(Egernia Cunninghami): Evidence from Allelic and Genotypic Analyses of Microsatellites

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Molecular Ecology (2001) 10, 867–878

BlackwellThe Science, Ltd impact of habitat fragmentation on dispersal of Cunningham’s skink (Egernia cunninghami): evidence from allelic and genotypic analyses of microsatellites

A. J. STOW, P. SUNNUCKS,* D. A. BRISCOE and M. G. GARDNER† Department of Biological Sciences, Macquarie University, NSW 2109, Australia

Abstract The effects of habitat fragmentation on processes within and among populations are important for conservation management. Despite a broad spectrum of lifestyles and the conservation significance of many reptiles, very little work on fine-scale population genetics has been carried out on this group. This study examines the dispersal patterns of a rock crevice-dwelling lizard, Cunningham’s skink (Egernia cunninghami), in a naturally vegetated reserve and an adjacent deforested site. Both genotypic and genic approaches were employed, using microsatellite loci. The spatial organization of individuals with respect to pairwise relatedness coefficients and allele frequencies, along with assignment tests, were used to infer dispersal characteristics for both sexes in a natural and a cleared area. The distribution of relatedness in both habitats was spatially structured, with E. cunninghami showing high pairwise relatedness within their rocky retreat sites. Analysis of relatedness over different spatial scales, spatial autocorrelation of alleles and assignment tests, all indicated that both sexes in the cleared area show less dispersal than their counterparts in the reserve. Furthermore, deforestation may inhibit female dispersal to a greater extent than that of males. The geographical structuring of allele frequencies for adults in the cleared area, but not the reserve, indicates that habitat fragmentation has the potential to alter at least the microevolution of E. cunninghami populations. Keywords: dispersal, Egernia cunninghami, microsatellite DNA, relatedness, sex-bias, spatial autocorrelation Received 7 June 2000; revision received 5 November 2000; accepted 5 November 2000

(e.g. how fragmentation affects mate choice and associated Introduction inbreeding) have received relatively little attention. Beha- Increasing fragmentation of native habitats necessitates viours such as multiple mating, kin-avoidance and sex- greater understanding of the factors that influence both biased dispersal are suggested mechanisms for inbreeding short- and long-term persistence of wild populations. avoidance (e.g. Olsson et al. 1994; Bull & Cooper 1999). Some effects of habitat fragmentation have been well Inbreeding in many species has been demonstrated to researched. In particular, isolation of habitat patches can reduce individual and population fitness, to be associated result in loss of genetic diversity within, and increased with elevated rates of population extinction and to decrease differentiation among them, through reduced dispersal evolutionary responsiveness (Frankham 1995, 1998; Madsen and consequent genetic drift (e.g. Sarre et al. 1990). However, et al. 1996; Olsson et al. 1996; Lacy 1997; Saccheri et al. 1998; effects of habitat fragmentation that could systematically Eldridge et al. 1999; Frankham et al. 1999). Knowledge alter the nature of within-population behaviour and genetics of the influence of habitat fragmentation on population processes associated with inbreeding is therefore important in conservation management. Correspondence: A. Stow. Fax: +61 29 850 8245; E-mail: Most genetic studies have investigated population [email protected] Present addresses: *Department of Genetics, La Trobe University, substructure using allele frequencies rather than genotypic Victoria 3083, Australia. †Evolutionary Biology Unit, South arrays, and consequently have not assessed genetically Australian Museum, North terrace, Adelaide, SA 5000, Australia. effective dispersal on the shortest timescales (Sunnucks

