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Population Genetics Between an Insular and Coastal Population of Gopher Tortoises (Gopherus polyphemus) in Southwest Florida
Colleen Winters, Julie Ross, and Phil Allman

Southeastern Naturalist, Volume 16, Issue 3 (2017): 369–382

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Southeastern Naturalist 369 C. Winters, J. Ross, and P. Allman 22001177 SOUTHEASTERN NATURALIST 1V6o(3l.) :1366,9 N–3o8. 23 Population Genetics Between an Insular and Coastal Population of Gopher Tortoises (Gopherus polyphemus) in Southwest Florida Colleen Winters1,*, Julie Ross2, and Phil Allman3 Abstract – Gopherus polyphemus (Gopher Tortoise) is a prominent species found in pine flatwoods, upland scrub, and coastal dunes of the southeast United States. Geoclimatic and anthropogenic sources of habitat changes have fragmented Gopher Tortoises into isolated populations that may reduce gene flow, promote inbreeding, and ultimately impact popula - tion viability. Rapid urbanization along the southwest Gulf Coast of Florida has degraded habitat, and fragmented insular and coastal populations. To assess the diversity in these vestige populations, we assessed the genetic structure of Gopher Tortoises on heavily developed Marco Island (n = 61) and the adjacent mainland within Rookery Bay National Estuarine Research Reserve (RBNERR) (n = 23) in Collier County, FL. Using microsatellite markers, we determined that the Marco Island tortoises are genetically distinct from the RBNERR tortoises. We identified unique alleles and reduced allelic richness in both populations, suggesting isolation has reduced gene flow. We therefore encourage careful management of the Marco Island Gopher Tortoises to maintain the uniqueness of the population while preventing further loss of diversity. Introduction Understanding the genetic variation among populations of a species is necessary to develop management actions that maintain the species’ genetic diversity. Genetic structuring among populations may result from the presence of barriers that reduce gene flow and promote drift and local selection (Futuyma 1998). Such barriers represent discontinuity in suitable habitat and loss of functional connectivity among population fragments (Kindlmann and Burel 2008). Subdividing populations may increase the probability of inbreeding and decrease the likelihood of population persistence (Lande 1987). In this paper, the genetic structures of 2 adjacent Gopherus polyphemus (Daudin) (Gopher Tortoise) populations in southwest Florida were investigated to better characterize genetic assemblages in the region. The Gopher Tortoise is a widespread species that utilizes pine flatwoods, upland scrub, and coastal dune habitats of the Coastal Plain in the southeastern United States from Louisiana to South Carolina (Auffenberg and Franz 1978). The species has existed in the region for 2 million years and serves keystone functions that increase community complexity (Franz and Quitmyer 2005). Their 1Department of Biological Sciences, Towson University, 8000 York Road, Towson, MD 21252. 2Department of Marine and Ecological Sciences, Florida Gulf Coast University, 10501 FGCU Boulevard, Fort Myers, FL 33965. 3Department of Biological Sciences, Florida Gulf Coast University, 10501 FGCU Boulevard, Fort Myers, FL 33965. *Corresponding author - Manuscript Editor: John Placyk Southeastern Naturalist C. Winters, J. Ross, and P. Allman 2017 Vol. 16, No. 3 370 fossorial behavior creates microhabitats in the form of a burrow and a mound of sand adjacent to the burrow’s entrance. Over 360 species of obligate or facultative commensals have been reported to utilize the burrows (Eisenberg 1983, Jackson and Milstrey 1989), including some that are legally protected (Kent et al. 1997, Witz et al. 1991). The burrow’s apron provides habitat for some rare herbaceous plants that consequently increases the community’s plant diversity (Kaczor and Hartnett 1990). Furthermore, the grazing behavior of Gopher Tortoises likely influences the floral community across the landscape as they function to disperse seeds (Boglioli et al. 2000). The significant loss of habitat associated with rapid urbanization, agricultural development, and mineral mining caused Gopher Tortoise populations to decline by 80% from 1880 to 1980 (Auffenberg and Franz 1982). Remaining populations now exist in fragmented patches of suitable habitat interwoven within a matrix of degraded habitat that restricts population recovery (McCoy et al. 2013, Mushinsky et al. 2006). Populations continue to decrease as urbanization and changes in land-use further reduce remaining habitat and populations (Brady and Abbott 2014, McCoy et al. 2006). The Gopher Tortoise