The Tales of Two Geckos: Does Dispersal Prevent Extinction In

Molecular Ecology (2007) 16, 3299–3312 doi: 10.1111/j.1365-294X.2007.03352.x

BlackwellThe Publishing Ltd tales of two geckos: does dispersal prevent extinction in recently fragmented populations?

M. HOEHN,*† S. D. SARRE† and K. HENLE* *Helmholtz Centre for Environmental Research — UFZ, Department of Conservation Biology, Permoserstrasse 15, 04318 Leipzig, Germany, †Applied Ecology Research Group, University of Canberra, ACT 2601, Australia

Abstract Although habitat loss and fragmentation threaten species throughout the world and are a major threat to biodiversity, it is apparent that some species are at greater risk of extinction in fragmented landscapes than others. Identification of these species and the characteristics that make them sensitive to habitat fragmentation has important implications for conservation management. Here, we present a comparative study of the population genetic structure of two arboreal gecko species (Oedura reticulata and Gehyra variegata) in fragmented and continuous woodlands. The species differ in their level of persistence in remnant vegetation patches (the former exhibiting a higher extinction rate than the latter). Previous demographic and modelling studies of these two species have suggested that their difference in persistence levels may be due, in part, to differences in dispersal abilities with G. variegata expected to have higher dispersal rates than O. reticulata. We tested this hypothesis and genotyped a total of 345 O. reticulata from 12 sites and 353 G. variegata from 13 sites at nine micro-

satellite loci. We showed that O. reticulata exhibits elevated levels of structure (FST = 0.102 vs. 0.044), lower levels of genetic diversity (HE = 0.79 vs. 0.88), and fewer misassignments (20% vs. 30%) than similarly fragmented populations of G. variegata, while all these parameters

were fairly similar for the two species in the continuous forest populations (FST = 0.003 vs. 0.004, HE = 0.89 vs. 0.89, misassignments: 58% vs. 53%, respectively). For both species, genetic structure was higher and genetic diversity was lower among fragmented populations than among those in the nature reserves. In addition, assignment tests and spatial autocorrelation revealed that small distances of about 500 m through fragmented landscapes are a barrier to O. reticulata but not for G. variegata. These data support our hypothesis that G. variegata disperse more readily and more frequently than O. reticulata and that dispersal and habitat specialization are critical factors in the persistence of species in habitat remnants. Keywords: continuous, dispersal, effective population size, geckos, generalist, habitat fragmentation, microsatellites, population genetics, specialization Received 9 December 2006; revision accepted 20 March 2007

landscapes than others. Identification of these species and Introduction the characteristics that make them sensitive to habitat Habitat loss and fragmentation threaten species through- fragmentation has important implications for the manage- out the world and are a major threat to biodiversity ment of species as well as contributing to our understanding (Groombridge 1992; WCMC 1992). When formerly of ecological and evolutionary theory (Sarre et al. 1995; contiguous vegetation becomes fragmented through land MacNally et al. 2000; Davies et al. 2000, 2001, 2004; Henle clearing, small isolated populations will result for many et al. 2004a, b; Schmuki et al. 2006). species. Emerging empirical evidence suggests that some Dispersal between an individual’s birthplace and that of species are at greater risk of extinction in fragmented its offspring is one of the most important life-history traits in species persistence (Koenig et al. 1996; Clobert et al. 2001; Correspondence: Marion Hoehn, Fax: +49 341 235 3191; E-mail: Sumner et al. 2001). Dispersal will usually result in gene [email protected] flow, the movement and integration of alleles from one

