Green Sea Turtles (Chelonia mydas) of Everglades National Park: Habitat Associations and Genetic Analyses

Kristen M. Hart¹, Eugenia Naro-Maciel², Caroline P. Good³, and Carole C. McIvor¹

¹US Geological Survey, Florida Integrated Science Center, St. Petersburg, FL, USA; ²Center for Biodiversity and Conservation and Center for Conservation Genetics, American Museum of Natural History, New York City, NY, USA; ³Nicholas School of the Environment and Earth Sciences Marine Laboratory, Duke University, Beaufort, NC, USA

Fig. 1. Study site in South Florida, Everglades National Park. [larger image]

• Discovering the relationships among sea turtle populations is a global research priority. By determining the connectivity of green sea turtles (Chelonia mydas) of Everglades National Park (ENP), our research will enable practitioners to better understand the range of the populations they manage, recognize distinct populations and identify regional management partners, and further understand green sea turtle population biology.
• Due to their highly migratory nature, sea turtles at their feeding or nesting areas are likely connected to other groups, although in many cases the nature of these relationships is insufficiently understood. We employ genetic, mark-recapture, and satellite telemetry technologies to elucidate information about these linkages.
• Previous genetic studies of juvenile green sea turtles in the region have postulated that dispersal may be mediated by juvenile natal homing behavior and ocean currents (Bass and Witzell 2000; Luke et al. 2004; Bass et al. 2006; Naro-Maciel et al. 2007). However, unusually small turtles have been observed in the ENP study site (K. Hart, pers. observ.), and this may indicate a stronger role for ocean currents than behavior, underscoring the significance of investigating connectivity.
• In this project we address the following questions: What is the genetic composition at multiple loci of green sea turtles at the ENP? Do population size, geographic distance, natal homing, and/or ocean currents affect their genetic composition? Is there significant temporal variation? Do results differ depending on whether mtDNA or microsatellites are examined, and if so, why? Is the ENP connected to other nesting or feeding grounds? From which rookeries are the turtles found at this site primarily drawn, and is there a role for international collaboration in their management? How much time do satellite-tagged individuals spend in mangrove-lined creeks and bays within the boundaries of ENP, and where are “hotspots” of turtle activity?

Thus far, we have captured one individual (Fig. 2) and recorded unusual observations of green sea turtles ranging from 10 to 60 cm carapace length in the mangrove tidal creeks of the BSC. We have also collected GPS coordinates of sightings for 36 different green sea turtles (Fig. 3).—many of these have been in the headwater regions, approximately two kilometers from the Gulf coast. In this headwater habitat, there is a surprising amount of submerged algae-covered logs that are remnants of old red mangroves (Rhizophora mangle) (Fig. 2) and clear, salt-water seeps (Fig. 3):

Fig. 2. Photos of the first green turtle captured as part of this study (captured Nov. 15, 2006, 31cm SCL) and submerged algae covered logs in the headwater regions of one of the creeks in the study site. [click on images above to view larger versions]

Fig. 3. Green sea turtle sightings and seep locations in the Big Sable Creek complex, Everglades National Park. Inset photos: close-up of a seep in the headwaters, red mangrove-lined creek upstream, and pound net setup in the mouth of the complex. [larger image]

Currently, our capture methods include using dipnets and pound nets (Fig. 3) to capture green sea turtles. We take standard straight and curved carapace measurements, administer inconel flipper tags and PIT tags, and take samples for diet and genetic analysis (Fig. 4):

Sample collection Blood FTA card Laboratory analysis Sequencing mtDNA dloop Primers: Abreu et al. (2006) Genotyping nuclear microsatellites 10-15 loci Data analysis Genetic diversity (Nei 1987) ARLEQUIN (Excoffier et al. 2005) Haplotype network TCS (Clement et al. 2000) Exact test of population differentiation Raymond and Rousset (1995) ARLEQUIN (Excoffier et al. 2005) Mixed Stock Analysis BAYES (Pella and Masuda 2001) Microsatellites: Assignment STRUCTURE (Pritchard et al. 2000) Fig. 4. Flowchart of the methods of DNA analysis.

• Genetic samples collected from these turtles are being sequenced at the mtDNA control region and genotyped at microsatellite loci (Fig. 4). In this approach our objectives are to elucidate levels of genetic differentiation between the ENP and other Atlantic green turtle populations, as well as among temporal periods at the study site. We will also elucidate natal origins and potentially uncover dispersal mechanisms of sampled turtles.
• Blood samples are collected from green sea turtles in the Everglades and laboratory work and analyses are carried out at the Institute for Comparative Genomics at the American Museum of Natural History.
• It has been recommended that genetic studies of sea turtle connectivity assay various markers to avoid “disastrously incorrect” management decisions (Bowen et al. 2005, p. 2390). Therefore, in this research, we analyze mitochondrial DNA control region sequences and nuclear microsatellite genotypes. We also investigate the possibility raised by Abreu-Grobois et al. (2006), that examining longer mtDNA control region segments could increase the resolution of population genetic studies.

• To date, one blood sample has been collected. From this sample, we successfully amplified an mtDNA dloop segment (862 bp; n=1) that completely matched (100% identity) two sequences posted on GENBANK. These were the CM-A1 haplotype (Encalada et al. 1996; Z50124.1; 486bp), and sequence AJ543731 (Azanza-Ricardo 2002) which overlapped with ours along 599 bp.
• We compared our sequence to the 10 longer segments posted on GENBANK that most closely matched our sample (Table 1), and found that all of differences among our haplotypes occurred within the 486 bp. region previously amplified by other studies using primers designed by or modified from Allard et al. (1994).

• At this initial stage of our study very little can be said based on the single sample collected, although we anticipate obtaining over 100 samples in our ongoing research.
• The CM-A1 sequence that matched our sample had previously been reported at rookeries in Florida and Mexico (Encalada et al. 1996), as well as the following feeding grounds: Barbados (Luke et al. 2004), Bahamas (Lahanas et al. 1998), Florida (Bass and Witzell 2000), and North Carolina (Bass et al. 2006).
• The longer sequence that completely matched our samples (as well as CM-A1) was reported from the 'Peninsula de Guanahacabibes' nesting area, Pinar del Rio, Cuba (Azanza-Ricardo 2002; AJ543731.1). We explored whether analysis of longer segments could enhance the resolution of our population genetic study by uncovering additional variation outside of the previously characterized region, as hypothesized by Abreu et al. (2006). Although comparing our sequence to the longer control region segments did not detect additional variation, the sample sizes were small and the utility of examining longer sequences certainly bears further investigation.

We thank the US Fish and Wildlife Service, the US Geological Survey, Everglades National Park, American Museum of Natural History, the Marathon Sea Turtle Hospital, Betsy Boynton, Gary L. Hill, Noah Silverman, Lisa Eby, Paula Gillikin, Selina Heppell, BJ Reynolds, and Adam Brame.

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