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2000). Short-term dispersal has frequently been assessed priori whether anthropogenic interference will have an using direct ecological methods such as radio telemetry influence on the genetic structure of E. cunninghami. While and capture−mark−recapture (CMR). However, new extensive clearance for agriculture has often resulted in analytical methods have been developed to exploit the scattered islands of rock outcrops in a ‘sea’ of grass, suitable information on short-term and fine-scale processes that habitat is naturally patchy owing to the spatial distribution can be extracted from individual genotypes based on of outcrops. This study examines the impact of land clearing hypervariable markers (reviews in Luikart & England on the dispersal patterns of E. cunninghami by examining 1999; Sunnucks 2000). Assignment tests can identify the genetic structure of male and female adults and immature migrants by using genotypic data to determine the popu- animals using six polymorphic microsatellite loci, in an area lation with which an individual has the highest likelihood cleared for grazing and an adjacent, naturally vegetated of belonging (reviews in Waser & Strobeck 1998; Davies reserve area. Hypotheses on dispersal are tested using the et al. 1999). Patterns of dispersal can also be inferred by assignment test and by analysing the spatial distribution examining the spatial organization of pairwise relatedness of relatedness and alleles. coefficients between individuals (relatedness structure). For example with sex-biased dispersal, individuals of the Materials and methods philopatric sex in close proximity to one another will have high pairwise relatedness relative to members of the Study species dispersing sex, and declining pairwise relatedness with distance (Taylor et al. 1997; Knight et al. 1999). By testing Egernia cunninghami is a diurnal, scincid lizard that dispersal hypotheses over a range of distance classes, grows to 250 mm snout–vent length and 350 g in weight. subtle differences in dispersal patterns may be recognized. It is viviparous, usually giving birth to 2–6 offspring. Adults Both Mantel tests (Mantel 1967) and spatial autocorrelation are omnivorous with their adult diet consisting largely of (Sokal & Oden 1978a,b) can be employed for this purpose. vegetation (Barwick 1965). Throughout much of its range The new molecular analyses outlined above make in eastern Australia, the species is predominantly confined several contributions over and above those of direct to rocky areas where it utilizes rock crevices as retreat sites. ecological methods. Molecular methods can be applied to In the elevated country of the north, central and southern species with dispersal rates too low to be observed in the tablelands of New South Wales (NSW), E. cunninghami are field, but still high enough to affect genetic structure. often abundant, with cohorts of different ages residing Observed dispersal may be irrelevant, in an evolutionary together in deep fissures within granite outcrops. A previous context, if the disperser does not breed (Johnson & Gaines CMR study indicated very localized movements in 1990). Furthermore, molecular methods may be more E. cunninghami, with the longest movement recorded in appropriate for use with threatened species because less 4 years being 70.1 m (Barwick 1965). It was also suggested invasive approaches, such as remote sampling of genetic that fallen logs provide a bridging habitat between rock material, can be employed (e.g. Sloane et al. 2000; review in outcrops, but that E. cunninghami avoids dense tree cover, Taberlet & Luikart 1999). because this leads to debris in crevices and restricted Suggested reasons why many vertebrates disperse include basking opportunities. inbreeding avoidance (e.g. Pusey & Wolf 1996) and reduc- tion in resource competition (e.g. Greenwood 1980, 1983). Sample collection, DNA extraction and microsatellite Sex-biased dispersal reduces inbreeding as it separates typing siblings of the opposite sex before mating (e.g. Pusey 1987). Models for sex-biased dispersal have been based largely A total of 189 lizards was sampled from March 1998 to on data for mammals and birds, with a paucity of data on October 1999, at one location on the central tablelands of reptiles (Doughty et al. 1994). Sex-biased dispersal has, NSW (33°33′ S, 149°55′ E). Three different ‘habitats’ were however, been demonstrated for some reptiles. For example, sampled; a site cleared of forest then grazed by sheep and male-biased dispersal has been shown in the side-blotched cattle for at least 100 years, one forested area that was also lizard (Uta stansburiana) using CMR (Doughty et al. 1994), grazed, and adjacent Evans Crown Nature Reserve (Fig. 1). and in the Galápagos marine iguana (Amblyrhynchus cristatus) The present genetic analysis is based on samples from 68 using contrasting nuclear DNA (nDNA) and mitochondrial adults and 75 immature animals from the cleared site and DNA (mtDNA) differentiation among islands (Rassman adjacent reserve, with populations of animals in the grazed et al. 1997). The importance of understanding the impact of forested area included only as possible source populations habitat fragmentation on inbreeding avoidance behaviours in assignment tests. Lizards were either captured by hand prompted us to assess this impact on dispersal patterns of or using unbaited magnum-sized Elliot Traps placed at the Cunningham’s skink (Egernia cunninghami). This lizard entry of rock fissures. Lizards were not observed in every lives in large groups within rock crevices. It is not clear a rock outcrop, nor were all occupied retreat sites sampled

