Acta Chiropterologica, 16(2): 293–300, 2014 PL ISSN 1508-1109 © Museum and Institute of Zoology PAS doi: 10.3161/150811014X687251 Population genetics of the Mauritian flying fox, Pteropus niger PETER A. LARSEN1, 6, CORINNE E. HAYES1, MARY A. WILKINS1, YANN GOMARD2, RAJENDRAPRASAD SOOKHAREEA3, ANNE D. YODER1, and STEVEN M. GOODMAN4, 5 1Department of Biology, Duke University, Durham, North Carolina 27798, USA 2Centre de Recherche et de Veille sur les Maladies Emergentes dans l’Océan indien (CRVOI), Ste Clotilde, La Réunion, France 3National Parks and Conservation Service, Réduit, Mauritius 4The Field Museum of Natural History, 1400 South Lake Shore Drive, Chicago, Illinois, USA 5Association Vahatra, BP 3972, Antananarivo 101, Madagascar 6Corresponding author: E-mail: [email protected] The Mauritius flying fox Pteropus niger is distributed on the islands of Mauritius and La Réunion in the western Indian Ocean. Although recent studies have examined the phylogenetics and systematics of this genus, relatively few have assessed the population genetics of species distributed on oceanic islands and no study has focused on the demographics of P. niger. Here, we present mitochondrial DNA sequence data from 39 individuals of P. niger collected from four main colonies distributed throughout Mauritius. Our results indicate that the Mauritian population of P. niger is likely panmictic, with moderate to high levels of gene flow occurring among colonies distributed across the island. Collectively, our sequence data suggest moderate levels of genetic variation within the population. These findings will help to inform ongoing conservation and disease surveillance initiatives. Key words: genetic structure, Mauritius, phylogeography, Pteropus niger INTRODUCTION The Mauritian flying fox Pteropus niger is re- stricted in its distribution to the Mascarene Islands In part associated with their wing structure and (Mauritius and La Réunion) in the southwestern size, nocturnal fruit bats of the Old World family Indian Ocean. On the basis of subfossil evidence, it Pteropodidae are notably strong flyers with respect is known that this species was once widespread on to other chiropterans (Norberg and Rayner, 1987). La Réunion, but was locally exterminated in the Non-forest dependent species are able to cross con- early 18th-century, almost certainly associated with siderable distances in search of food resources and local hunting pressure (Bergmans, 1999; Cheke and while dispersing. For example, some members of Hume, 2008). In 2007, a small colony of P. niger the genus Pteropus, which readily cross non- was identified on La Réunion, probably representing forested areas, are able to fly over 50 km in a given a recent recolonization (Roué and Probst, 2010; night (Palmer et al., 2000; Tideman and Nelson, Hutson and Racey, 2013) and attesting to the ability 2004). This capacity to cross substantial areas, in- of this species to disperse the ≈180 km of open cluding open water, has allowed members of this water between these two islands. On Mauritius, genus to colonize notably remote islands across the which has a surface area of 1,860 km2, this species Old World tropics. Surprisingly, even though these remains widespread and relatively common. The bats can disperse across considerable oceanic areas, local population was estimated in 2010 as 49,000– they nonetheless exhibit considerable levels of 56,000 individuals, and thus considered stable endemicity on islands and island groups, includ- (Sookhareea, 2011; Hutson and Racey, 2013). ing those in the Indian Ocean. Hence, patterns of Recent phylogenetic work on the genus Pteropus in- dispersal are by no means regular and different dicates that P. niger diverged less than 500,000 types of filters exist that provide mechanisms for years ago as part of a western Indian Ocean radia- local speciation (Chan et al., 2011; Almeida et tion (Almeida et al., 2014) and can be considered al., 2014). a relatively recent taxon. 294 P. A. Larsen, C. E. Hayes, M. A. Wilkins, Y. Gomard, R. Sookhareea, et al. In the context of ongoing zoonoses study of MATERIALS AND METHODS P. niger on Mauritius, as well as understanding aspects of this species’ population structure associ- Sample Collection ated with conservation actions, it was deemed im- portant to obtain molecular genetic data to examine With authorization from the Mauritian Government and in collaboration with National Parks and Conservation Service of- patterns of gene flow among populations and to gen- ficers and their designated hunters, several known day roost erally assess levels of genetic diversity. Prior to this sites of Pteropus niger were visited and a limited number of current study, DNA sequence data had been gener- samples per site were collected (Fig. 1). All roosts were sampled ated from only five individuals of P. niger (O’Brien in November of 2012 (Appendix). In the field immediately after et al., 2009; Almeida et al., 2014) and all genetic capture, the animals were measured, prepared as standard