This study builds upon the current understanding of green turtle population genetic structure in the Pacific that has largely been based on mitochondrial DNA (mtDNA), by examining nuclear DNA (nDNA) diversity, regional connectivity, and male-mediated gene flow. A total of 1,111 nesting green turtle samples were analyzed with 10 microsatellite markers from 20 Pacific rookeries. Population differentiation (F ST ) was significant (p <0.05) in all but 8 of 190 pairwise rookery comparisons. Pairwise F ST values and discriminant analysis of principal components (DAPC) revealed a defined East-West split consistent with mtDNA studies. Additionally, isolation-by-distance was evaluated with estimated effective migration surfaces (EEMS). The data indicated structure throughout the Pacific rookeries largely in agreement with stock structure defined by mtDNA studies, except for some areas on the Central American and Australian continental shelves, providing evidence of possible male-mediated gene flow. The series of analyses performed did indicate that male-mediated gene flow has likely occurred where breeding migration corridors of separate populations overlap with courtship areas. This may occur primarily along the margins of continents, including along Mexico and Central America in the East Pacific. Our study provides an ocean-wide baseline nDNA dataset for green turtle rookeries in the Pacific and reexamines the current thinking regarding the role of male turtles in the population dynamics of management units (MU) and to what extent nuclear gene flow occurs among designated MUs.

Longline fishing vessels, such as those that target tuna or billfish, also unintentionally catch endangered marine turtle species on the high seas. The stock composition of this bycatch is often unknown but potentially complex, with individuals coming from many possible origins on an ocean-basin scale. To better understand the stock composition of green turtle (Chelonia mydas) bycatch we obtained 46 turtles, 27-91 cm in curved carapace length, caught by Hawaii- and American Samoa-based pelagic longline fishing vessels across large areas of the North- and South-central Pacific. We genotyped these at nine microsatellite loci and one mitochondrial DNA marker, and used a baseline of 1,043 nesting female green turtles from beaches across the Pacific for population assignment and mixed-stock analysis. By analyzing both marker types jointly we were able to increase power and genetically resolve ten baseline stocks of nesting females with mean self-assignment and simulated accuracies of 75-97%. Above the Equator, green turtle bycatch was composed mostly of individuals from Hawaiian and Eastern Pacific stocks, with a small number from the Western Pacific. Below the Equator, the most common stocks in the bycatch were from Australia and the Coral Sea, American Samoa and French Polynesia, and the Galápagos Islands. Overall, turtles originating from East, West, and Central Pacific breeding populations were major components of the bycatch, suggesting that the geographic ranges of these populations overlap across large tracts of ocean during the pelagic life history stages.

Assessing population connectivity and identifying conservation units are critical to formulating effective management plans for marine turtles. We combined a multiplexed targeted enrichment capture technique and high-throughput sequencing to discover single nucleotide polymorphisms (SNPs) and to genotype hawksbill turtles ( Eretmochelys imbricata ) to examine population structure. Our approach discovered 455 potential informative SNPs for hawksbill turtles. We optimized 25 of the 455 SNPs discovered and used them to identify preliminary evidence of hawksbill population structure, suggesting that these markers will be informative in discerning conservation units when applied to larger sample sizes in future studies.

Understanding population dynamics in broadly distributed marine species with cryptic life history stages is challenging. Information on the population dynamics of sea turtles tends to be biased toward females, due to their accessibility for study on nesting beaches. Males are encountered only at sea; there is little information about their migratory routes, residence areas, foraging zones, and population boundaries. In particular, male leatherbacks (Dermochelys coriacea) are quite elusive; little is known about adult and juvenile male distribution or behavior. The at-sea distribution of male turtles from different breeding populations is not known. Here, 122 captured or stranded male leatherback turtles from the USA, Turkey, France, and Canada (collected 1997–2012) were assigned to one of nine Atlantic basin populations using genetic analysis with microsatellite DNA markers. We found that all turtles originated from western Atlantic nesting beaches (Trinidad 55%, French Guiana 31%, and Costa Rica 14%). Although genetic data for other Atlantic nesting populations were represented in the assignment analysis (St. Croix, Brazil, Florida, and Africa (west and south), none of the male leatherbacks included in this study were shown to originate from these populations. This was an unexpected result based on estimated source population sizes. One stranded turtle from Turkey was assigned to French Guiana, while others that were stranded in France were from Trinidad or French Guiana breeding populations. For 12 male leatherbacks in our dataset, natal origins determined from the genetic assignment tests were compared to published satellite and flipper tag information to provide evidence of natal homing for male leatherbacks, which corroborated our genetic findings. Our focused study on male leatherback natal origins provides information not previously known for this cryptic, but essential component of the breeding population. This method should provide a guideline for future studies, with the ultimate goal of improving management and conservation strategies for threatened and endangered species by taking the male component of the breeding population into account.