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Fig. 1 Sampling locations of Egernia cunninghami in three different habitats: reserve, cleared and grazed, and forested and grazed.

successfully. Sampled retreat sites were spaced such that lite loci. The microsatellite loci used were: ﬁve (AAAG)n lizards captured within 10 m of one another were assigned developed from Egernia stokesii (Est1, Est4, Est9, Est12, to one group. A total of 50 lizards was captured in the Est13, Gardner et al. 1999), and four cloned from Tiliqua

reserve area, and 93 in the cleared site. Lizards were rugosa (Cooper et al. 1997), one (AAAG)n (Tr3.2), a sex-linked weighed and snout–vent, head length and head width (AAC)n (Tr4.11) and two dinucleotides (CA)n (Tr5.21) and measurements taken. They were sexed by attempting to (TC)n(CA)n (Tr5.20). The sex-linked Tr4.11 was used to sup- extrude a hemipenis in each specimen; visualization of plement information on sex obtained anatomically (all a hemipenis positively identifies a male but failure is heterozygous individuals are female). Alleles were detected inconclusive evidence of sex. Lizards were implanted with as described in Gardner et al. (1999) with some differences in a passive transponder (TROVAN ID 100) for identification amplification conditions: we used Promega Taq polymerase to prevent resampling and to obtain repeat location data. A with polymerase chain reaction (PCR) cycling conditions tissue sample was taken from the tip of the tail and placed (‘touchdown’ programs) as follows: initial denaturation in 100% ethanol. Adults were defined on the basis of weight 94 °C 2 min, followed by one cycle of v °C 30 s, 72 °C 45 s, and snout–vent length following Barwick (1965). Only 94 °C 15 s; one cycle of w °C 30 s, 72 °C 45 s, 94 °C 15 s; one adult lizards were included for sex-based comparisons. cycle of x °C 30 s, 72 °C 45 s, 94 °C 15 s; one cycle of y °C DNA extraction was carried out using the salting-out 30 s, 72 °C 45 s, 94 °C 15 s; 30 cycles of z °C 30 s, 72 °C 45 s, procedure described in Sunnucks & Hales (1996) and all 94 °C 15 s; final extension 72 °C for 2 min. PCR program for individuals were typed for nine polymorphic microsatel- loci Est13, Tr4.11, Tr3.2: v = 62, w = 61, x = 59, y = 57 and

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870 A. J. STOW ET AL.

