were conducted in PAUP 4b10; we used 1,000 bootstrap replicates of the fast-search option.     We determined the model of sequence evolution that best fit the entire data set and each partition of the data (first, second, and third codon positions) using MrModeltest 2.3 . One hundred ML bootstrap replicates were conducted in Garli 1.0 under a GTR+I.+G model of sequence evolution. Partitioned Bayesian phylogenetic analyses were performed in MrBayes 3.1.2 . These consisted of three independent runs each with four incrementally heated chains sampled every 1,000 generations for 10 million generations. We verified adequate mixing within runs and convergence among runs in Tracer and discarded the first 1,001 samples from each run before summarizing trees.     To examine phylogeographic structure within species, we constructed a haplotype network within a parsimony framework in TCS 1.21 . We included all descendants of the most recent common ancestor for P. rufus and P. s. comorensis that were well-supported in the Bayesian phylogenetic inference. We used a 95% connection limit and treated gaps as a fifth state.    We assessed whether genetic diversity was best explained by within population variation or among population variation using an AMOVA executed in Arlequin 3.5 . Samples of P. s. comorensis from the Comoros were grouped by island (Grande Comore, Mohéli, Anjouan, and Mayotte) and P. rufus from Madagascar were separated into five groups based on the proximity of collection localities (Figure 1). […]

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