GENETIC VARIATION IN THE LITTLE RED FLYING-FOX Pteropus ...

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The little red flying-fox Pteropus scapulatus has a range exceeding 3.5 million km 2 during its seasonal migration in. Australia. Management of this species has ...
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Biological Conservation 76 (1996) 45 50 Copyright © 1996 Elsevier Science Limited Printed in Great Britain. All rights reserved 0006-3207/96/$15.00+.00

GENETIC VARIATION IN THE LITTLE RED FLYING-FOX Pteropus scapulatus (CHIROPTERA: PTEROPODIDAE)" IMPLICATIONS FOR MANAGEMENT E. A. Sinclair a*, N. J. Webb a, A. D. Marchant b* & C. R.

Tidemann

a

"Department of Forestry, School of Resource and Environmental Management, Australian National University, Canberra ACT 0200, Australia hResearch School of Biological Sciences, Australian National University', Canberra ACT 0200, Australia (Received 28 July 1994; accepted 26 May 1995)

become extinct, some in recent years, and many more species are in danger of extinction. These declines, together with the important ecological role of megachiropterans as vectors of plant propagules, indicated an urgent need for proactive management. These reviews, however, also highlighted the fact that management of many megachiropteran species was problematical because of insufficient information on their basic biology. Pteropus scapulatus is one such species. It is a medium-sized flying-fox, large males weighing about 550 g. Mating takes place in November-December and young are born after 5 months' gestation in AprilMay (O'Brien, 1993). P. scapulatus has by far the largest distribution of the four Pteropus species in Australia (Hall, 1987), extending over a total area in excess of 3.5 million km 2 (Fig. 1) and encompassing a range of climates from tropical to temperate (Castles, 1992). Since the study of Ratcliffe (1931) it has been known that P. scapulatus is present in the temperate, southern parts of its range only during the warmer months October-April. The extent of this migration varies greatly from year to year in terms of the numbers of animals undertaking the migration, the areas they occupy, and the composition of migrating groups (Ratcliffe, 1931; Nelson, 1965; Tidemann, unpublished data). Efforts to define the migration pattern by mark-release-recapture (Tidemann & Loughland, 1993) have so far been unsuccessful, due to only one animal, of 620 banded, being recovered (0.16%). This individual, banded at Grafton in January 1990 as a female in its first pregnancy, was recovered 21 months later 490 km to the north (Tidemann, unpublished data). Approaches to conservation and management have been greatly enhanced by the evolution of new and highly informative molecular techniques, which have often proved to be more revealing than traditional morphological or ecological methods. The taxonomies of some morphologically 'cryptic' species of Australian microbats have been determined by Adams et aL (1987a, b, 1988) and Baverst0ck (1988) and others using allozyme electrophoresis, and molecular techniques have also been used with a view to conserving genetic

Abstract

The little red flying-fox Pteropus scapulatus has a range exceeding 3.5 million k m 2 during its seasonal migration in Australia. Management of this species has been problematical because the range spans five States and Territories, each with its own system of managing wildlife. The results of an investigation of population structure by genetic analysis are presented. Allozyme electrophoresis and Random Amplified Polymorphic DNA (RAPD) analyses were used to determine genetic variation within and among six populations from widely separated locations on the continent. Both allozyrne and DNA techniques demonstrated very little genetic structuring among the subpopulation samples. Analysis of molecular variance on the RAPD data showed only 5% of variance among populations, although this difference was shown to be significant. A value of 0.028 for Wright's Fsr (a measure of the among-population component of variance in allele frequencies) suggested a similarly low degree of differentiation among subpopulations. The levels of gene flow detected by these genetic analyses indicate that P. scapulatus is effectively panmictic. Keywords." Pteropus, population genetics, allozyme electrophoresis, RAPD, panmictic.

INTRODUCTION

Mickleburgh et al. (1992) and Wilson and Graham (1992) reviewed the management and conservation of Old World fruit bats or flying-foxes (Megachiroptera: Pteropodidae), many of which have experienced range contractions and declines in numbers. Seven have *Present address: Department of Zoology, University of Western Australia, Nedlands, WA 6009, Australia +Present address: Agricultural Research Institute, Wagga Wagga, NSW 2650, Australia Correspondence to: C. R. Tidemann. Tel: 61 06 249 2582; Fax: 61 06 249 0746; e-mail: [email protected] 45

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E . A . Sinclair et al.

C3 t~

LismOte

Grallon

Fig. 1. Distribution of Pteropus scapulatus (Hall, 1987), (enclosed by dotted line) and sampling sites used in this study (solid squares). See text for dates of collections and sizes of samples.

information which would enable better informed decisions to be made about management of the species. P. scapulatus is not rare or endangered, but management of it (and other Pteropus spp.) has been problematical because its range spans five States or Territories, each with its own system of managing wildlife. Until 1994 P. scapulatus was treated in one State as vermin, because of its sometimes severe depredations on fruit crops, while in others it was protected as native fauna. Recent changes to legislation in Queensland (19 December 1994) mean that P. scapulatus is protected everywhere in Australia, although permits to destroy animals raiding fruit crops may still be issued in some areas (Vardon & Tidemann, in press). Clearly, it is important for effective management at the species level that the pattern of movements within the overall range of the population be understood.