Table 1 Number of alleles and size range (A) and heterozygosity estimates per locus in each habitat for Egernia cunninghami Analysis of relatedness Randomization tests were used for significance testing Locus A H H O E of differences in levels of relatedness, to overcome the Cleared nonindependent nature of pairwise comparisons. Mantel Est13 16 (164–244) 0.947 0.912 (1967) testing was used to test for the overall relationship Tr3.2 20 (169–269) 0.891 0.906 of relatedness with geographical distance using multreg Tr5.21 18 (79–145) 0.860 0.888 (Goudet 1999). Mantel tests were conducted with 10 000 Tr5.20 3 (146–152) 0.266 0.291 permutations, for immature animals and for adult males and Est9 8 (219–279) 0.419 0.446 females in both habitats. To examine the influence of factors Est1 20 (209–337) 0.957 0.913 including spatial scale and sampling density, pairwise Reserve Est13 17 (180–252) 0.868 0.926 relatedness coefficients were formulated into three data sets Tr3.2 20 (161–261) 0.961 0.928 (classes); (1) all values, (2) all values excluding within-group Tr5.21 15 (79–145) 0.887 0.875 comparisons, and (3) all values with pairwise distance > 250 m. Tr5.20 3 (146–152) 0.260 0.268 Class 3 was included as it yielded closer numbers of pairwise Est9 12 (215–267) 0.660 0.646 comparisons between habitats. In addition, comparison of Est1 20 (209–329) 0.940 0.883 slopes (b) of all individual regressions of R on log-transformed geographical distance was carried out (where an individual regression analyses pairwise R-values and the corresponding pairwise geographical distance between the ith individual z = 55; Est1, Est4, Est9, Est12, Tr5.20, Tr5.21: v = 55, w = 53, and all other individuals, Knight et al. 1999). This method x = 51, y = 49 and z = 47. allows the distribution of individual spatial relationships Of the nine loci typed, Est4 had a high proportion of null with pairwise R, within a particular class, to be tested directly alleles and another two (Est12 and Tr3.2) are in strong with another. These tests were conducted for each sex in linkage disequilibrium (LD) (A. Stow unpublished data). each habitat using the Mann−Whitney U-test, because some Given that high LD for these two loci is also seen in data could not be normalized. To compare pairwise R-values E. stokesii and confirmed by analysis of family data (Gardner for individuals belonging to the same group (i.e. captured et al. 2000) it is reasonable to assume close physical linkage within 10 m of each other), and to compare these values between the loci. As the following analyses require inde- with pairwise R-values between individuals captured in pendently segregating loci, Est12 was removed, as was different groups, randomization testing of the difference in Tr4.11 (sex-linked) and Est4 (nulls), leaving six loci for the means was conducted. This was carried out using the samp calculation of relatedness, assignment tests and spatial module of rt version 2.1 (Manly 1997) with 5000 permuta- autocorrelation of alleles. Characteristics of the loci used, in tions. These tests were applied to adults of each sex, and both habitats, are given in Table 1 together with hetero- immature E. cunninghami, in each habitat. zygosity estimates calculated using genepop version 3.1d (Raymond & Rousset 1995). All individuals gave a PCR Assignment tests product for all loci and expected and observed heterozygosity gave no indication of the presence of null alleles. Groupings of lizards were tested for migrants using an Of 62 common alleles (frequency > 5%), three were site assignment test. A maximum likelihood method developed specific. by Rannala & Mountain (1997) was used to calculate likelihoods-of-belonging using the program geneclass (Cornuet et al. 1999). This method was chosen as it takes Estimation of relatedness into account differences in diversity between two differ- The Macintosh program kinship version 1.2 (Goodnight ent source populations and the sampling error associated et al. 1998) was used to calculate the relatedness coefficient with estimating allele frequencies. The group with the (R) for all possible pairwise comparisons within cleared highest likelihood of being the source of each individual and natural habitats. To compare within-habitat processes, was determined (using a priori groupings of lizards with such as the relationship between pairwise relatedness individual to be assigned not included in its capture- and distance, relatedness coefficients were calculated with group for calculation). For individuals assigned to a group respect to allele frequencies in the sample of adults from other than that in which they were captured, the distance the target habitat. To compare absolute within-group between the capture site and inferred natal site was differences in relatedness of individuals in the two habitats, calculated. Mann−Whitney U-tests were used to test the the frequencies used to calculate R came from the sample null hypothesis of no difference in these distances between of lizards from both habitats combined. sexes and habitats.

