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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

Project Description

maps showing study site in South Florida, Everglades National Park
Fig. 1. Study site in South Florida, Everglades National Park. [larger image]
The Everglades National Park (ENP), USA, is an ecosystem of internationally recognized importance utilized by many endangered or threatened species, including the green sea turtle. The Park has been designated an International Biosphere Reserve, a World Heritage Site, and a Wetland of International Importance in light of its ecological significance. However, relatively little is known about the ecology of sea turtles in the Everglades, especially in mangrove ecosystems. We therefore recently initiated a comprehensive program focusing on mark-recapture, satellite tracking, foraging ecology, health, and genetic research in the Big Sable Creek complex in the southwest coastal Everglades (Fig. 1).

Introduction

  • 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?

Project Update

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):

photo of captured green turtle photo of left side of green turtle's head photo of submerged algae-covered logs in creek
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]

map showing green sea turtle sitings and seep locations in the Big Sable Creek complex; photos of a seep in the headwaters, red mangrove-lined creek, and pound net setup
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]

Methods

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
arrow pointing towards the right Laboratory analysis
  • Sequencing
    • mtDNA dloop
    • Primers: Abreu et al. (2006)
  • Genotyping
    • nuclear microsatellites
    • 10-15 loci
arrow pointing towards the right 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 analyses:

  • 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.

Results

  • 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).
Table 1. Characteristics of the 10 longer mtDNA haplotypes that most closely resembled the sequence collected in the Everglades National Park.
GENBANK ID OTHER ID % Identity Alignment length Reference
AJ543731.1 CMY543731
100
599
Azanza-Ricardo 2002
AB012104.1 -
99.88
826
Kumazawa and Nishida 1995
M98394.1 CEZMTTGP
99.83
601
Allard et al. 1994
AJ543733.1 CMY543733
99.83
599
Azanza-Ricardo 2002
AJ543732.1 CMY543732
99.83
599
Azanza-Ricardo 2002
AJ543729.1 CMY543729
99.83
599
Azanza-Ricardo 2002
AJ543735.1 CMY543735
99.67
599
Azanza-Ricardo 2002
AJ543734.1 CMY543734
99.67
599
Azanza-Ricardo 2002
AF366258.1 CM-A29
99.63
537
Bjorndal and Bolten 2001 [GENBANK]
AJ543730.1 CMY543730
99.01
605
Azanza-Ricardo 2002
U40660.1 CMU40660
98.82
510
Dutton et al. 1996

Initial Conclusions

  • 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.

Acknowledgements

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.

Literature Cited

Abreu-Grobois A, Horrocks JA, Formia A, Dutton P, LeRoux R, Velez-Zuazo X, Soares L, Meylan P, 2006. New mtDNA dloop primers which work for a variety of marine turtle species may increase the resolution of mixed stock analyses. In: Proceedings of the Twenty-Sixth Annual Symposium on Sea Turtle Biology and Conservation, Crete, Greece.

Allard MW, Miyamoto MM, Bjorndal KA, Bolton AB, Bowen BW, 1994. Support for natal homing in green turtles from mitochondrial DNA sequences. Copeia 1: 34-41.

Azanza-Ricardo J, 2002. Marcaje y caracterizacion genetica de la tortuga verde que anida en la Peninsula de Guanahacabibes, Pinar del Rio, Cuba. Thesis, Department of Centro de Investigaciones Marinas, Universidad de La Habana, Ciudad de La Habana, Cuba.

Bass AL and Witzell WN, 2000. Demographic composition of immature green turtles (Chelonia mydas) from the east central Florida coast: evidence from mtDNA markers. Herpetologica 56:357-367.

Bass AL, Epperly SP, Braun-McNeill J, 2006. Green turtle (Chelonia mydas) foraging and nesting aggregations in the Caribbean and Atlantic: impact of currents and behavior on dispersal. Journal of Heredity 97:346-354.

Clement M, Posada D, Crandall KA, 2000. TCS: a computer program to estimate gene genealogies. Molecular Ecology 9:1657-1659.

Dutton PH, 1996. Methods for collection and preservation of samples for sea turtle genetic studies. In: Proceedings of the International Symposium on Sea Turtle Conservation Genetics, Miami FL, 12-14 September 1995 (Bowen BW and Witzell WN, eds). Miami, Florida: NOAA Technical Memorandum NMFS-SEFSC-396; 17-24.

Encalada SE, Lahanas PN, Bjorndal KA, Bolten AB, Miyamoto MM, Bowen BW, 1996. Phylogeography and population structure of the Atlantic and Mediterranean green turtle Chelonia mydas: a mitochondrial DNA control region sequence assessment. Molecular Ecology 5:473-483.

Excoffier L, Laval G, Schneider S, 2005. Arlequin version. 3.0: An integrated software package for population genetics data analysis. Evolutionary Bioinformatics Online 1:47-50.

Kumazawa Y, Nishida M, 1995. Complete Mitochondrial DNA Sequences of the Green Turtle and Blue-Tailed Mole Skink: Statistical Evidence for Archosaurian Affinity of Turtles. Molecular Biology and Evolution 16(6):784-792.

Lahanas PN, Bjorndal KA, Bolten AB, Encalada SE, Miyamoto MM, Valverde RA, Bowen BW, 1998. Genetic composition of a green turtle (Chelonia mydas) feeding ground population: evidence for multiple origins. Marine Biology 130:345-52.

Luke K, Horrocks JA, Le Roux RA, Dutton PH, 2004. Origins of green turtle (Chelonia mydas) feeding aggregations around Barbados, West Indies. Marine Biology 144:799-805.

Naro-Maciel E, Becker JH, Lima EHSM, Marcovaldi MA, DeSalle R, 2007. Testing dispersal hypotheses in foraging green sea turtles (Chelonia mydas) of Brazil. Journal of Heredity 98(1): 29-39.

Nei M, 1987. Molecular Evolutionary Genetics Columbia University Press: New York, NY.

Pella J and Masuda M, 2001. Bayesian methods for analysis of stock mixtures from genetic characters. Fishery Bulletin 9:151-167.

Pritchard JK, Stephens M, Donnelly P, 2000. Inference of Population Structure Using Multilocus Genotype Data. Genetics 155:945-959.

Raymond M, Rousset F, 1995. An exact test for population differentiation. Evolution 49:1280-1283.


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