3300 M. HOEHN, S.D. SARRE and K . HENLE population into another. Gene flow protects small popula- stochasticity also show contrasting expectations for the tions from processes such as reduction in the number of two species (Sarre et al. 1996; Wiegand et al. 2001, 2002). alleles, reduction in heterozygosity, and reduction in Whereas rates of persistence observed were lower for O. genetic diversity through genetic drift and inbreeding reticulata than those predicted by the population viability (Frankham et al. 2002; Keller & Waller 2002). Although model, the opposite was the case for G. variegata. One of dispersal used to be difficult to study, genetic markers the reasons for the discrepancies between predicted and provide a powerful tool for obtaining indirect estimates of observed distributions may be that the models did not dispersal and gene flow in natural populations. Recent include an expectation of dispersal between remnants for advances in statistical techniques and the analysis of micro- either species. While the dispersal capability of O. reticulata satellite data promise to simplify studies of dispersal and is believed to be low, pitfall trapping and anecdotal evidence colonization. For instance, assignment testing can improve suggest that movement by G. variegata is also infrequent the resolution of dispersal patterns by assigning individu- (Sarre et al. 1995; R. How, personal communication). als to their most likely population of origin (Paetkau et al. Nevertheless, occasional longer-distance dispersal is 1995; Rannala & Mountain 1997; Cornuet et al. 1999; Pritchard known for G. variegata (Moritz 1987; K. Henle & B. Gruber, et al. 2000). Like F-statistics, the basis of assignment protocols unpublished data.). Sarre et al. (1996) suggested that the is an estimate of the allele frequency within populations discrepancy between the observed and modelled persistence and therefore requires an a priori identification of popula- rates could be best explained by greater rates of movement tion boundaries. In contrast, analytical methods that do not for G. variegata than for O. reticulata. require information about population boundaries, such as To investigate further this dichotomy of responses to isolation-by-distance analysis and spatial autocorrelation, fragmentation, we used microsatellite DNA markers to can use genetic and geographical information to describe compare the genetic structure and rates of dispersal in the dispersal and genetic spatial structure in a two-dimensional two gecko species. Our expectation is that the high level of landscape (Rousset 1997; Peakall & Smouse 2001; Double persistence exhibited by G. variegata is a result of a higher et al. 2005). dispersal rate compared with O. reticulata. Thus, we pre- Here, we present a comparative study of the genetic dicted higher levels of genetic differentiation and lower rates structure of two gecko species in fragmented and continuous of dispersal among O. reticulata than among G. variegata woodlands. The species occur sympatrically, but have populations. We surveyed the genetic structure in fragmented responded differently to the fragmentation of their wood- and continuous populations of these two species to test our land habitat. Gehyra variegata (tree dtella) is abundant and hypotheses. In addition, we expected a loss of genetic widespread throughout the southern half of Australia. It is diversity and a higher level of genetic structure for both a habitat generalist, and can be found on trees, logs, fallen species in habitat fragments. timber, shrubs, rocks, and in highly disturbed habitat. Oedura reticulata (reticulated velvet gecko) is endemic to Materials and methods the southwest of Western Australia and is a habitat specialist. It is limited in its habitat range, being exclusively arboreal Study area and sampling and restricted to smooth-barked Eucalyptus woodlands. Both species have been subjected to severe population The study area was located between Kellerberrin and fragmentation in the Western Australian wheatbelt, where Trayning in the Western Australian wheatbelt (Fig. 1). numerous very small populations persist in patches of Large areas of native vegetation have been removed from woodland surrounded by land used for wheat crops. In the region and replaced by agricultural crops, pastures, summer, the intervening matrix is often reduced to sparse and livestock. Since 1900, approximately 93% of the original stubble or even predominantly bare earth (How & Kitchener vegetation has been cleared and the remnant vegetation is 1983; Kitchener et al. 1988; Sarre 1995a, b, 1998; Sarre et al. distributed over thousands of patches of varying size 1995). (Saunders & Hobbs 1991; Hobbs 1993; Saunders et al. 1993). In a previous empirical study, Sarre et al. (1995) demon- Tissue samples were collected from the two gecko spe- strated that G. variegata showed a markedly higher persistence cies during the summer months, from November 2000 than O. reticulata in habitat remnants (97% remnant occu- until March 2001, in December 2003, and in November pancy vs. 72%). In addition, studies 10 years apart (1991 2005. A total of 345 Oedura reticulata individuals from six and 2001) documented extinction of O. reticulata in three of habitat fragments and six locations in two nature reserves 33 habitat remnants, whereas G. variegata has persisted at and a total of 353 Gehyra variegata individuals from seven comparable population sizes in all 33 remnants over the habitat fragments and six locations in two nature reserves same time period (M. Hoehn, unpublished data). Individual- were sampled for genetic analysis. The majority of contin- based population viability models developed for both uous habitat has been cleared in the area, but three sites per species and including demographic and environmental species were located within 1 km in the North Bandee

DISPERSAL AND EXTINCTION IN FRAGMENTED GECKO POPULATIONS 3301

Fig. 1 Map of the study area and location of sites in the Western Australian wheatbelt. Oedura reticulata populations inhabit fragments labelled ORF1-6 and continuous sites labelled ORC1-3 (North Bandee Nature Reserve), Gehyra variegata fragments are labelled GVF1-7 and continuous sites GVC1-3 (North Bandee Nature Reserve). Korrelocking Nature Reserve with sites labelled ORC4-6 and GVC4-6 are not shown.

Nature Reserve, which comprises 174 ha of continuous sampling strategies (semi-experimental approach) for woodland habitat. Another three populations of each studying assignment and dispersal between pairs of habi- species where located within 1.2 km in the Korrelocking tat patches on a fine geographical scale. We selected three Nature Reserve (259 ha). pairs of O. reticulata populations, separated by 150, 550, Lizards were located at night using head-torches and and 580 m, and three pairs of G. variegata populations, sep- captured by hand. The tip of the tail of each individual was arated by 150, 300, and 1000 m (Table 1). In most cases the removed and stored in liquid nitrogen. In all fragments, distance to other extant populations was large and we con- 25–30 samples were collected with the exception of frag- sider that dispersal from individuals of other populations ment population 7 (N = 13) for G. variegata, where no fur- into the sample populations was likely to be very low. ther individuals could be located. The sample populations However, two neighbouring O. reticulata populations (ORF4 were labelled independently for each species, with one and ORF1) were close to the same roadside vegetation habitat fragment (or ‘patch’ or ‘remnant’) equivalent to one (150 m and 230 m distant), which could harbour a potential sample population. The O. reticulata populations were source population of O. reticulata or function as a corridor. labelled ORF1-6, and the G. variegata populations GVF1-7 Two neighbouring G. variegata populations were also close (Table 1). In two cases, a habitat remnant was used as a to roadside vegetation (0 m and 350 m distant), but in this sample population for both species, that is G. variegata popu- case the roadside vegetation did not create a connection lation GVF3 inhabited the same fragment as O. reticulata between the neighbouring habitat fragments. All other population ORF5, and similarly, G. variegata population sample populations, for both species, were separated from GVF6 occupied the same habitat patch as O. reticulata roadside vegetation by distances exceeding 400 m. In the population ORF4. All other sample populations were from North Bandee Nature Reserve, we selected two pairs of separate fragments, with no overlap between the two populations separated by 400 m and 650 m and in the species. Continuous woodland sites were used as sample Korrelocking Nature Reserve, two pairs of populations populations for both species and were numbered ORC1-3 separated by 700 m and 500 m for each species. and GVC1-3 in the North Bandee Nature Reserve and The census population size was determined with the ORC4-6 and GVC4-6 in the Korrelocking Nature Reserve. program capture (Otis et al. 1978) for another study (Hoehn The presence and absence of O. reticulata and G. variegata 2006). The values ranged from 33 to 197 individuals for populations in the study area was determined in previous O. reticulata and from 18 to 266 for G. variegata and we selected surveys (Sarre et al. 1995; M. Hoehn, unpublished data). populations so that the mean population size (124 and 95, This distribution data was used to design the following respectively) would be similar between species.