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Table 3 Spatial autocorrelation indices (II) and significance values Spatial autocorrelation analysis for adult male, adult female and immature Egernia cunninghami at Approaches based on arrays of loci, such as assignment tests the cleared and reserve site and relatedness structure, illuminate different aspects of Female Male Immature population processes to those based on single-loci (Sunnucks 2000). In order to test for habitat differences in the spatial II P II P II P structure of allele frequencies, spatial autocorrelation (SA) analysis was applied. SA has the advantage over Mantel Cleared testing of providing results on the shape of the spatial 1 0.3462 < 0.005 0.075 < 0.005 0.231 < 0.005 relationship. SA of alleles was calculated using the pro- 2 –0.0967 < 0.005 –0.054 < 0.005 –0.051 < 0.005 Reserve gram aida (Bertorelle & Barbujani 1995). Presence/absence 1 –0.051 n.s –0.002 n.s 0.141 < 0.005 matrices of allele spatial distributions were constructed 2 –0.112 < 0.05 –0.103 n.s –0.091 < 0.005 with homozygotes weighted = 2 and heterozygotes = 1. AIDA coefficients analogous to Moran’s I (II) are presented Class 1, within-group; Class 2, all data excluding within-group (Bertorelle & Barbujani 1995). Data were placed in two comparisons. classes: (i) individuals within the same group, (ii) individuals in different groups. Confidence intervals were calculated separately for these two distance classes using a random- and immatures P < 0.0002, all tests remain significant at the ization procedure with 1000 permutations. 5% level when type 1 error levels are corrected for multiple Following Sunnucks et al. (1997), we refer to data, tests, Rice 1989). Given that R-values are scaled such that approaches and results based on single loci as ‘genic’, and mean R = 0 in each habitat and theoretical R for second those based on arrays of loci as ‘genotypic’. order relatives is 0.25 and first order relatives is 0.5 (Queller & Goodnight 1989; Blouin et al. 1996), mean (± SD) within- group R-values in the cleared site (0.253 ± 0.274) and the Results reserve site (0.264 ± 0.247) demonstrate a high level of within-group relatedness. Egernia cunninghami lives in groups of highly related individuals Land clearing is associated with reduced dispersal in adult Within both cleared and reserve sites, Egernia cunninghami E. cunninghami lived as groups of highly related individuals. Genotypic similarity (pairwise R) was significantly higher within Adult lizards sampled within the cleared site showed more than among groups for adults of each sex and immature geographically localized genetic structure, and thus lower animals in both habitats (Table 2; cleared area adult levels of inferred dispersal, than those within the reserve females and immatures P < 0.0002, adult males P = 0.0094; site. This was evident in both genotypic and genic data. reserve area adult females P = 0.031, adult males P = 0.0024 First, adult lizards in the cleared site showed significantly elevated allelic similarity within groups and significant allelic dissimilarity among groups (Table 3). In comparison, adult male lizards in the reserve did not show significant Table 2 Mean relatedness (R) and SD between pairs of adult allelic dissimilarity among groups, and neither sex showed male, adult female and immature Egernia cunninghami within and among groups for each habitat, cleared and reserve. Relatedness allelic similarity within groups in the reserve (Table 3). This coefficients were calculated from habitat-specific allele frequencies. is in contrast to the high within-group genotypic similarity N = number of pairwise comparisons in each class (pairwise R) evident in the reserve (Table 2), implying that dispersal has been sufficiently high to prevent significant Within-group Among-group genic structuring. Consistent with this, genotypic similarity Lizard and in the reserve did not decline significantly with distance habitat category (N) Mean (R)SD (N) Mean (R)SD between individuals, whereas this relationship was sig- Cleared nificant in the cleared area (Table 4; class 1, Fig. 2a,b,d,e). Males 37 0.101 0.219 314 0.016 0.195 Finally, lower levels of dispersal in the cleared site compared Females 9 0.383 0.210 127 –0.064 0.180 with the reserve site are also suggested by movements Immature 132 0.290 0.758 999 –0.014 0.190 inferred from assignment tests. Adults in the cleared site Reserve show smaller distances (verging on significance, Z = –1.89, Males 11 0.161 0.235 94 –0.011 0.165 P = 0.056) between their inferred natal site and subsequent Females 7 0.115 0.116 48 0.003 0.101 capture sites (± SE) (251.3 ± 51.2 m) than adults in the Immature 64 0.372 0.368 243 0.028 0.171 reserve (477.2 ± 8 1.5 m).

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Fig. 2 Relationship between pairwise R and corresponding distance values for all male and female adults and immature Egernia cunninghami in each habitat, cleared and reserve. R-coefﬁcients were calculated using habitat speciﬁc allele frequencies. (a) Adult females in the cleared site. (b) Adult males in the cleared site. (c) Immature animals in the cleared site. (d) Adult females in the reserve site. (e) Adult males in the reserve site. (f) Immature animals at the reserve site.

HABITAT FRAGMENTATION AND LIZARD DISPERSAL 873

Table 4 Mantel testing of pairwise relatedness values (R) with groups in the cleared site and nonbiased dispersal in the geographical distance reserve.

