Campbell J. Nairn

Mitogenomic sequences better resolve stock structure of southern Greater Caribbean green turtle rookeries

Analyses of mitochondrial control region polymorphisms have supported the presence of several demographically independent green turtle (Chelonia mydas) rookeries in the Greater Caribbean region. However, extensive sharing of common haplotypes based on 490‐bp control region sequences confounds assessment of the scale of natal homing and population structure among regional rookeries. We screened the majority of the mitochondrial genomes of 20 green turtles carrying the common haplotype CM‐A5 and representing the rookeries of Buck Island, St. Croix, United States Virgin Islands (USVI); Aves Island, Venezuela; Galibi, Suriname; and Tortuguero, Costa Rica. Five single‐nucleotide polymorphisms (SNPs) were identified that subdivided CM‐A5 among regions. Mitogenomic pairwise φST values of eastern Caribbean rookery comparisons were markedly lower than the respective pairwise FST values. This discrepancy results from the presence of haplotypes representing two divergent lineages in each rookery, highlighting the importance of choosing the appropriate test statistic for addressing the study question. Haplotype frequency differentiation supports demographic independence of Aves Island and Suriname, emphasizing the need to recognize the smaller Aves rookery as a distinct management unit. Aves Island and Buck Island rookeries shared mitogenomic haplotypes; however, frequency divergence suggests that the Buck Island rookery is sufficiently demographically isolated to warrant management unit status for the USVI rookeries. Given that haplotype sharing among rookeries is common in marine turtles with cosmopolitan distributions, mitogenomic sequencing may enhance inferences of population structure and phylogeography, as well as improve the resolution of mixed stock analyses aimed at estimating natal origins of foraging turtles.

Geographic Patterns of Genetic Variation in a Broadly Distributed Marine Vertebrate: New Insights into Loggerhead Turtle Stock Structure from Expanded Mitochondrial DNA Sequences

Previous genetic studies have demonstrated that natal homing shapes the stock structure of marine turtle nesting populations. However, widespread sharing of common haplotypes based on short segments of the mitochondrial control region often limits resolution of the demographic connectivity of populations. Recent studies employing longer control region sequences to resolve haplotype sharing have focused on regional assessments of genetic structure and phylogeography. Here we synthesize available control region sequences for loggerhead turtles from the Mediterranean Sea, Atlantic, and western Indian Ocean basins. These data represent six of the nine globally significant regional management units (RMUs) for the species and include novel sequence data from Brazil, Cape Verde, South Africa and Oman. Genetic tests of differentiation among 42 rookeries represented by short sequences (380 bp haplotypes from 3,486 samples) and 40 rookeries represented by long sequences (∼800 bp haplotypes from 3,434 samples) supported the distinction of the six RMUs analyzed as well as recognition of at least 18 demographically independent management units (MUs) with respect to female natal homing. A total of 59 haplotypes were resolved. These haplotypes belonged to two highly divergent global lineages, with haplogroup I represented primarily by CC-A1, CC-A4, and CC-A11 variants and haplogroup II represented by CC-A2 and derived variants. Geographic distribution patterns of haplogroup II haplotypes and the nested position of CC-A11.6 from Oman among the Atlantic haplotypes invoke recent colonization of the Indian Ocean from the Atlantic for both global lineages. The haplotypes we confirmed for western Indian Ocean RMUs allow reinterpretation of previous mixed stock analysis and further suggest that contemporary migratory connectivity between the Indian and Atlantic Oceans occurs on a broader scale than previously hypothesized. This study represents a valuable model for conducting comprehensive international cooperative data management and research in marine ecology.

Tetranucleotide markers from the loggerhead sea turtle ( Caretta caretta ) and their cross-amplification in other marine turtle species

The loggerhead sea turtle (Caretta caretta) is a federally threatened species and listed as endangered by the World Conservation Union (IUCN). We describe primers and polymerase chain reaction (PCR) conditions to amplify 11 novel tetranucleotide microsatellite loci from the loggerhead sea turtle. We tested primers using samples from 22 females that nested at Melbourne Beach, Florida (USA). Primer pairs yielded an average of 11.2 alleles per locus (range of 4–24), an average observed heterozygosity of 0.83 (range 0.59–0.96), and an average polymorphic information content of 0.80 (range 0.62–0.94). We also demonstrate the utility of these primers, in addition to primers for 15 loci previously described, for amplifying microsatellite loci in four additional species representing the two extant marine turtle families: olive ridley (Lepidochelys olivacea), hawksbill (Eretmochelys imbricata), green turtle (Chelonia mydas), and leatherback (Dermochelys coriacea).