is listed as federally threatened in the western portion of their range from Louisiana to the Mobile and Tombigbee rivers in Alabama, and are state listed as threatened in Georgia, Florida, and Louisiana, endangered in South Carolina and Mississippi, and protected as a non-game species in Alabama (Enge et al. 2006). The consequences of habitat loss on Gopher Tortoises extend beyond loss of individuals, as it also impacts recovery potential. Fragmenting populations disrupts natural movement patterns and may result in significant loss of genetic variation and evolutionary potential (Frankham et al. 2010, Madsden et al. 1999, Saccheri et al. 1998). Such fragmentation may cause inbreeding depression and loss of genetic heterozygosity. Inbreeding is linked to increased mortality in young animals, reduced reproductive success, and lower resistance to disease, predation, and stress (Keller and Waller 2002). A reduction in reproductive fitness negatively affects the ability of a population to recover (Frankham 1998, Keller and Waller 2002, Reed and Frankham 2003). The negative influence fragmentation has on recovery potential may be exacerbated for insular populations because resources are more limited on islands (Diamond 1976). Florida Fish and Wildlife Conservation Commission’s current Gopher Tortoise Management Plan permits the relocation of Gopher Tortoises that are potentially impacted by development (FFWCC 2013). Moving individuals to disparate populations can lead to outbreeding and impact local adaptive processes occurring at recipient sites (Edmands 2007, Weeks et al. 2011). Management decisions must therefore balance relocation efforts to prevent inbreeding depression while allowing for local divergence. Until recently, there has been a paucity of genetic data available to determine the impacts of such relocation ef forts. Schwartz and Karl (2005) identified 8 genetic subpopulations of Gopher Tortoise from 19 locations across Florida and southern Georgia. They identified evidence of anthropogenic bottlenecks in 5 populations, and a pattern of greater divergence north to south and weaker separation east to west. The authors recommended a Southeastern Naturalist 371 C. Winters, J. Ross, and P. Allman 2017 Vol. 16, No. 3 compilation of genetic records for populations so that relocated tortoises can be moved to locations that minimize negative impacts. Unfortunately, the authors were unable to sample tortoise populations in the extreme southwest portion of their range. In this study, the genetic diversity of tortoises in 2 adjacent, but separated, populations in Collier County in southwestern Florida was analyzed. Genetic structuring and inbreeding within each population was explored as well as divergence between the 2 populations. Field-Site Description We sampled 2 sites on Marco Island and 1 site on Rookery Bay National Estuarine Research Reserve (RBNERR) (Fig. 1). Marco Island (25°56'N, 81°69'W) is a 3100-ha urbanized barrier island in southwest Florida containing primarily coastal dune, coastal scrub, and mangrove habitats. The Calusa people inhabited the island as early as 500 AD and were then displaced by Spanish explorers as early as 1500 (Waitley 1993). The island remained relatively undeveloped until the island’s second automobile bridge was built in 1969. Marco Island is currently home to ~18,000 people with a density of 573 individuals/km2 (USCB 2015). Gopher Tortoises are found in 2 residential neighborhoods, in the southeast and northwest portions of the island, separated by 6.3 km of commercial development (C. Winters, pers. observ.). We captured by hand a total of 61 tortoises on the island. Figure 1. Map of Florida. Inset shows areas of Gopher Tortoise sampling: Rookery Bay National Estuarine Research Reserve RBNERR (cross) and Marco Is land (diamonds). Southeastern Naturalist C. Winters, J. Ross, and P. Allman 2017 Vol. 16, No. 3 372 Rookery Bay National Estuarine Research Reserve (RBNERR) (26°01'N, 81°42'W) protects 45,000 ha of estuary, mangroves, and uplands on the mainland directly north of Marco Island. A small population of Gopher Tortoises is found within the 2500 ha of coastal upland habitats C. Winters, pers. observ.). The property is currently separated from Marco Island by an expanse of mangrove estuaries and a channel, but the 2 locations were connected via a land bridge as recently as 7000 years ago (Barry 1983). Prior to that in the Pleistocene, glaciers of North America made 4 major advances and retreats. Sea levels were as much as 133 m below those of today, causing Florida’s peninsula to be about twice the size it is today (Hulbert 2001, Miller 1998). During the glacial periods of this time, the coastal plains of southern United States