Females Males Immature Relatedness among potential breeding partners NP NP N P Adult male and female E. cunninghami of the same group Cleared in the cleared area were on average more related than their 1 136 0.003 351 0.043 1176 0.0002 counterparts in the natural reserve area, but this difference 2 127 0.041 314 0.088 1044 0.054 was not significant (P = 0.281; Table 5). 3 82 0.017 184 0.058 584 0.037 Reserve 1 45 0.419 91 0.560 325 0.004 Low levels of dispersal in immature E. cunninghami 2 38 0.695 81 0.875 261 0.990 3 35 0.469 68 0.841 231 0.966 Immature lizards in the reserve site showed much lower levels of inferred dispersal than did adults, as demonstrated Significance values are for a negative correlation of R with by strong declining genotypic relatedness with distance for geographical distance and 10 000 permutations. R coefficients immature animals, but not for adults (Table 4; class 1). were calculated from habitat-specific allele frequencies. Strong site fidelity for immature animals relative to that N = number of pairwise comparisons in each class. 1, all values; of adults at the reserve was also demonstrated by genic 2, all values excluding within-group comparisons; 3, all values with pairwise distance > 250 m. data. Only immature animals in the reserve site showed significant allelic similarity within groups, and strongly significant allelic dissimilarity among groups (Table 3). The finding of increasing pairwise R with distance for Land clearing inhibits female dispersal to a greater extent immature animals in the reserve, when the within-group than that of males class is removed (Table 4, class 2 and 3) was unexpected Lower levels of female dispersal than that of males in the and requires further investigation. There was no strong cleared site can be seen in several analyses. First, there was evidence for differences in dispersal for immature lizards significantly higher pairwise R within groups for adult between the cleared and reserve site. In both sites immature females in the cleared site than those in the reserve site lizards show strong allelic structure (Table 3) and there (P < 0.001; Table 5) while the difference for males was is no significant difference in pairwise R of individuals not significant (P = 0.152; Table 5). Second, the slopes for sharing a group (P = 0.846; Table 5), thus showing that the individual regression lines differed significantly between dispersal differences between the two sites mostly arise females in the cleared site and those in the reserve, showing through movements of individuals older than those we a stronger decline in pairwise R over distance for females have classed as immature. in the cleared site (Z = –2.134, P = 0.032; Table 6, Fig. 3a,b). This difference in regression slopes was not apparent for Effects of sample size differences between sites males (Z = –0.332, P = 0.740; Table 6, Fig. 3a,b). Third, there was a greater disparity between male and female dispersal Field observations suggest that lizard abundance in the in the cleared site compared with the reserve site. This is reserve site is lower than that in the cleared site (A. Stow shown by female lizards having significantly higher within- unpublished data). Accordingly, sample size is lower in the group pairwise R than males in the cleared site (P = 0.003, reserve site. Differing sample size is unlikely to account for Table 5), but not in the reserve site (P = 0.647, Table 5). In the between-site differences observed. The strongest effect addition, females showed a significantly stronger decline associated with deforestation is reduced female dispersal. of pairwise R with distance than did males (Z = –2.784, This is evident from comparisons of within-group pairwise P = 0.005; Table 6, Fig. 3a), whereas there was no such R, based on similar sample sizes, nine in the cleared site vs. significant difference between males and females in the seven in the reserve. To ensure that sample size was not reserve (Z = –0.217, P = 0.828; Table 6, Fig. 3b). Moreover, biasing the result, the nine pairwise R-values in the cleared females in the cleared site had significantly smaller site were randomly subsampled 10 times to seven values, natal−capture distances (± SE) (114.2 ± 14.2 m) inferred resulting in substantially higher mean within-group R from assignment tests than did males (313.8 ± 65.0 m; Z = (0.398 ± 0.026 SD) than that attained for females in the –2.021, P = 0.042), whereas there was no such difference reserve (mean R = 0.086, Table 5). The seven lowest within- between sexes in the reserve site (males 467.5 ± 77.7 m, group pairwise R-values for adult females in the cleared females 482.9 ± 125.2 m; Z = –0.214, P = 0.786). Finally, the site, compared with reserve values, still result in significantly pattern of within-group pairwise R (Table 7, Fig. 4a,b) higher within-group relatedness for females in the cleared indicates the generality of inferred female philopatry for site (P < 0.001). Furthermore, there is no reason to believe