MATERIALS AND METHODS

Population sampling resources in cetaceans (Hoelzel, 1992) and some Australian flora and fauna (Hopper & Coates, 1990). Recent modifications by Williams et al. (1990) to the Polymerase Chain Reaction (PCR), originally developed by Mullis et al. (1986), have allowed rapid genetic analyses of large numbers of individuals in a relatively short time. Random Amplified Polymorphic DNAs (RAPDs) are fragments of D N A produced when genomic DNA is amplified using short (___ 10 base pairs), random sequence primers during a PCR reaction. The RAPD technique has the advantages that it uses small quantities of DNA, requires no prior knowledge of the genome and provides a large number of markers (Hadrys et al., 1992). The major disadvantage of the RAPD technique is that it is not readily possible to identify the products of different loci, and the markers are genetically dominant, thus the data cannot be analysed using existing formulae developed for more conventional markers (Lynch & Milligan, 1994). The technique has been used for a number of applications including the construction of linkage maps (Carlson et al., 1991), parentage analysis (Scott et al., 1992) and population studies (Dawson et al., 1993; Huff et al., 1993) The work described here forms one part of an overall investigation of population structure and genetic variation in Pteropus spp. in Australia (Webb & Tidemann, in press; Webb & Tidemann, in prep.). The objective of the present study was to determine the degree of genetic mixing within the population of P. scapulatus, to ascertain which of the three models of population structure, outlined by Richardson et al. (1986), best described the data. The three models considered were: (1) panmixia; (2) discrete subpopulations and (3) isolation-by-distance. Allozyme electrophoresis and RAPD PCR were used to determine genetic variation within and between subpopulations. The aim was to provide

Six subpopulations of P. scapulatus were sampled (Fig. 1): Grafton, January 1991 (n=20) and January 1993 (n=20); Dubbo, April 1993 (n=20); Lismore, January 1992 (n=17); Fergusson River, December 1992 (n=20); Mary Valley, September 1993 (n=20). Most samples were collected with a harp trap (Tidemann & Loughland, 1993), but animals from Dubbo were captured by hand and those from Fergusson River were shot. one millilitre of blood was collected in 150 ~1 acid-citratedextrose (ACD) solution for allozyme analysis. Blood samples were kept on ice and centrifuged in the field and were then transported to the laboratory in a portable freezer at -20°C. In the laboratory samples were stored at -70°C until required. Punches of wing membrane (6 mm in diameter) were kept in 5 M NaCl solution for subsequent DNA analysis. Ailozyme electrophoresis Samples were prepared from frozen red blood cells. Cells were lysed in an equal volume of distilled water containing 1 mg/ml NADP and then centrifuged for 20 min to remove the cell debris. Aliquots of solution were prepared for each sample, labelled and kept at -70°C until required. Thirty-five loci were examined for polymorphisms, using Titan (Helena Laboratories, Beaumont, TX) gel electrophoresis. Standard electrophoretic procedures were used, as outlined by Richardson et al. (1986).

Polymerase chain reaction Total genomic DNA was extracted from wing punches using a technique modified from Jackson et al. (1991). The PCR reactions were carried out using a protocol modified from Williams et al. (1990). Samples were prepared in 25 ~1 aliquots using 2.5 ~1 of 10X PCR buffer (150 mM ammonium sulphate, 500 mM Tris HC1 (pH 8.0), 20 mM magnesium chloride, 2 mg/ml BSA, 4.5% triton × 100, 2 mM dNTPs), 0-4 /~m primer, 1 p3 of 1 × 10~ M tetramethylammonium chloride (TMAC),

Genetic variation in Pteropus scapulatus in Australia 1 unit of Taq polymerase (Pharmacia, Uppsala, Sweden), 0.5 /zl D N A (100 ng//zl) and 20.5 /~1 of sterile water. Mixtures were overlayed with mineral oil. The cycle consisted of a 5-min initial denaturation at 94°C, 30 s at 94°C, 1 min at 35°C and 5 min at 72°-C for 5 cycles, then 30 s at 94°C, 1 min at 40°C and 5 min at 72°C for 40 cycles. The cycles were performed in a Perkin-Elmer Cetus (Norwalk, CT) thermal-cycler. R A P D extraction R A P D 10-mer primers (Kit O, Operon Technologies Inc., Alameda, CA) were used to extract RAPDs. PCR amplification products were separated in 1-5% agarose gels stained with ethidium bromide and viewed under UV light. After initial screening primers which produced clear, polymorphic bands were selected for the analysis. All individuals were scored for the presence or absence of specific PCR products. PCR reactions from the same two individuals were run on the outer tracks of all gels to aid in the scoring of bands between different gels. A D N A size marker (Spp-1 cut with EcoR1) was also run on each gel. Only those bands that were clear and consistent were scored, and in cases where amplification products from one or more primers were absent, these animals were excluded from the analyses. R A P D results are only presented for the four subpopulations that were extracted and amplified at the same time (Grafton 1, Grafton 2, Lismore and Fergusson River) since the technique is known to be very sensitive to slight changes in PCR conditions that can occur between experiments (Hadrys et al., 1992). Data analysis Biosys-1 (Swofford & Selander, 1981) was used to analyse the electrophoretic data. Genotype frequencies were tested for deviations from Hardy-Weinberg equilibrium. Wright's F-statistic was calculated to determine population structure and a U P G M A cluster analysis was performed using similarity matrices derived from

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Nei (1972). The number of migrants per generation (N,,) was calculated using the FsT value (Slatkin, 1985). Simple regression was used to examine the relationship of genetic distance and geographic distance between subpopulations. Euclidean distance matrices were generated from the R A P D band presence/absence data for individual primers separately and for the whole data set. The distance matrices were analysed using W l N A M O V A 1.5 (Excoffier et al., 1992) which provides an analysis of molecular variance, calculating variance components within and between subpopulations (Huff et al., 1993). The significance of components was calculated by a non-parametric permutational procedure.

RESULTS Allozyme electrophoresis Ten of the 35 loci examined were variable, with only four satisfying the criteria of being interpretable and having the most common allele at a frequency