Deeper Mitochondrial Sequencing Reveals Cryptic Diversity and Structure in Brazilian Green Turtle Rookeries

Abstract Genetic markers are often used to designate population units for management and conservation, but widespread sharing of mitochondrial DNA control-region haplotypes defined from short (< 500 base-pair [bp]) sequences often limits inferences of population connectivity in marine turtles. Haplotype CM-A8, defined from 490-bp sequences, dominated the haplotype profiles of the 3 major green turtle (Chelonia mydas) rookeries in Brazil. Previous analyses based on 490-bp haplotypes did not detect differentiation between the northern rookeries of Atol das Rocas and Fernando de Norohna, but did indicate differentiation of the northern rookeries from Trindade Island in the south. We reexamined the stock structure of the Brazilian green turtle rookeries using 817-bp control region and mitochondrial short tandem repeat (mtSTR) sequences. Nine 490-bp haplotypes were subdivided into 41 haplotypes by combining 817-bp and mtSTR sequences. Eight of the 14 CM-A8 turtles from Fernando de Noronha carried mtSTR haplotypes that were not detected in the larger rookeries. Pairwise exact tests indicated that the northern Brazilian green turtle rookeries of the Rocas Atoll and Fernando de Noronha are discrete populations with respect to female natal homing. Moreover, several apparently endemic markers in the 3 Brazilian green turtle nesting populations should improve resolution of future mixed-stock analyses. Comparable data are needed from green turtle rookeries in the central and eastern Atlantic to assess structure and connectivity at the ocean basin scale.

Loggerhead turtle eggshells as a source of maternal nuclear genomic DNA for population genetic studies.

Tagging studies on nesting beaches are commonly used to estimate nesting frequency, remigration interval and nesting population size for marine turtle rookeries. Estimates of these demographic parameters from tagging projects may be biased because of the small scale of tagging efforts relative to female nest site fidelity and the logistical difficulty of intercepting all nesting females. Therefore, alternative and supplemental means of individual identification of nesting females are required. We demonstrate that maternal nuclear microsatellite DNA can be isolated from unincubated eggshells of the loggerhead sea turtle (Caretta caretta) through comparison of DNA extracted from 59 eggs collected within 15 h of oviposition and DNA derived from skin samples from respective nesting females. Scorable microsatellite genotypes were produced in 897 of 994 (90.2%) single‐locus egg amplifications attempted. Among eggs from known females, 730 of 748 (97.6%) single‐locus, egg‐derived genotypes matched the respective skin‐derived genotypes. Allelic dropout was the most common type of error, followed by the presence of nonmaternal, presumably paternal, alleles. Genotypes derived from unincubated eggshells permit individual assignment of nests and therefore demographic parameter estimates for loggerhead turtle nesting populations, despite genotyping errors that require further optimization. Although sampling unincubated eggs is destructive, this technique is noninvasive to nesting females and is applicable in marine turtle population genetics studies when individual resolution is required but direct interception of nesting females is undesirable or logistically infeasible.

Characterization of a subtropical hawksbill sea turtle (Eretmocheyles imbricata) assemblage utilizing shallow water natural and artificial habitats in the Florida Keys.