contained extensive grasslands that allowed for browsers like bison, horses, and tortoises to thrive (see Myers and Ewel 1990). The last glacial peak occurred about 10,000 years ago and was followed by interglacial warming that increased sea level to current levels around 7000 years ago (Barry 1983). Tortoises living on Marco Island would have been separated from mainland populations at that time. We sampled for tortoises within the xeric scrub and scrub palmetto habitats of the reserve. The presence of non-native tortoises indicate animals may occasionally be released within RBNERR, but it is difficult to know if this has occurred with Gopher Tortoises. Materials and Methods We sampled 61 Gopher Tortoises on Marco Island and 23 from RBNERR. Individuals were captured through opportunistic sightings while conducting site surveys on foot or by bucket trapping (Florida Fish and Wildlife Conservation Commission 2013). Using both methods allowed capture of tortoises from both sexes and all age classes. We conducted searches from May 2008 through August 2009 on Marco Island and from October 2010 through June 2011 in RBNERR. Cagle’s (1939) method of notching unique combinations of the marginal scutes was used on all captured tortoises. We collected the scute shavings in sterile collection bags (Nasco Whirl-Pak, Fort Atkinson, WI) for storage until DNA extraction. Drill bits used for marking individuals were sterilized with 80% ethanol before and after each use. We extracted DNA from the scute shavings following Dawes et al. (2008). Briefly, shavings were frozen in liquid nitrogen and then ground into a powder. The powdered shell was decalcified by incubation in 0.5 M pH 8.0 ethylenediaminetetraacetic acid (EDTA) for 3–5 days while shaking at 37 °C. Following decalcification, we purified DNA from the samples following the directions for the Qiagen DNeasy Blood and Tissue Kit (Qiagen, Valencia, CA). We determined individual genotypes using 9 species-specific microsatellite loci (Schwartz et al. 2003) and conducted polymerase chain reaction (PCR) in 15-ml volumes under the following conditions: 1.5 μl 10X PCR buffer with 15 mM MgCl2 (Gene Choice, Continental Lab Products, San Diego, CA), 0.2 mM dNTPs (Roche, Indianapolis, IN), 0.2 μm of each primer (GP15, GP19, GP26, GP30, GP55, GP61, GP81, GP96, GP102) (Schwartz et al. 2003), 1.25 units of Taq polymerase (Gene Choice), and 30 ng of template DNA. The PCR thermocycling program was 94 °C Southeastern Naturalist 373 C. Winters, J. Ross, and P. Allman 2017 Vol. 16, No. 3 for 3 min; 35 cycles of 94 °C for 30 s, 60 °C for 30 s, and 72 °C for 45 s; followed by a 5 min extension at 72 °C. PCR products were verified by gel electrophoresis on a 1% agarose gel. We labeled the forward primer for each locus with a fluorescent dye (Sigma Proligo, St. Louis, MO) to facilitate fragment analysis on a CEQ8000 Genetic Analysis System (Beckman Coulter, Fullerton, CA). Statistical analyses were performed with the genotype data gathered from the 9 microsatellite loci. We determined null allele frequencies for each locus in each population using the Expectation Maximization (EM) algorithm (Dempster et al. (1977) in FreeNA (Chapuis and Estoup 2007). We examined linkage disequilibrium and deviations from Hardy-Weinberg equilibrium (HWE) in GENEPOP using the likelihood ratio test (Raymond and Rousset 1985, Slatkin and Excoffier 1996) with 1000 dememorization steps and 100 batches with 1000 replicates per batch. Alpha level was adjusted by sequential Bonferroni correction (Rice 19 89). We inferred population structure by Bayesian clustering with STRUCTURE v2.3.4 (Falush et al. 2003, 2007; Hubisz et al. 2009; Pritchard et al. 2000) and assignment in GenAlEx 6.5 (Peakall and Smouse 2006, 2012). STRUCTURE analysis was first used to determine if the 2 sites on Marco Island were separate populations. Further analysis included both the Marco Island and RBNERR tortoises. In STRUCTURE, we analyzed for number of populations (K), with K values from 1 to 10, using a burn-in of 50,000 with 100,000 Markov chain Monte Carlo (MCMC) repeats and 20 iterations at each K assuming admixture and correlated allele frequencies. STRUCTURE output was summarized using CLUMPAK (Kopelman et al. 2015) and optimum K determined following methods used by Evanno et al. (2005). We also tested population assignment utilizing GenAlEx 6.5 with bias correction for population frequency. GenAlEx employs log-likelihoods to calculate assignment of individuals following the method of Paetkau et al (2004). We estimated the number of alleles or allelic richness in each population and corrected sample sizes by rarefaction using HP-RARE (Kalinowski 2005). An estimation of the effective number of migrants, Nm, was