3302 M. HOEHN, S.D. SARRE and K . HENLE

Table 1 Habitat fragments and continuous forest sites sampled (C1-3 North Bandee Nature Reserve, C4-6 Korrelocking Nature Reserve) and reference number (ref) referring to the study of Sarre et al. (1995). The number of individuals genotyped (samples), fragment size (size in ha), population size (pop size) calculated by the program capture (Hoehn 2006), distances to neighbouring fragments (distance NF) and the road side vegetation (distance RSV) and, approximate time (years) since isolation from Sarre (1995a)

Fragment Ref Species Samples Size Pop size Distance NF Distance RSV Isolation

ORF1 170 Oedura 30 0.5 197 580 m 230 m 90 ORF4 — Oedura 27 0.4 35 580 m 150 m 90 ORF2 s85 Oedura 30 0.8 168 550 m 1000 m 60 ORF3 s84 Oedura 27 0.4 33 550 m 500 m 60 ORF5 442 Oedura 30 5.4 167 150 m 450 m 90 ORF6 — Oedura 30 2 141 150 m 450 m 90 ORC1 — Oedura 30 — — 400 m — — ORC2 — Oedura 17 — — 400 m/650 m — — ORC3 — Oedura 30 — — 650 m — — ORC4 — Oedura 31 — — 700 m — — ORC5 — Oedura 32 — — 700 m/500 m — — ORC6 — Oedura 31 — — 500 m — — GVF1 171 Gehyra 30 0.3 — 300 m 350 m 90 GVF2 — Gehyra 30 1.4 76 300 m 0 m 90 GVF3 442 Gehyra 30 5.4 266 150 m 450 m 90 GVF4 — Gehyra 30 2 127 150 m 450 m 90 GVF5 169 Gehyra 30 0.3 50 1000 m 400 m 80 GVF6 s143 Gehyra 25 0.5 30 1000 m 1000 m 80 GVF7 168 Gehyra 13 0.6 18 400 m 250 m 80 GVC1 — Gehyra 30 — — 400 m/650 m — — GVC2 — Gehyra 24 — — 650 m — — GVC3 — Gehyra 29 — — 700 m — — GVC4 — Gehyra 33 — — 700 m/500 m — — GVC5 — Gehyra 23 — — 500 m — — GVC6 — Gehyra 27 — — 400 m — —

Laboratory methods locus — were calculated using fstat 2.9.3 (Goudet 1995, 2001). Global tests for deviation from Hardy–Weinberg DNA was extracted from the tip of the tail of each indi- were performed. In addition, we tested heterozygote deficits vidual using the Chelex extraction method (Walsh et al. per locus and habitat fragment employing a sequential 1991). We genotyped individuals of O. reticulata using Bonferroni correction (fstat 2.9.3). nine tetranucleotide microsatellite loci developed from an The population genetic structure was investigated by enriched library for this species (OR205, OR220, OR266, estimating F using the method of Weir & Cockerham OR6F4, OR10H7, OR11G3, OR12D7, OR12D9, OR14A7) ST (1984) in the program fstat 2.9.3. The significance of mean (Hoehn & Sarre 2005). For G. variegata, we genotyped F-statistics was assessed by constructing 95% confidence individuals using nine tetranucleotide microsatellite markers intervals (CI) by jackknifing across loci using genetix 4.01 cloned from an enriched library for this species (GV1C5, (Belkhir et al. 2001). For the comparison of mean F GV1C10, GV1F1, GV3B5, GV3C6, GV3E10, GV4B6, GV4G6, ST between species and between landscapes, t-tests for statis- GV4C9) (Hoehn & Sarre 2006). Polymerase chain reaction tical significance were performed. Interpretation of genetic (PCR) amplification and genotyping using the Beckman differentiation values between species is often problematic Coulter CEQ 8000 were performed according to conditions because of their dependence on the level of genetic variation. described in Hoehn & Sarre (2005) and Hoehn & Sarre To address this, we applied a standardized measure of (2006). genetic differentiation (Hedrick 1999, 2005). The standardized ′ GST, G ST is defined as: Statistical analysis G G′ = ST (1) Descriptive statistics — the allelic richness (An) (number of ST G alleles corrected by sample size, based on a minimum ST(max) sample size of 13 individuals for both species) and the where GST is an estimate of FST for a locus with multiple observed (HO) and expected (HE) heterozygosities per alleles and