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Table 5 Mean and SD of R, at both habitats, for pairs of adult for conservation management. Similar effects have rarely male, adult female and opposite sex pairs from the same group been investigated, although they were reported in experi- mentally fragmented root vole (Microtus oeconomus) popu- Lizard and lations (Jon & Rolf 1999). For that species, it was found habitat category N Mean (R)SD that the presence of habitat corridors significantly facili- Males tated female dispersal, while male dispersal was inde- Cleared 37 0.133 0.219 pendent of the presence or absence of habitat corridors. It Reserve 11 0.061 0.182 was suggested that habitat corridors may allow reproduc- Females tive females to transfer and thereby increase survival prob- Cleared 9 0.396 0.174 abilities of extinction-prone demes of root voles. Reserve 7 0.086 0.116 In this study, genetic structuring was more pronounced Opposite sex Cleared 42 0.160 0.298 and inferred dispersal was lower for adult E. cunninghami Reserve 18 0.118 0.144 in the cleared site than in the reserve. Effects of reduced Immature dispersal after habitat fragmentation have been inferred Cleared 132 0.315 0.287 for the reticulated velvet gecko (Oedura reticulata), by Reserve 64 0.358 0.264 lower mtDNA haplotype diversity in fragments relative to reserve areas (Sarre 1995). The impact of habitat frag- R coefficients were calculated using allele frequencies from both mentation on populations of mammals has received more habitats combined. N = number of pairwise comparisons. attention than its effects on reptiles. For mammals, reduced dispersal between fragments has been reported in the pika (Ochotona princeps), prairie dog (Cynomys ludovicanus) and Table 6 Mean and SD for slope values (b) for regressions of R on meadow vole (Microtus pennsylvanicus) among others, with distance for individual male and female adult Egernia cunninghami reduced dispersal often attributed to risks of predation at both the cleared and reserve site (Peacock & Smith 1997 and references therein). Assessment of why dispersal differences between the cleared and Habitat and reserve site are evident for adult E. cunninghami requires lizard category (N) Mean (b)SD data not collected for this study (e.g. predation levels). Cleared While a higher predation risk for E. cunninghami in the Male 27 –0.014 0.036 cleared site has intuitive appeal, other differences may also Female 17 –0.046 0.041 affect inferred dispersal patterns. These differences may Reserve include factors such as inbreeding avoidance and resource Male 14 –0.010 0.041 availability such as food or thermoregulatory constraints Female 10 –0.010 0.053 placed by shading from overhanging trees. Other than being nonreproductive, immature lizards may be less affected by R coefficients were calculated using habitat-specific allele frequencies. N = number of individuals. such differences than adults. Thermoregulatory constraints could differ as size may affect solar absorbance (e.g. Christian et al. 1996). Foraging characteristics and food availability may also be dissimilar due to dietary differences that low sample size would systematically underestimate between juvenile and adult E. cunninghami, with a shift pairwise R. from carnivory as juveniles to predominantly herbivory as adults (e.g. Barwick 1965; Brown 1983). In addition, potential effects of structural differences between the two sites need Discussion to be resolved by repeating these comparisons at another Although Egernia cunninghami has a naturally fragmented location. Other than deforestation, grazing and other habitat of rocky areas harbouring highly related individuals, anthropogenic impacts, the two sites differ topographic- clearing of vegetation is associated with greater isolation of ally, with the reserve sampling sites generally being habitat patches and differences in the organization of located on steeper terrain with equivalent structure of within-group relatedness. Lowered dispersal between rock outcrops. As E. cunninghami can competently climb groups is evident in both sexes in the cleared site. However, vertical rock faces, its hard to imagine that slope would be clearing of vegetation was associated with lower female than important to dispersal. Topography may also influence male dispersal, resulting in male-biased dispersal in the thermal qualities due to aspect, however, in this respect, cleared site. canopy cover may be supposed to be of more importance. Sex-based differences in the effects of habitat fragmenta- Lower levels of dispersal in the cleared site have resulted tion in E. cunninghami could have important implications in differences in the organization of relatedness within

HABITAT FRAGMENTATION AND LIZARD DISPERSAL 875

Fig. 3 Frequency distribution of slope values (b) of relatedness on distance for male and female adults. Negative slope values indicate declining pairwise R with distance. (a) Cleared site. (b) Reserve site.