determined following Slatkin (1985) in GENEPOP. We determined allele frequencies and observed (Ho) and expected (He) heterozygosity in GenAlEx 6.5 across all loci for each population. Wright’s FIS using Weir and Cockerham estimator θ (Weir and Cockerham 1984) was calculated in GenAlEx; P values were obtained after 1000 permutations and 18,000 randomizations. We calculated genetic distance between the populations by FST using GenAlEx 6.5 and DEST using the R package of DEMEtics (Gerlach et al. 2010, R Development Core Team 2009). Since FST value estimates are impacted by the level of genetic variation within populations (Hedrick 2005, Neigel 2002), FST was also normalized relative to a theoretical maximum value given the observed variation (Hedrick 2005, Meirmans 2006) using RecodeData v0.1 (Meirmans 2006). We used BOTTLENECK to evaluate heterozygosity excess in each population (Piry et al. 1999). A 2-phase mutation model (TPM) was run with the default settings of 1000 iterations, variance of 30% and a 70% proportion of stepwise mutations using a 2-tailed Wilcoxon sign rank test with sequential Bonferroni correction (Rice 1989). Southeastern Naturalist C. Winters, J. Ross, and P. Allman 2017 Vol. 16, No. 3 374 Results A total of 84 individuals were genotyped, 61 from Marco Island and 23 from RBNERR, at 9 polymorphic loci. Null allele frequencies ranged from 0.00 to 0.33 (Table 1). Null alleles may be due to individuals missing genotype data for loci, the low number of alleles present, or the sample size (Gagneux et al. 1997, Garcia de Leon et al. 1998, Kwok et al. 1990). Eight individuals from Marco Island were missing data for a single locus (GP26, GP19, or GP102), 1 individual from Marco Island was missing data from 2 loci (GP96 and GP61), and 2 individuals from RBNERR were missing data for locus GP102. All of the following analyses were run with and without these individuals and no significant difference in results was observed. STRUCTURE results analyzed by CLUMPAK assigned the Marco tortoises to 3 clusters, but with less than 80% probability for all individuals. GenAlEx analysis placed all tortoises from Marco Island (n = 61) into a single genetic cluster. Further analysis with all samples (n = 84) separated the tortoises into 2 distinct populations, Marco Island and RBNERR (Fig. 2). GenAlEx log-likelihood-based assignment also clustered the tortoises into 2 separate populations (Fig. 3). Both the Marco Island and RBNERR populations were polymorphic at all 9 loci. None of the loci deviated from HWE. One locus pair (GP26–GP30) in the Marco Island population showed linkage disequilibrium after sequential Bonferroni correction (P < 0.05). Mean number of alleles was 6.67 (Marco Island) and 4.11 (RBNERR) (Table 2). Allelic richness was 3.15 in the Marco Island population and 2.58 in RBNERR. The percentage of private alleles was 57% (34 private alleles/60 total alleles) in the Marco Island population and 30% (11/37) in the RBNERR population (Table 2). The effective number of migrants, Nm, was estimated to be 1.41. Pairwise estimates of genetic differentiation between Marco Island and RBNERR were 0.14 (FST uncorrected), 0.46 (FST corrected), and 0.24 (DEST) (P < 0.001 for all estimates). Table 1. Measurement of genetic diversity—number of alleles, number of individuals with missing alleles and estimated null allele frequencies—in 2 Gopher Tortoise populations by locus. Loci described by Schwartz and Karl (2003). Number of individuals Estimated null Number of alleles missing alleles allele frequencies Locus Marco Island RBNERR Marco Island RBNERR Marco Island RBNERR GP15 8 6 0 0 0.15 0.00 GP96 6 2 1* 0 0.13 0.04 GP26 3 3 2 0 0.16 0.20 GP30 7 3 0 0 0.12 0.07 GP61 9 4 1* 0 0.06 0.16 GP81 12 8 0 0 0.30 0.15 GP19 4 3 2 0 0.33 0.27 GP55 6 4 0 0 0.07 0.19 GP102 6 4 4 2 0.13 0.10 *One individual missing alleles for both loci. Southeastern Naturalist 375 C. Winters, J. Ross, and P. Allman 2017 Vol. 16, No. 3 FIS was 0.42, P < 0.001 (Marco Island) and 0.38, P < 0.001 (RBNERR) (Table 2). Analysis of the Marco Island samples with the TPM model in BOTTLENECK did not detect heterozygote excess in either population, and both populations had normal L-shaped distributions. Figure 2. (A) Population assignment inferred by STRUCTURE for K values 1 to 10. Optimum K determined by using the Evanno (ΔK) transformation. Bars on the x-axis represent individual Gopher Tortoises. The y-axis is the likelihood of assignment to a given cluster K represented by different shades of gray. (B) Estimated natural log probability (L[K]). (C) ΔK calculated from the log probability using the Evanno transformation (Evanno et al. 2005). Figure 3. Population assignment by GenAlEx. Positive log-likelihood of