(kH −− 11)( ) information about the potential source populations was G = S (2) ST(max) −+ incorporated, admixture was assumed and the model with kH 1 S correlated allele frequency was selected. For some popula- where k is the number of equal-sized subpopulations tion pairs, structure 2.1 failed to confidently assign any and HS is the average subpopulation Hardy–Weinberg individual to a single genetic population. Thus, in a second ′ heterozygosity. Pairwise G ST values were calculated for step of the analysis, runs were performed at K = 1–5 to ′ each species. The significance of G ST between species and assess if some of the paired sites actually constitute a single between landscapes was assessed by calculating the mean panmictic population. ′ G ST and performing a t-test. The Bayesian method of likelihood-based assignment Isolation by distance was tested through linear regres- tests were implemented using geneclass 1.0.02 (Rannala sion of FST/(1 – FST) against the geographical distance & Mountain 1997; Cornuet et al. 1999). In a first step, indi- between pairs of habitat fragments (Rousset 1997). The viduals were directly assigned to the population for which Mantel test option in fstat 2.9.3 was used to assess the their probability of belonging is highest. In a second step, significance of correlation between matrices of genetic dif- a probability that the individual belongs to each population ferentiation and geographical distance between sampled was simulated and individuals were assigned to the popu- populations. Geographical straight-line distance between lation for which their probability of belonging is highest, the edges of the fragments within the study area was deter- and where the arbitrary threshold value is above P = 0.05. mined using the map generated by M. G. Brooker, CSIRO, Division of Wildlife and Ecology, Perth and Department of Results Agriculture, Western Australia. We applied spatial autocorrelation (SA) analysis, A total of 345 Oedura reticulata from 12 sites (six fragments implemented in genalex 6.0 (Peakall & Smouse 2001), and six continuous forest sites) and 353 Gehyra variegata as an additional method to estimate if dispersal and gene from 13 sites (seven fragments and six continuous forest flow are limited. Unlike traditional SA (Sokal & Oden 1978), sites) were genotyped at nine microsatellite loci. Four of this technique employs a multivariate approach that the 108 tests for Hardy–Weinberg equilibrium in O. reticulata strengthens the spatial signal and reduces the noise. Using and six of the 117 tests in G. variegata showed significant pairwise geographical and pairwise squared genetic distance deviation from expected genotype frequencies after matrices, it generates an autocorrelation coefficient, r, Bonferroni correction; all were due to a deficiency of which is closely related to Moran’s I (Sokal & Oden 1978). heterozygotes. The heterozygote deficiency may be due to The autocorrelation coefficient provides a measure of the null alleles; although there does not appear to be any genetic similarity between pairs of individuals whose geo- consistent pattern of Hardy–Weinberg deviation among graphical separation falls within the specified distance class. loci, or populations. No linkage disequilibrium was detected Significant positive autocorrelation implies that individuals between loci within populations at any locality and there- within a particular distance class are more genetically similar fore independence among loci has been assumed in the than expected by random. Tests for statistical significance subsequent analyses. were performed by random permutation with a Monte Carlo simulation performed with 1000 permutations and Genetic diversity 1000 bootstraps for estimation of 95% confidence intervals.

We performed this analysis separately for habitat fragments In O. reticulata, allelic richness (An) per site and locus ranged and nature reserves using the even-distance class option from 4.00 to 19.26 in fragments and from 6.00 to 17.53 in (using 25 distance classes, 100 m each). continuous forest sites. We detected a highly significant To provide an index of dispersal rates between popula- difference in the mean allelic richness between fragmented tions, assignments were conducted using the programs and continuous forest sites (mean 7.97 and 11.36, respectively, structure 2.1 (Pritchard et al. 2000) and geneclass 1.0.02 Mann–Whitney U-tests: P < 0.001) (Fig. 2a). In G. variegata,

(Cornuet et al. 1999). Using the Bayesian clustering method allelic richness (An) per site and locus ranged from 4.74 to implemented in structure 2.1, six populations (three 16.00 in fragments and from 7.44 to 16.42 in continuous population pairs) in the fragments and six populations (four forest sites. The difference in the allelic richness between population pairs) in the continuous forest were analysed fragments and continuous forest sites was significant (mean for each species. Population GVF7 was omitted from this 9.95 and 10.77, respectively, P < 0.05). Our analysis showed analysis because of the small sample size (N = 13) and that fragmented populations of O. reticulata have significantly because dispersal was assessed between pairs of popula- lower allelic richness than fragmented populations of G. tions. For every population pair, five independent runs of variegata (P < 0.001), while there is no significant difference K = 2 were performed at 200 000 Markov chain Monte in allelic richness between populations of the two species Carlo repetitions and 100 000 burn-in periods. No prior in the nature reserves (P = 0.48) (Fig. 2b).

Fig. 2 Comparison of allelic richness (An), expected (H ) heterozygosity, F and ′ E ST G ST estimates (a) between fragments and continuous forest site for Oedura reticulata (above) and Gehyra variegata (below) and (b) between Oedura reticulata and Gehyra variegata in fragments (above) and continuous forest site (below).

