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The green turtle (Chelonia mydas) haplotype CM-A13 has been recorded in the Greater Caribbean region as well as in the Mediterranean Sea (Bagley 2003; Encalada et al. 1996). Phylogeographic analyses placed CM-A13 within the northern Caribbean haplogroup and prompted the hypothesis that the Mediterranean nesting aggregation was founded through colonization from the Caribbean (Bowen et al. 1992; Encalada et al. 1996). CM-A13 accounted for nearly all females sampled in the nesting populations of Turkey and Cyprus (Bagda et al. 2012; Encalada et al. 1996) but was not known from rookeries outside the eastern Mediterranean. Therefore the presence of CM-A13 among juveniles foraging at northern Greater Caribbean sites (Florida, the Bahamas and Texas) invoked the possibility of dispersal from the Mediterranean into the western Atlantic (Anderson et al. 2013; Bagley 2003; Bjorndal & Bolten 2008). However the recent detection of CM-A13 at low frequency in the Florida nesting aggregation suggested that local origins for foraging juveniles was a more parsimonious alternative (Shamblin et al. 2015). All CM-A13 turtles analyzed using expanded (~800 base pair) control region sequences on both sides of the Atlantic were identical (CM-A13.1) (Bagda et al. 2012; Shamblin et al. 2015), so determining the origins of the Greater Caribbean juveniles required application of additional markers.

The dinucleotide microsatellite repeat array (mtSTR) that occurs at the 3’ end of the mitochondrial control region has proven useful in uncovering additional mitochondrial variation (Tikochinski et al. 2012). In Atlantic green turtles, this array was partitioned into four separate loci by point mutations and non-repetitive sequences that interrupt strings of ‘AT’ repeats (Fig. 1). Therefore assessing the number of ‘AT’ repeat units present in each of the four loci permits subdivision of point mutation-defined haplotypes into multiple variants. Despite nearly complete fixation of haplotype CM-A13 in the Mediterranean (Bagda et al. 2012), analysis of individuals representing the Israeli rookery and strandings along the Israeli coast detected 33 mtSTR haplotypes (Tikochinski et al. 2012). Several variants recorded among the strandings were absent in the Israeli nesting population, suggesting that some degree of structure likely occurs among rookeries comprising the Mediterranean nesting aggregation.

Figure 1. Sequence chromatogram illustrating the four ‘AT’ repeat unit loci in the Atlantic green turtle mitochondrial genome.

We sequenced the mtSTR array in 15 CM-A13 turtles from Florida: 12 nesting females and three foraging juveniles (Fig. 2). Nesting samples were those previously analyzed in Shamblin et al. (2015) as well as new females sampled at Melbourne Beach in 2011 (n = 2) and 2012 (n = 1). Foraging juveniles were those previously identified as CM-A13 in an analysis of foraging aggregations in developmental habitats in east central Florida (Bagley 2003). We generated 817-base pair control region haplotypes using primers LCM15382 and H950 as previously described (Shamblin et al. 2012). We conducted polymerase chain reactions and sequencing of the mitochondrial repeat as described by Tikochinski et al. (2012), except that primer CMD1 was used for sequencing the forward strand.

Figure 1. Sampling locations for CM-A13 green turtles in central eastern Florida, USA. Stars indicate rookeries. Triangles represent juvenile foraging sites. This figure was created using the SEATURTLE.ORG Maptool (2002).

All 15 individuals nesting and foraging in Florida carried the same mitochondrial repeat haplotype: 5-7-7-4. This was not one of the 33 repeat haplotypes detected in the Israeli rookery or among individuals stranded along the Israeli coast (Tikochsinki et al. 2012). These results suggest that the CM-A13 juveniles foraging in Florida most likely originated within the Greater Caribbean region, although more thorough sampling on both sides of the Atlantic are required to determine if 5-7-7-4 represents the sole repeat haplotype present in the western Atlantic and is diagnostic of Greater Caribbean origin. Local sourcing of CM-A13 in the western Atlantic is consistent with ocean circulation modeling in which virtual hatchlings released from green turtle rookeries in Turkey, Cyprus, and Israel were retained in the eastern Mediterranean (Putman & Naro-Maciel 2013).
The large disparity in repeat haplotype diversity observed between Caribbean and Mediterranean turtles provides further support for the hypothesis that the current Florida CM-A13 nesters represent the remnant of a much larger lineage that recently passed through a severe bottleneck. Shamblin et al. (2015) suggested that CM-A13 might have been the primary lineage of the once immense Bermuda rookery that was extirpated in the 18th century (Parsons 1962). Bermuda’s position proximal to the Gulf Stream and downstream of the other Greater Caribbean rookeries may have facilitated transport of large numbers of oceanic juveniles into the Mediterranean Sea (Shamblin et al. 2015). Analysis of expanded loggerhead turtle (Caretta caretta) control region sequences and more extensive sampling in the Mediterranean suggested multiple colonization events and earlier colonization than previously hypothesized (Clusa et al. 2013). If colonization by green turtles occurred in a similar manner, the high repeat haplotype diversity for CM-A13 in the Mediterranean may have arisen through multiple colonization events as well as in situ diversification within the Mediterranean during the thousands of years following colonization.

The CM-A13 story highlights important lessons that should be considered in interpreting marine turtle connectivity analyses. First, incomplete baseline data from rookeries may yield misleading results with respect to mixed stock analysis. In addition to ensuring that all potentially contributing rookeries have been represented, rookery sampling should be designed to capture any geographical structure that might be present and be sufficiently deep to detect rare haplotypes. Second, traditionally used control region sequences may provide limited resolution for assessing stock structure and migratory connectivity at regional and even ocean basin scales. Use of nuclear and additional mitochondrial markers have improved stock resolution in some cases (Dutton et al. 2013; Shamblin et al. 2012; Tikochinski et al. 2012). Finally, the strongest inferences of connectivity often arise from combining multiple methodologies (Godley et al. 2010; Stewart et al. 2013). In our case, the genetic results were congruent with previous biophysical modeling, suggesting that the connection between CM-A13 green turtles in the Greater Caribbean and Mediterranean Sea reflects an historical colonization signature rather than contemporary migratory connectivity.

Acknowledgements. We thank R. Erik Martin, Niki Desjardin, and Chris Johnson for providing nest samples from Hutchinson Island, Hobe Sound National Wildlife Refuge, and Tequesta for an earlier study. We also thank the many students who have participated in the University of Central Florida’s Marine Turtle Research Group that assisted with sample collection. This research was funded by a grant awarded from the Sea Turtle Grants Program. The Sea Turtle Grants Program is funded from proceeds from the sale of the Florida Sea Turtle License Plate (www.helpingseaturtles.org).

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