Gopher Tortoise Genetics from Moody Air Force Base, Georgia David Mederos, Department of Biology Faculty Sponsors: Dr. J. Mitchell Lockhart and Dr. John F. Elder, Department of Biology Co-Author: Gregory Lee, Moody Air Force Base ABSTRACT Gopher tortoises (Gopherus polyphemus) are a threatened species in Georgia and a basic understanding of natural history and population genetic parameters is vital for successful management of the species. Knowledge of population genetics can provide important information concerning normal movement and artificial translocation of animals and perhaps diseases they may carry. DNA was isolated from the blood of 15 gopher tortoises from different colonies found on Moody Air Force Base, Georgia. Using microsatellite analysis from nine different VNTR loci we were able to establish the gene flow between colonies, genetic distance, and map out phylogenetic trees between different sub-populations. Data from an additional 80 animals will be generated and included in the final analysis. INTRODUCTION and BACKGROUND Moody AFB is required by Department of Defense and Department of Air Force regulations and federal law to protect rare, threatened, and endangered (RTE) species. As part of this mandate, the installation is occasionally required to translocate RTE species that will be impacted by the military mission. This work will help provide evidence to determine if the prevalence of disease within various populations of an RTE species (the gopher tortoise, Gopherus polyphemus – see Figure 2) are naturally occurring or are from potential animal translocations. Moody AFB consists of 10,913 acres in Lowndes and Lanier Counties, Georgia, and is divided into two main administrative units: 1) Main Base, where the airfield and cantonment areas are located, and, 2) Grand Bay Range, where the Grand Bay weapons range and buffer areas are located. Of the 10,913 acres, 7,943 acres (73%) are forested. The majority of the forested area (4,848 acres) is classified as forested wetlands, consisting of cypress domes and Carolina bays. The remainder of the forested area (3,095 acres) is comprised of upland forest stands, primarily consisting of typical pine flatwoods habitat with numerous small, isolated, seasonally wet shallow ponds and depressions. A Natural Heritage Inventory of the installation was conducted in 1994 and included characterization of installation ecosystems and surveys for indigenous species, including specific surveys for gopher tortoises. Seven gopher tortoise colonies, consisting of 204 active burrows were identified on Moody AFB (see Map – Figure 1), with three colonies on Main Base and four colonies on Grand Bay Range. Copies of the Natural Heritage Inventory and applicable Air Force regulations and policies are available for review in the Moody AFB Natural Resources Office. 71CPARCSBF CP AR CS BF Figure 2. Gopher tortoise, Gopherus polyphemus and sign indicating its natural history. METHODS DNA samples were extracted from 0.25 ml homogenized, clotted gopher tortoise blood using a 4 M Guanidinium Isothiocyanate (GIT) and Phenol/chloroform extraction process followed by an ethanol precipitation in 2 volumes of cold 95% ethanol (Turner et al.,1989). Microsatellite loci in the template DNA were amplified using polymerase chain reaction (PCR) amplification techniques. PCR primers were synthesized (Tonja Schwartz, University of South Florida) that amplify 9 different loci. Primers were combined to produce two multiplexing reactions in order to amplify multiple loci at once. Multiplex mix 1 contained 15 pmols of the primers GP15F, GP15R-6-FAM, and 10 pmols of the primers GP30F-TET, GP30R, GP55F-TET, GP55, GP26F, GP26R-TET. Multiplex mix 2 contained 10 pmols of the primers GP96F-6-FAM, GP96R, GP61F-6-HEX, GP61R, GP19F-FAM, GP19R and 15 pmols of primers GP102F-TET, GP102R, GP81F, GP81R-6-FAM. Basic PCR components protocol concentrations of MgCl, polymerase, and other components were proprietary standards of the EPICENTRE FailSafe 2X PreMix Kit. PCR temperature cycling was done using a Amplitron II PCR thermocycler (Barnstead/Thermolyne Corporation, Dubuque, IA). Thermal programs were identical for both multiplex reactions. PCR cycle parameters were: 2 minutes at 94°C followed by, 35 cycles of 94°C for 30 sec, 55°C for 30 sec, and 72°C for 30 sec. A final extension completion period of 30 min was done at 72°C. Samples were analyzed by electrophoresis using Agarose SFR (AMRESCO, Solon, OH). Sixteen ul of each PCR product were combined with 0.5μl of ethidium bromide and 5μl of 0.15% glycerol/ Bromophenol Blue loading dye. Samples were electrophoresed in 3 % gels at 60 volts for 2 hours prior to documentation by photography under UV light (see Figure 3 for example of optimization gel). RESULTS and DISCUSSION Preliminary results were produced at USF for all nine loci in 15 individual tortoise samples. Allelic microsatellite data was analyzed at VSU using the maximum parsimony and maximum likelihood algorithms of PHYLIP (Felsenstein, 2004). Genetic distances (Figure 4) were produced for population samples in order to demonstrate the potential utility of these microsatellite loci for detecting gopher tortoise population structure. Subsequently, PCR amplification of all microsatellite loci was optimized for data acquisition in Dr. Mitch Lockhart’s lab at VSU. Data produced at USF was replicated to demonstrate competence with methodology prior to data collection for the Moody Air Force Base study. Further data collection has begun on a larger scale population sampling for the same area. Figure 4. Nei’s Genetic Distance Matrix generated for trial analysis of gopher tortoise microsatellite data. Figure 3. FailSafe PCR optimization gel for tortoise microsatellite analysis. Note DNA ladder in 3 bottom lanes and various PCR optimization conditions in the remaining lanes. FUTURE RESEARCH We would like to complete microsatellite analysis for all Tortoises collected from Moody AFB, including colony analysis and genetic relatedness studies. We hope to be able to determine if certain animals (i.e. disease carriers) may have been imported or Artificially translocated onto base property. REFERENCES PHYLIP – see Felsenstein, 2004 at Turner, B.J., John F. Elder, Jr. and Thomas F. Laughlin DNA Fingerprinting Of Fishes: A General Method Using Oligonucleotide Probes. Fingerprint News (Cambridge). 1(4):15-16.