Average expected heterozygosity (HE) observed in O. 0.39). Tests showed that there was a significant difference reticulata ranged from 0.69 to 0.87 in fragments (mean HE: between species (t-test, P < 0.01). All other test followed 0.79) and from 0.85 to 0.92 in continuous forest sites (mean the trend shown by the FST values (Fig. 2b). HE: 0.89). In G. variegata, expected heterozygosity (HE) Most pairs of sites exhibited a fairly similar range of FST averaged across loci ranged from 0.85 to 0.90 in fragments values, although for O. reticulata population, ORF3 had

(mean HE: 0.88) and from 0.88 to 0.91 in continuous forest larger values on average than the other sites. In addition, sites (mean HE: 0.89). Populations of both species showed ORF3 seemed to have lower allelic richness and heterozy- a significant difference in heterozygosity between fragments gosity. Therefore, we performed all calculations without and continuous forest sites (Fig. 2a). However, the level of the outlier population and found that differences between significance for O. reticulata (P < 0.001) was considerably the species in An and HS and FST were still significant higher than that for G. variegata (P < 0.05). Furthermore, (P < 0.001) with the exception that the standardized ′ tests revealed that fragment populations of O. reticulata value of G ST was only approaching significance (t-test, have significantly lower heterozygosity than fragment P = 0.06) compared with P < 0.05 when all populations are populations of G. variegata (P < 0.001), while there is no sig- included. nificant difference between populations of either species in the continuous forests (P = 0.42) (Fig. 2b). Isolation by distance A positive association between genetic differentiation Population differentiation (FST/1 – FST) and the ln of geographical distance separating Both gecko species showed significant genotypic differ- samples (P < 0.01) was observed for G. variegata. But no entiation among fragment populations (P < 0.01). However, such pattern was apparent for O. reticulata with or without

FST measures revealed higher levels of subdivision among the outlier population of ORF3 when total geographical fragmented populations of O. reticulata (FST = 0.102; 95% CI distance was taken into account, suggesting that distance 0.086–0.119) than among fragmented populations of G. between remnants had no influence on the genetic structure variegata (FST = 0.044; 95% CI 0.037–0.050, t-test: P < 0.001). in this species (Fig. 3). Conversely, no significant difference in subdivision was observed between species in the continuous woodland

(O. reticulata: FST = 0.003; 95% CI 0.001–0.005; G. variegata: FST = 0.004; 95% CI 0.001–0.007, t-test: P = 0.26) and there was no significant genotypic differentiation among continuous populations (Fig. 2b).

In O. reticulata, pairwise FST estimates ranged from 0.041 to 0.163 among fragment populations and from 0.001 to 0.007 among populations in the continuous nature reserves. The level of differentiation was significantly higher in fragmented (FST = 0.109; 95% CI 0.079–0.140) compared with continuous populations (FST = 0.003; 95% CI 0.001–0.005, t- test: P < 0.001) (Fig. 2a). The same trend was apparent in G. variegata with pairwise FST estimates ranging from 0.007 to 0.081 among fragment populations and from 0.000 to 0.009 among populations in the continuous woodland. Differen- tiation among fragmented populations (FST = 0.025; 95% CI 0.010–0.041) appeared to be significantly higher than among continuous populations (FST = 0.004; 95% CI 0.001– 0.007, t-test: P < 0.01) (Fig. 2a). For the previous comparison, we included pairwise FST estimates only from fragment populations that were in close proximity to each other. The mean geographical distance between these fragments was 670 m for O. reticulata and 650 m for G. variegata compared with a mean geographical distance of 750 m among the continuous forest sites for both species.

Using the standardized values of GST, the level of differ- Fig. 3 Relationship between the logarithms of geographical entiation was also higher in O. reticulata (G′ = 0.45, 95% CIs: ST distances and genetic differentiation estimated as FST/(1 – FST) for ′ Oedura reticulata (above) and Gehyra variegata (below). 0.40–0.49) than in G. variegata (G ST = 0.34, 95% CIs: 0.29–

Fig. 4 (a) Spatial autocorrelation correlograms for fragmented Oedura reticulata populations (above) and fragmented Gehyra variegata populations (below). (b) Spatial autocorrelation correlograms for continuous Oedura reticulata populations (above) and continuous Gehyra variegata populations (below). Distances are in metres and only populations from the Korrelocking Nature Reserve are shown. The permuted 95% confidence interval (dashed lines) and the bootstrapped 95% confidence error bars are also shown. The autocorrelation coefficient, r, provides a measure of the genetic similarity between pairs of individuals and significant positive autocorrelation implies that individuals within a particular distance class are more genetically similar than expected by random.