Table 7 Mean relatedness (R) between pairs of adult male (m), adult female (f) and immature (I) Egernia cunninghami within sample locations (L)

Cleared Reserve

Mean Mean Mean Mean Class L N R L N R L N R L N R

m 1 5 0.108 5 0.080 1 3 0.382 0 f 1 7 3 0.312 2 0.354 7 0 I 10 0.183 8 0.165 1 4 0.387 m 2 2 0.307 0 2 3 0.130 0 f 2 0.457 8 1 3 0.170 8 1 I 6 0.369 3 0.232 8 0.202 2 0.289 m 3 2 –0.009 4 –0.030 3 2 0.233 1 f0 900 90 I 8 0.505 4 0.474 8 0.612 0 m 4 1 0 4 3 –0.011 0 f 1 10 2 0.134 1 10 0 I 3 0.329 0 0 1 m 5 4 0.171 3 0.386 5 2 0.038 f 3 0.490 11 1 0 I1 1 0 m60 1 60 f 1 12 2 0.404 3 –0.019 I 3 0.410 2 0.494 2 0.085

Relatedness coefﬁcients were calculated from habitat-speciﬁc allele frequencies. N = Number of adult individuals for each sex and immature lizards per group.

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Fig. 4 Relatedness (R) between pairs of adult male, adult female and immature Egernia cunninghami within each group, for both habitats. Relatedness coefﬁcients were calculated from habitat-speciﬁc allele frequencies. (a) Cleared site. (b) Reserve site.

groups of E. cunninghami. A greater accumulation of close with altered dispersal patterns in E. cunninghami at one female kin is associated with the removal of vegetation, as site, and we will replicate this natural experiment else- is male-biased dispersal. Male-biased dispersal may reflect where. Further studies will also assess the impact of habitat inbreeding avoidance (e.g. Pusey & Wolf 1996) due to fragmentation on other possible inbreeding avoidance higher levels of within-group adult relatedness in the strategies, as well as learning more about the biology of cleared site (Table 5). The effect of habitat fragmentation on this interesting species. other within-group processes, such as other inbreeding avoidance behaviours or the mating system, is yet to be Acknowledgements assessed in E. cunninghami. Inbreeding avoidance by choosing unrelated mates through kin recognition has been This is publication number 322 of the Key Centre for Biodiversity documented in the monogamous sleepy lizard (Tiliqua and Bioresources. The animals were handled in accordance to rugosa) and the monogamous Gidgee skink (Egernia stokesii) Macquarie University animals ethics committee recommendations and their collection licensed by the NSW National Parks and (Bull & Main 1996; Bull & Cooper 1999; MG Gardner, CM Wildlife Service. We thank Roy and Muriel Stephens for access to Bull, SJB Cooper, G Duffield 2001). Multiple mating, their property, Fara Pelarek and Sam Stow for assistance in the another inbreeding avoidance strategy, may dilute the field and Steve Cooper, Andrea Taylor and Sam Banks for helpful deleterious impacts of breeding with related individuals comments on an earlier draft of this manuscript. and has been documented in sand lizards (Lacerta agilis) along with male-biased dispersal in juveniles, to increase the fitness of offspring (Olsson et al. 1994, 1996). References E. cunninghami provides a model with which to study Barwick RE (1965) Studies on the scincid lizard Egernia cunninghami. the effects of habitat fragmentation on a species with a PhD Thesis. The Australian National University, Australia. naturally fragmented and highly localized range. We have Bertorelle G, Barbujani G (1995) Analysis of DNA diversity by demonstrated that the clearing of vegetation is associated spatial autocorrelation. Genetics, 140, 811–819.

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