assignment given on axes. Southeastern Naturalist C. Winters, J. Ross, and P. Allman 2017 Vol. 16, No. 3 376 Discussion We explored genetic variation of 2 adjacent but isolated Gopher Tortoise populations in southwest Florida to determine if they form genetically distinct populations or a single continuous population. The 2 populations were found to be genetically distinct at multiple analyses. Populations with low genetic diversity are less able to adapt to environmental change (Reed et al. 2002) and are at a greater risk of extinction (Saccheri et al. 1998). Both inbreeding and genetic drift can reduce genetic diversity; however, each influences diversity differently. Inbreeding will decrease heterozygosity but has no direct effect on allelic richness as alleles are maintained through successful reproduction (England et al. 2003, Frankham et al. 2002, Hartl and Clark 1997, Lande 1988, Reed and Frankham, 2003). Genetic drift resulting from allele loss impacts allelic richness and heterozygosity, with the strongest effect found in small populations with limited gene flow (Crow and Kimura 1970, Wright 1931). The long lifespan and overlapping generations of the Gopher Tortoises may offer protection against genetic drift (Davy and Murphy 2014, Ennen et al. 2011). Both Marco Island and Rookery Bay tortoises exhibit reduced allelic richness with a high number of private alleles, which is expected under mutation drift equilibrium (Crow and Kimura 1970). The inbreeding coefficient, FIS, for both populations is significantly higher than zero (0.42 Marco Island, 0.38 RBNERR; P < 0.001; Table 2), suggesting heterozygote deficiency in both populations. Inbreeding and genetic drift can occur when small populations become isolated due to habitat fragmentation or during a bottleneck. Insular populations typically suffer increased inbreeding relative to mainland populations due to reduced population sizes associated with the restricted resources and space (Frankham 1998). Marco Island is a barrier island in southwest Florida with a relict population of 300 or more individuals that have been isolated from the mainland population for ~7000 years (Barry 1983). Archeological evidence indicates the Calusa, the first human inhabitants of the island, primarily consumed shellfish and fish, but Gopher Tortoise shells have been discovered within Indian mounds located on the Table 2. Measurements of genetic diversity of Gopher Tortoises from 2 populations at 9 microsatellite loci: observed heterozygosity (Ho), expected heterozygosity (He), total number of alleles, mean number of alleles, number of private alleles and percentage of total, allelic richness after rarefaction, and inbreeding coefficient FIS results shown for Marco Island and Rookery Bay National Estuarine Research Reserve (RBNERR). Diversity measure Marco Island RBNERR Ho 0.341 0.295 He 0.582 0.469 Total alleles 60 37 Mean alleles 6.67 4.11 Allelic richness 3.15 2.58 No. private alleles/% total 34 (57%) 11 (30%) FIS 0.417, P < 0.001 0.377, P < 0.001 Southeastern Naturalist 377 C. Winters, J. Ross, and P. Allman 2017 Vol. 16, No. 3 island (MacMahon and Marquardt 2004). Spanish explorers arrived in the 1500s, but urbanization did not occur until the 1970s when the island was developed into a tourist destination (Waitley 1993). The US Census records indicate the human population increased on Marco Island from 4700 in 1980 to 18,000 in 2015 (USCB 2015). Gopher Tortoises only remain on 2 portions of the island that have become isolated due to residential and commercial development (C. Winters, pers. observ.). We analyzed Marco tortoises separately from the RBNERR tortoises to determine if they were genetically separate populations. Evanno analysis in CLUMPAK found optimal K = 3, but likelihoods for individual assignments were below 80%, and genetic distance between the clusters was not significant. GenAlEx assignment placed all Marco individuals in a single cluster, and when further analyzed, the 3 proposed clusters from STRUCTURE overlapped extensively. It is important to note that the northwest site yielded only 3 tortoise samples, with the remaining 58 coming from the southeast site. These sites on Marco Island have only been physically isolated from one another within the last 40 years, ~1 tortoise generation, and have not yet had sufficient time to genetically differentiate. Studies of chelonians isolated by habitat fragmentation and population decline have failed to detect evidence of bottleneck or genetic drift (e.g., Davy and Murphy 2014, Marsack and Swanson 2009). It is possible that the long life span of chelonians may delay the effects genetic isolation and decline (e.g., Davy and Murphy 2014, Marsack and Swanson 2009). Moreover, it