potential than isolation-by-distance analysis for identifying Spatial autocorrelation structure at a genetic fine-scale level. Nevertheless, the The outcome of the spatial autocorrelation analysis of the analysis of the continuous forest populations showed no two gecko species is presented in Fig. 4. The correlogram sign of significant positive correlation. produced for the fragment populations in G. variegata indicates a significant positive correlation in the first Assignment three distance classes, 100 m (r = 0.070, P = 0.01), 200 m (r = 0.027, P = 0.01) and 400 m (r = 0.033, P = 0.01), with an Both the Bayesian clustering method (structure 2.1) and intercept of 951 m (Fig. 4a). An autocorrelation focusing on the direct Bayesian method (geneclass 1.0.02) produced the nature reserve populations revealed no significant spatial consistent results that could be used to estimate dispersal structure in any of the distance classes. For convenience, probabilities. Figure 5 shows the results from the Bayesian Fig. 4(b) only shows the results from the populations in clustering method (structure 2.1). We observed a higher the Korrelocking Nature Reserve, but results from the percentage of misassignments in fragmented populations North Bandee Nature Reserve are comparable. The spatial of G. variegata (mean of 30% for both structure 2.1 and autocorrelation of fragment populations for O. reticulata geneclass 1.0.02 methods, 0.070 and 0.045 standard error showed contrasting results to the isolation-by-distance (SE), respectively, subsequent data always presented in analysis. The analysis revealed a significant positive genetic this order) than of O. reticulata (mean of 15% and 20%, structure up to a distance of 518 m, with positive r in the 0.047 and 0.096 SE, respectively). This occurred even distance classes, 100 m (r = 0.15, P = 0.01) and 200 m (r = 0.03, though the populations of O. reticulata were in slightly P = 0.01). Spatial autocorrelation seems to have a higher closer proximity to each other. For O. reticulata, the

Fig. 5 Average probability for the individuals of (a) Oedura reticulata and (b) Gehyra variegata populations having originated in the source population or a neighbouring population derived from data of the Bayesian clustering method (structure 2.1). Neighbouring population pairs are ORF1/ORF4; ORF2/ORF3; ORF5/ORF6 for Oedura reticulate and GVF1/GVF2; GVF3/GVF4; GVF5/GVF6 for Gehyra variegata.

assignment tests revealed that the two closest populations rate between 17% and 27%. Populations GVF5 and GVF6 (ORF5 and ORF6: 150 m) had the highest percentage of were 1000 m apart and showed relatively high misassign- misassigned individuals (27% and 50%, respectively). ment rates (between 8% and 40%). In the continuous forest Populations ORF1 and ORF4 and populations ORF2 and populations, we observed a higher percentage of misassign- ORF3 are isolated by 580 m and 550 m, respectively, and ments for both species (e.g. Korrelocking Nature Reserve: the assignment tests revealed that between 6% and 19% O. reticulata: mean of 50% and 58%, 0.00 and 0.038 SE, were misassigned for the first population pair, and between respectively; G. variegata: mean of 50% and 53%, 0.00 and 0 and 4% for the second. For G. variegata, the highest 0.063 SE, respectively) than in the fragmented populations. percentage (between 40% and 50%) of misassigned indi- Data for the North Bandee Nature Reserve is corresponding. viduals was between the two populations GVF3 and GVF4, Using the Bayesian approach with a threshold level of which were closest in distance (150 m). Populations GVF1 P ≤ 0.05 for the habitat fragments, only 77 of 174 individuals and GVF2 are isolated by 300 m and had a misassignment of O. reticulata and even fewer individuals of G. variegata

(25 of 171) could be assigned. Thus, this level of constraint of about 500 m are a barrier to O. reticulata but not for G. provided little information: we identified only nine of 77 variegata. We also observed an isolation-by-distance effect assigned O. reticulata as dispersers (mean = 3.0 per popu- in FST values in G. variegata but not in O. reticulata, which lation pair) and six of 25 assigned G. variegata (mean = 2.0 together with spatial autocorrelation analysis suggests that per population pair). The majority of the O. reticulata G. variegata can disperse on a scale approaching a kilometre dispersers (five) were identified between the two closest rather then the few hundred metres achieved by O. reticulata. populations (ORF5 and ORF6) whereas the six G. variegata Overall, O. reticulata exhibits lower levels of heterozygosity, dispersers were more evenly distributed among the three lower allelic richness, elevated levels of structure, fewer population pairs. In the Korrelocking Nature Reserve, only misassignments than similarly fragmented populations of seven of 12 O. reticulata were assigned as dispersers (mean G. variegata, while all these parameters were fairly similar = 3.5 per population pair) and four of four G. variegata were in the continuous forest populations. These findings are of assigned (mean = 2.0 per population pair). ecological significance to the distribution of populations For population pairs ORF5/ORF6 and GVF3/GVF4, throughout the landscape of the Kellerberrin wheatbelt area. and for all the population pairs in the two nature reserves, From these results recommendations for possible manage- structure 2.1 failed to confidently assign any individual ment action to prevent extinctions can be derived. Here, we to a single population. Thus, we estimated the number of discuss these patterns and their methodological limitations. clusters (K) for these populations. Log-likelihood values were found to be similarly low for K = 1 and K = 2 or lower Structure in fragmented and nonfragmented populations for K = 1. For comparison, we also calculated the number of K for other population pairs, where log-likelihood Our analyses, incorporating comparisons among fragmented values were low for K ≥ 2, but substantially higher for and nonfragmented populations and between species K = 1. This result indicates that the fragments, which are with contrasting levels of persistence and speciality, reveal in close geographical distances, and the two nature reserves several clear patterns concerning the impact of frag- constitute a single panmictic population for both species. mentation upon these two species. First, in continuous and relatively pristine habitat (nature reserves), the genetic structure and the percentage of misassignments of the Discussion two species are indistinguishable from each other on a Dispersal is a key trait of species that avoid extinction geographical scale of 1–1.2 km. On this scale, genetic following habitat fragmentation. When populations become structure was very low for both species (FST = 0.003 OR; fragmented, dispersal between patches can provide a 0.004 GV). Spatial autocorrelation might be a better approach ‘top-up’ or ‘rescue effect’ for small, resident populations, for the detection of fine-scale genetic structure when other which reduces the probability of local extinction (Brown methods fail to identify genetic patterns (Double et al. 2005). & Kodric-Brown 1977; Hanski 1999). Understanding the However, there was no significant positive correlation impacts of human-induced fragmentation upon dispersal between any of the nature reserve populations. In addition, as opposed to the impacts of other environmental changes the number of populations in the nature reserve was is problematic because of the nearly universal lack of estimated with the program structure 2.1 and the result prefragmentation data in naturally occurring populations indicated that the populations in each of the two nature (Srikwan & Woodruff 2000; Sumner et al. 2004). In this reserves are approaching panmixis. Second, genetic structure context, comparative molecular approaches that contrast in both species was clearly influenced by fragmentation fragmented and nonfragmented populations, combined through land clearance for agriculture. For both species, with detailed field and modelling studies, provide one of levels of FST were higher and the percentage of mis- the few ways of exploring such an important topic (Young assignments was lower among fragmented populations ′ & Clarke 2000; Stow et al. 2001; Caizergues et al. 2003; than among those in the nature reserves. While G ST was Segelbacher et al. 2003; Williams et al. 2003; Stow & Sunnucks twice as high in O. reticulata than in G. variegata, the 2004a, b). impacts of fragmentation were still evident in G. variegata