In order to provide information to better inform management decisions and direct further research, vessel-based visual transects, snorkel transects, and in-water capture techniques were used to characterize hawksbill sea turtles in the shallow marine habitats of a Marine Protected Area (MPA), the Key West National Wildlife Refuge in the Florida Keys. Hawksbills were found in hardbottom and seagrass dominated habitats throughout the Refuge, and on man-made rubble structures in the Northwest Channel near Cottrell Key. Hawksbills captured (N = 82) were exclusively juveniles and subadults with a straight standard carapace length (SSCL) ranging from 21.4 to 69.0cm with a mean of 44.1 cm (SD = 10.8). Somatic growth rates were calculated from 15 recaptured turtles with periods at large ranging from 51 to 1188 days. Mean SSCL growth rate was 7.7 cm/year (SD = 4.6). Juvenile hawksbills (<50 cm SSCL) showed a significantly higher growth rate (9.2 cm/year, SD = 4.5, N = 11) than subadult hawksbills (50–70 cm SSCL, 3.6 cm/year, SD = 0.9, N = 4). Analysis of 740 base pair mitochondrial control region sequences from 50 sampled turtles yielded 12 haplotypes. Haplotype frequencies were significantly different compared to four other Caribbean juvenile foraging aggregations, including one off the Atlantic coast of Florida. Many-to-one mixed stock analysis indicated Mexico as the primary source of juveniles in the region and also suggested that the Refuge may serve as important developmental habitat for the Cuban nesting aggregation. Serum testosterone radioimmunoassay results from 33 individuals indicated a female biased sex ratio of 3.3 females: 1 male for hawksbills in the Refuge. This assemblage of hawksbills is near the northern limit of the species range, and is one of only two such assemblages described in the waters of the continental United States. Since this assemblage resides in an MPA with intensive human use, basic information on the assemblage is vital to resource managers charged with conservation and species protection in the MPA.

Isolation and characterization of tetranucleotide microsatellites from the leatherback turtle (Dermochelys coriacea)

The leatherback turtle (Dermochelys coriacea) is a globally endangered marine species. Numerous questions regarding life history and demographics that are of conservation interest remain and many of these can be addressed through the use of highly polymorphic nuclear markers. We describe primers and polymerase chain reaction conditions to amplify 19 tetranucleotide microsatellite loci from the leatherback turtle. The primers were tested on samples from 22 females that nested at Archie Carr National Wildlife Refuge, Melbourne Beach, Florida, USA. The primers developed in this study yielded an average of 9.4 alleles per locus (range of 5–19) and an average observed heterozygosity of 0.84 (range 0.36–1.00). These markers should prove useful in supplementing existing markers for individual and population level analyses.

Microsatellite markers for eastern hemlock (Tsuga canadensis)

We describe polymerase chain reaction primer pairs and reaction conditions for amplification of 15 microsatellite loci from eastern hemlock (Tsuga canadensis). The primers were tested on 23 individuals from a natural population in southwestern North Carolina, USA. These primers yielded an average of 5.9 alleles per locus (range of 2–14), an average observed heterozygosity of 0.45 (range 0.14–0.73), and an average polymorphic information content of 0.54 (range 0.28–0.86). In addition, eight of the primer pairs were found to amplify microsatellite loci in one or more additional species of Tsuga.

Microsatellite Markers for Population Studies of the Salt Marsh Species Juncus roemerianus (Juncaceae)

Premise of the study: Juncus roemerianus (Juncaceae) is a foundational species and ecosystem engineer of salt marshes in the Gulf of Mexico. These ecosystems provide coastal flood attenuation, nurseries for important species, and other ecosystem services, but are experiencing significant decline. Nuclear microsatellite markers were developed for J. roemerianus to study genetic diversity and population structure for conservation and restoration efforts. Methods and Results: Illumina NextSeq high-throughput sequencing was used to develop a panel of 19 polymorphic microsatellite markers that were tested across individuals from three populations on the Gulf Coast. All markers were polymorphic, with observed and expected heterozygosities ranging from 0.212 to 0.828 and from 0.362 to 0.873, respectively. Allelic richness ranged from two to 13 alleles per locus with an average of 5.737. Conclusions: The 19 microsatellite markers are useful for population studies throughout the range of J. roemerianus. Three loci cross-amplified in the related taxon J. effusus.

Isolation and characterization of tetranucleotide microsatellite loci from the seaside sparrow (Ammodramus maritimus)

The seaside sparrow (Ammodramus maritimus; SESP) includes seven extant subspecies ranging from the northeastern coast of the United States to the Gulf of Mexico. Genetic diversity of SESP may be impacted since coastal marsh habitats are highly fragmented, thereby limiting gene flow among adjacent populations. Genetic analysis of SESP populations will provide insight for development of conservation and management strategies for the species. We describe development and characterization of 11 novel microsatellite markers for SESP. These loci will be useful for individual identification as well as studies of fine-scale genetic relatedness within populations and subspecies of SESP.