AbstractFisheries that operate at large spatial scales and with high intensity have the potential to impact highly migratory species, and it is important to characterize threats to specific breeding populations of these species. We used many‐to‐many mixed‐stock analysis (MSA) (n = 408) and microsatellite assignment testing (n = 397) to determine source populations for leatherback turtles (Dermochelys coriacea) caught as bycatch in the U.S. pelagic longline fishery from 2002 to 2012 in the western North Atlantic. Within the United States, we had bycatch samples from the majority of statistical fishing areas: Gulf of Mexico (GOM), Northeast Distant (NED), Caribbean (CAR), Florida East Coast (FEC), Mid Atlantic Bight (MAB), Northeast Coastal (NEC), South Atlantic Bight (SAB) and Sargasso (SAR). We determined the proportions of turtles from each of nine nesting stocks in the Atlantic in each of the sampled areas. These nesting stocks included Brazil, Costa Rica, Florida, Trinidad, French Guiana, St. Croix, Ghana, Gabon, and South Africa. The MSA revealed that the NED had a lower relative proportion of turtles from Costa Rica than other areas and that the GOM had the highest relative proportion of turtles from Costa Rica. No turtles were assigned to the African rookeries, lending further evidence that turtles from that region forage elsewhere and therefore may not be affected by western North Atlantic fisheries. This work contributes to the ongoing assessment of threats to leatherback turtles in the Regional Management Unit (RMU) of the western North Atlantic, and draws attention to the disproportionate number of turtles from Costa Rica being caught in the Gulf of Mexico; Costa Rica is one of the only populations in the northern Atlantic that is not experiencing significant increases in nest numbers. This approach should be useful in determining population‐specific threats to other highly migratory protected species that may depend on segregated foraging areas either within or among species.

A set of nuclear single nucleotide polymorphisms (SNPs) and microsatellite markers was used to detect genetic stock structure among 5 Pacific green turtle Chelonia mydas nesting populations. We sampled populations in the Galapagos Islands, Ecuador (n = 57), Colola, Mexico (n = 75), French Frigate Shoals, Hawaii (n = 141), Yap, Micronesia (n = 73), and Wan-an, Taiwan (n = 57), to represent eastern, central, and western Pacific regions. A combination of 29 single independent SNPs and linked SNPs combined as haplotypes were used for a total of 20 independent markers. In addition, 8 polymorphic microsatellite markers were applied to the same sample set. Both sets of nuclear markers confirmed significant differentiation between all sampled populations in the 3 Pacific regions (p <= 0.001). The use of these SNPs and microsatellites resulted in sufficient power to detect small population differences not seen in previous studies using smaller numbers of nuclear markers. Our results suggest that male-mediated gene flow between regional nesting stocks is more limited than previously believed, allowing the potential to delineate stocks more clearly. Finally, we discuss the value of SNP markers as an alternative or complement to other nuclear markers such as microsatellites for the examination of stock structure.

Summary Investigating migratory connectivity between breeding and foraging areas is critical to effective management and conservation of highly mobile marine taxa, particularly threatened, endangered, or economically important species that cross through regional, national and international boundaries. The leatherback turtle ( Dermochelys coriacea , Vandelli 1761) is one such transboundary species that spends time at breeding areas at low latitudes in the northwest Atlantic during spring and summer. From there, they migrate widely throughout the North Atlantic, but many show fidelity to one region off eastern Canada, where critical foraging habitat has been proposed. Our goal was to identify nesting beach origins for turtles foraging here. Using genetics, we identified natal beaches for 288 turtles that were live‐captured off the coast of Nova Scotia, Canada. Turtles were sampled (skin or blood) and genotyped using 17 polymorphic microsatellite markers. Results from three assignment testing programs ( ONCOR , GeneClass2 and Structure) were compared. Our nesting population reference data set included 1417 individuals from nine Atlantic nesting assemblages. A supplementary data set for 83 foraging turtles traced to nesting beaches using flipper tags and/or PIT tags ( n = 72), or inferred from satellite telemetry ( n = 11), enabled ground‐truthing of the assignments. We first assigned turtles using only genetic information and then used the supplementary recapture information to verify assignments. ONCOR performed best, assigning 64 of the 83 recaptured turtles to natal beaches (77·1%). Turtles assigned to Trinidad (164), French Guiana (72), Costa Rica (44), St. Croix (7), and Florida (1) reflect the relative size of those nesting populations, although none of the turtles were assigned to four other potential source nesting assemblages. Our results demonstrate the utility of genetic approaches for determining source populations of foraging marine animals and include the first identification of natal rookeries of male leatherbacks, identified through satellite telemetry and verified with genetics. This work highlights the importance of long‐term monitoring and tagging programmes in nesting and high‐use foraging areas. Moreover, it provides a scientific basis for evaluating stock‐specific effects of fisheries on migratory marine species, thus identifying where coordinated international recovery efforts may be most effective.