Our fine-scale microsatellite DNA analysis of two species with significantly higher levels of FST among fragmented of gecko (Oedura reticulata and Gehyra variegata) demon- populations than among nature reserve populations. strates the usefulness of such an approach. Clear hypotheses emerged from long-term field and modelling studies Dispersal as a function of genetic and geographical and we have demonstrated that the genetic structure of distance fragmented O. reticulata populations is significantly higher than in G. variegata. In addition, assignment tests revealed Isolation by distance. The relationship between genetic dif- that G. variegata has higher rates of interpatch dispersal ferentiation and geographical distance contributes to our compared with O. reticulata suggesting that small distances understanding whether genetic differentiation is a result of

© 2007 The Authors Journal compilation © 2007 Blackwell Publishing Ltd DISPERSAL AND EXTINCTION IN FRAGMENTED GECKO POPULATIONS 3309 limited dispersal or more complex demographic factors Assignment. The benefit of the first analysis in our study, (Leblois et al. 2000; Brouat et al. 2003). In general, it has been the most-likely option, is that gecko individuals do not argued that if dispersal is limited by distance, at equilibrium necessarily need to be assigned with a high accuracy to between mutation, migration, and drift genetic and obtain an overall estimate of the dispersal probability. In geographical distance should be positively correlated. A a second approach for identifying an exact number of negative correlation or no isolation by distance is usually dispersers, the assignment test was then performed at a interpreted as evidence that dispersal is not limited by threshold of P ≤ 0.05. We were able to estimate the percentage distance, such that the sampled region functions effectively of misassignments, which was on average lower for O. as one large population (Rousset 1997, 2001; Leblois et al. reticulata than for G. variegata, suggesting that the number 2000; Brouat et al. 2003). However, there are at least two of dispersers in G. variegata is nearly twice that in O. reticulata. other reasons why a lack of a significant pattern of isolation For both species, the percentage of misassignments was on by distance may occur. First, gene flow may be very low average higher between the continuous forest populations over the distance sampled such that populations are than between the fragments. essentially isolated, and allele frequencies are determined With the semi-experimental approach, we were also able by drift. This was recently demonstrated in the alpine to determine a rough distance of separation. For O. reticulata butterfly, Parnassius smintheus, because of reduced con- a distance of 150 m through cleared habitat did not appear nectivity of habitat (Keyghobadi et al. 2005). Second, the to represent a barrier. Movement between the two pairs of Mantel test might not have enough power to detect genetic distant populations (ORF1/ORF4 and ORF2/ORF3) was structure at a fine scale. substantially lower than for those separated by only 150 m On the basis of isolation-by-distance analysis alone, we indicating that 500–600 m is sufficient to prevent even would interpret that the correlation between geographical modest dispersal. Three of four individuals detected as and genetic distance for G. variegata but not for O. reticulata immigrants between these distant pairs were detected as suggests that G. variegata moves between remnants and having moved between population pair ORF1/ORF4. These O. reticulata does not. The alternative explanation, that two patches are adjacent to woodland roadside vegetation O. reticulata moves on a scale of 10 s of km, is contradicted that may act as a corridor for O. reticulata movement. This by the lack of high numbers of misassigned individuals possibility deserves future research as it suggests a potential among closely located populations of O. reticulata. In role for corridors in the conservation of species. contrast, genetic differentiation is positively correlated For G. variegata, the isolation of remnants by 150–300 m of with total geographical distance in G. variegata, which cleared matrix did not stop migrants from moving through conforms to the theoretical expectations that the species is the modified landscape. At least one individual covered dispersing, but that dispersal is limited by distance. distances of up to 1000 m between habitat patches. Data from the isolation-by-distance and spatial autocorrelation Spatial autocorrelation. The potential contribution of spatial analyses suggest that movement over 1 km still occurs. Dis- autocorrelation analysis in combination with hypervariable persal requires individuals to pass through rural landscapes DNA markers has been overlooked in animal studies in which the native vegetation has been removed and replaced (Peakall et al. 2003; Double et al. 2005). While the isolation- by crops or stubble after harvest. The environment contains by-distance analysis failed to identify a landscape-related a high density of introduced predators, and a scarcity of genetic pattern in O. reticulata, the autocorrelation showed suitable hiding places (even rocks and fences) that may be that genetic structure is present at two distance classes, used as temporary shelter for the geckos. Hence, successful 100 m and 200 m. The intercept occurs at a relatively short dispersal under these circumstances is likely to be a rare event. distance of 518 m, which indicates a low level of dispersal We discovered that the number of identified dispersers and a small neighbourhood size (Vignieri 2005). Still, this by assignment test where a threshold was established was result is in rough agreement with the conclusions from similar for the two gecko species or even slightly higher in the isolation-by-distance and assignment analysis. The O. reticulata similar in fragmented and continuous land- performance of the isolation-by-distance analysis might scapes. Theoretical and empirical research imply that there have been compromised by the clumping of the samples. is a positive relationship between the level of differenti-