mu- analyses of the species have focused entirely on the seum fluid preserved specimens, and pectoral muscle samples saved in lysis buffer. Juveniles clearly associated with their systematics and phylogeography of the genus, and mothers were only included in genetic analyses if sequence data in some cases, with special reference to relation- was unavailable from the mother (Appendix). Voucher speci- ships among species distributed throughout the mens are preserved and deposited in the Field Museum of western Indian Ocean (O’Brien et al., 2009; Chan Natural History, Chicago. For all specimens, organ tissue sam- et al., 2011; Almeida et al., 2014). The limited ples were conserved in liquid nitrogen for zoonoses studies. Research involving live animals followed the guidelines for the distribution of P. niger coupled with habitat loss capture and handling of mammals approved by the American of native forest on Mauritius and hunting pressure Society of Mammalogists (Gannon et al., 2007). associated with consumption of commercially im- portant fruit crops (Nyhagen, 2004; Nyhagen et al., Molecular Methods 2005; Price, 2013) has contributed to the classifi- cation of the species as vulnerable to extinction Genomic DNA was extracted from muscle tissues using the (Hut son and Racey, 2013). The Mauritian Gov - DNeasy Blood and Tissue Kit (Qiagen Inc., Chatsworth, CA, ernment has not conducted to date any culling of USA). The mitochondrial D-loop and COI genes were amplified using primers reported in Brown et al. (2011). We used the P. niger (Florens, 2012) even given pressure from local fruit growers and exporters for this action to be taken. These different concerns underline the impor- tance of understanding aspects of the population genetics of this species, which is the focus of this current study. Our analyses utilize genetic variation in the mi- tochondrial control region (D-loop), a marker that has been frequently used to examine the genetic variation within natural populations of bats, includ- ing species of Pteropus (Salgueiro et al., 2004; Russell et al., 2008; Fleming et al., 2009; Brown et al., 2011; Guevara-Chumacero et al., 2013). In addition to D-loop DNA sequence information, we generated sequence data from the mitochondrial cytochrome c oxidase subunit I (COI) gene from representative samples. DNA sequence data of the COI gene is frequently used for DNA barcoding initiatives, and prior to this study, no COI data had been available from P. niger. Collectively, the genetic data presented herein provides important insight into the population structure of this spe- cies across an island landscape that is about 45 km in width and 65 km in length. The patterns of genetic variation determined herein will con- tribute to ongoing conservation efforts, and will FIG. 1. Map of Mauritius showing the four main localities from serve to inform future initiatives concerning the which specimens of P. niger were collected. Genetic analyses were performed on 39 individuals (Cascavelle: n = 16, Petite monitoring of emerging infectious diseases on Case Noyale: n = 4, Riche en Eau: n = 6, Fayence Mountain: Mauritius. n = 13) Population genetics of Pteropus niger 295 Phusion High-Fidelity PCR Kit (New England Biolabs, (Fig. 1). Of the 39 sequences, 20 haplotypes were Ipswich, MA, USA) for all PCR amplifications, which were observed and several haplotypes were shared among performed with the following thermal profile: 98°C for 1 min, the four collecting localities (Tables 1 and 2, Figs. 2 followed by 30 cycles of denaturation at 98°C for 10 sec, an- nealing at 50°C for 30 sec, and extension at 72°C for 45 sec with and 3). Average pairwise nucleotide diversity of the a final extension of 72°C for 10 min. PCR products were puri- 39 D-loop sequences was 0.02 and 47 polymorphic fied using the QIAquick PCR Purification kit (Qiagen Inc., base pairs were observed across the entire D-loop Chatsworth, CA, USA). DNA sequencing was performed using alignment (Table 1). The Tamura 3-parameter model ABI Big Dye version 3.1 chemistry and fragments were elec- of evolution was identified as the best fitting model trophoresed on an ABI 3700 Genetic Analyzer at the Duke Center for Genomic and Computational Genome Sequencing for our data and was implemented in Arlequin. The facility. Sequences were verified, assembled, and aligned using AMOVA identified 0.05% of the overall genetic the Geneious software package (version 7.1) and the MAFFT variation as occurring among the four populations alignment plugin (version 7.017). All sequence data reported (d.f. = 3, sum of squares [SS] = 14.49, variance = herein was deposited in GenBank under the following accession 0.00), while 99.95% of the genetic variation oc- numbers KP404015–KP404061. curred within populations (d.f. = 35, SS = 168.41, ϕ variance = 4.81). The overall pairwise ST statistic Genetic Diversity and Demographic Analyses was 0.00 and this value was not significant MEGA (version 6.0 — Tamura et al., 2013) software was (P = 0.43) (Table 3).