This study presents a comprehensive genetic analysis of stock structure for leatherback turtles (Dermochelys coriacea), combining 17 microsatellite loci and 763 bp of the mtDNA control region. Recently discovered eastern Atlantic nesting populations of this critically endangered species were absent in a previous survey that found little ocean-wide mtDNA variation. We added rookeries in West Africa and Brazil and generated longer sequences for previously analyzed samples. A total of 1,417 individuals were sampled from nine nesting sites in the Atlantic and SW Indian Ocean. We detected additional mtDNA variation with the longer sequences, identifying ten polymorphic sites that resolved a total of ten haplotypes, including three new variants of haplotypes previously described by shorter sequences. Population differentiation was substantial between all but two adjacent rookery pairs, and F (ST) values ranged from 0.034 to 0.676 and 0.004 to 0.205 for mtDNA and microsatellite data respectively, suggesting that male-mediated gene flow is not as widespread as previously assumed. We detected weak (F (ST) = 0.008 and 0.006) but significant differentiation with microsatellites between the two population pairs that were indistinguishable with mtDNA data. POWSIM analysis showed that our mtDNA marker had very low statistical power to detect weak structure (F (ST) < 0.005), while our microsatellite marker array had high power. We conclude that the weak differentiation detected with microsatellites reflects a fine scale level of demographic independence that warrants recognition, and that all nine of the nesting colonies should be considered as demographically independent populations for conservation. Our findings illustrate the importance of evaluating the power of specific genetic markers to detect structure in order to correctly identify the appropriate population units to conserve.

Buccal and cloacal swabs have been used for genetic sampling for a variety of reptiles but not for marine turtles to date. We evaluated whether this method offers a simple and quick way to sample cells from live marine turtles in the wild when it is not feasible to obtain blood or skin. Good-quality DNA was obtained for genetic analyses from both buccal and cloacal swabs. Although we recommend blood and skin sampling whenever possible to collect the highest quality DNA, buccal and cloacal swabs do represent a useful alternative for genetic sampling when these preferred methods are not feasible.

Fourteen microsatellites were isolated and characterized from a small-insert genomic DNA library from the leatherback turtle Dermochelys coriacea enriched for dinucleotide microsatellite motifs. We tested primers on 207 leatherbacks sampled from St. Croix, US Virgin Islands. Primer pairs yielded an average of 5.7 alleles per locus, an average observed heterozygosity of 0.47, and average polymorphic information content of 0.43. Cross species amplification of these markers was performed on the six other extant species of marine turtles: olive ridley (Lepidochelys olivacea), Kemp's ridley (Lepidochelys kempii), hawksbill (Eretmochelys imbricata), loggerhead (Caretta caretta), green (Chelonia mydas), and flatback (Natator depressus) turtles. Eleven of the markers worked in at least one of the species, and seven of these were polymorphic. These leatherback-specific microsatellite markers will facilitate population genetic and ecological studies to aid in the conservation of this divergent species of marine turtle, and provide additional markers for the other species of cheloniids.

We present data on 29 new single nucleotide polymorphism assays for the green sea turtle, Chelonia mydas. DNA extracts from 39 green turtles were used for two methods of single nucleotide polymorphism discovery. The first approach employed an amplified fragment length polymorphism technique. The second technique screened a microsatellite library. Allele-specific amplification assays were developed for high-throughput single nucleotide polymorphism genotyping and tested on two Pacific C. mydas nesting populations. Observed heterozygosities ranged from 0 to 0.95 for a Hawaiian population and from 0 to 0.85 for a Galapagos population. Each of the populations had one locus out of Hardy-Weinberg equilibrium, SSCM2b and SSCM5 for Hawaii and Galapagos, respectively. No loci showed significant genotypic linkage disequilibrium across an expanded set of four Pacific nesting populations. However, two loci, SSCM4 and SSCM10b showed linkage disequilibrium across three populations indicating possible association.

The green sea turtle, Chelonia mydas, was used as a case study for single nucleotide polymorphism (SNP) discovery in a species that has little genetic sequence information available. As green turtles have a complex population structure, additional nuclear markers other than microsatellites could add to our understanding of their complex life history. Amplified fragment length polymorphism technique was used to generate sets of random fragments of genomic DNA, which were then electrophoretically separated with precast gels, stained with SYBR green, excised, and directly sequenced. It was possible to perform this method without the use of polyacrylamide gels, radioactive or fluorescent labeled primers, or hybridization methods, reducing the time, expense, and safety hazards of SNP discovery. Within 13 loci, 2547 base pairs were screened, resulting in the discovery of 35 SNPs. Using this method, it was possible to yield a sufficient number of loci to screen for SNP markers without the availability of prior sequence information.