The observed spatial pattern in G. variegata implies that the ation (FST) and the ability to correctly assign individuals fragmented landscape is also a barrier to this species, but to their natal population (Eldridge et al. 2001; Berry et al. that dispersal occurs at a larger scale than in O. reticulata. 2004). We suggest that the assignment test was more likely The spatial autocorrelation remains positive up to 400 m to correctly identify the dispersers for O. reticulata, which and then begins to decline with an intercept of 951 m. In had an FST of 0.10, than for G. variegata with an FST of 0.04. both species, no significant genetic structure has been Therefore, we assume that in the species G. variegata, the actual identified in the continuous woodlands suggesting that the number of dispersers is likely to be higher than estimated. populations are panmictic. The same might be true for the estimated number of

© 2007 The Authors Journal compilation © 2007 Blackwell Publishing Ltd 3310 M. HOEHN, S.D. SARRE and K . HENLE dispersers between population pairs ORF5/6 and GVF3/4, Berry O, Tocher MD, Sarre SD (2004) Can assignment tests meas- which are in close proximity and between the population ure dispersal? Molecular Ecology, 13, 551–561. pairs in the continuous forest. The differentiation between Branch LC, Clark A-M, Moler PE, Bowen BW (2003) Habitat spe- cificity, fragmented landscapes, and the phylogeography of these populations is lower and consequently the number of three lizards in Florida scrub. Conservation Genetics, 4, 199–212. dispersers could be underestimated. In addition, further Brouat C, Sennedot F, Audiot P, Leblois R, Rasplus JY (2003) Fine- structure 2.1 analysis showed that these populations scale genetic structure of two carabid species with contrasted consist of one (K = 1) panmictic population. levels of habitat specialization. Molecular Ecology, 12, 1731–1745. Brown JH, Kodric-Brown A (1977) Turnover rates in insular biogeography: effect of immigration on extinction. Ecology, 58, Conclusion and conservation implications 445–449. Caizergues A, Ratti O, Helle P (2003) Population genetic structure In the study area, five other terrestrial gecko species of male black grouse (Tetrao tetrix L.) in fragmented vs. continuous (Crenadactylus ocellatus, Diplodactylus granariensis, D. maini, landscapes. Molecular Ecology, 12, 2297–2305. D. pulcher, and D. spinigerus) have already become extinct Clobert J, Danchin E, Dhondt AA (2001) Dispersal. Oxford Univer- in most remnant woodlands, presumably as a result of habitat sity Press, Oxford. clearing and agriculture. We suggest that the differences Cornuet JM, Piry S, Luikart G, Estoup A, Solignac M (1999) New in the persistence of the two remaining gecko species are methods employing multilocus genotypes to select or exclude mainly attributable to their different dispersal ability through populations as origins of individuals. Genetics, 153, 1989–2000. Davies KF, Margules CR, Lawrence KF (2000) Which traits a matrix of habitat more suitable for G. variegata than for O. of species predict population declines in experimental forest reticulata. Similarly, in western Japan, it was demonstrated fragments? Ecology, 81, 1450–1461. that the house-dwelling gecko Gekko japonicus had higher rates Davies KF, Melbourne BA, Margules CR (2001) Effects of within- of gene flow between habitat fragments than its sympatric and between-patch processes on community dynamics in a counterpart, Gekko tawaensis, a species that is not present fragmentation experiment. Ecology, 82, 1830–1846. in human constructions. It was argued that this was pro- Davies KF, Margules CR, Lawrence JF (2004) A synergistic effect moted by human-mediated transport (Toda et al. 2003). The puts rare, specialized species at greater risk of extinction. Ecology, 85, 265–271. few available comparative genetic studies of two or more Double MC, Peakall R, Beck NR (2005) Dispersal, philopatry, and sympatric species support our conclusions (lizards: Branch infidelity: dissecting local genetic structure in superb fairy- et al. 2003; small mammals: Matocq et al. 2000; Ehrich et al. wrens (Malurus cyaneus). Evolution, 59, 625–635. 2001a, b; insects: Monaghan et al. 2002; Brouat et al. 2003). 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