is likely that gradual land-use change and urbanization over the past 40 years has led to a slow decline in the population size instead of a rapid decline typically associated with a bottleneck event. Tests for genetic bottlenecks are reported to have limited ability to detect them in populations with low sample numbers (less than 30 individuals) and loci (less than 10) or for long-lived species like Gopher Tortoises (Davy and Murphy 2014, Peery et al. 2012, Piry et al. 1999). The total number of Gopher Tortoises on Marco Island is difficult to determine since many reside on private properties; thus, knowing what proportion of the population is represented by the 61 tortoises collected makes it difficult to determine if this factor impacted the BOTTLENECK results. It is possible that the Marco Island tortoises may be experiencing a diffuse genetic bottleneck with immediate impacts that are difficult to identify. RBNERR tortoises display a similar reduction in allelic richness, low inbreeding coefficient, and a high level of unique alleles as found in the Marco Island population. This may indicate evidence of a past or current bottleneck; however, the sample number was low (23) and may not be enough for a conc lusive result. This is the first study to analyze genetic divergence of an island population from mainland Gopher Tortoises. The populations have diverged significantly by evidence of the large number of private alleles and the multiple significant tests of genetic differentiation. The FST estimate is higher than what has been reported for other Gopher Tortoise populations using the same loci—0.03 at Kennedy Space Center (Sinclair et al. 2010) and 0.24 across Florida and Georgia (Schwartz and Karl 2005)—and similar to the findings (FST = 0.00–0.54) of Clostio et al. (2012) using 5 of the same loci combined with 5 additional loci. Furthermore, the large Southeastern Naturalist C. Winters, J. Ross, and P. Allman 2017 Vol. 16, No. 3 378 number of private alleles may indicate local adaptation to environmental conditions that vary across populations (Sjostrand et al. 2013), and should be considered for conservation purposes of protecting unique populations (Petit e t al. 1998). Current permitting guidelines in Florida allow Gopher Tortoises to be translocated within 160 km north or south of the original population, but no restriction is given for movement east and west (Florida Fish and Wildlife Conservation Commission 2013). Well-informed translocations can help to maintain gene flow and prevent genetic isolation while also preserving the unique genotypes present in the populations. Schwartz and Karl (2005) identified 8 genetic subpopulations of Gopher Tortoises across Florida and southern Georgia, but the extreme southwest region of Florida was not included. Our study of Marco Island and RBNERR expands the genetic record for Gopher Tortoises in the state of Florida. In light of the significant genetic differentiation between the Marco Island and RBNERR populations, it is likely that the Marco Island population is genetically distinct from peninsular Florida populations. Recent studies of Gopher Tortoises across their range (Clostio et al. 2012, Ennen et al. 2012) have emphasized the need to identify unique populations as distinct management units (sensu Moritz 1994) to inform management decisions. This study has identified the Marco Island tortoises as genetically isolated, and proper management and protection of this species should preserve the uniqueness of the population while minimizing further population reduction. Translocations off the island will increase the loss of alleles leading to genetic drift; therefore, solutions that will retain individuals on the island should be explored. Tortoises on RBNERR may also require careful management, but genotyping of additional individuals and genetic comparison to nearby peninsular populations should be done prior to translocations into or out of the reserve. Acknowledgments A special thanks to members of the Florida Gulf Coast Herpetology Research Lab for their time in the field and reviewing earlier versions of the manuscript. We are grateful to Russ Burke, Bridgette Hagerty, and Rich Seigel for reviewing earlier versions of the manuscript. We also thank Cheryl Metzger and the staff at Rookery Bay National Estuarine Research Reserve for logistical support. Matt Finn provided the GPS and access to tortoises for sampling. All methods were completed under Florida Fish and Wildlife Conservation Commission permit #WV08181 (Julie Ross) and Florida Gulf Coast University IACUC #0708-05 (Phillip Allman). Literature Cited Auffenberg, W. and R. Franz. 1978. Gopherus polyphemus. 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