Amy Frey

Targeted multiplex next-generation sequencing: advances in techniques of mitochondrial and nuclear DNA sequencing for population genomics

Next‐generation sequencing (NGS) is emerging as an efficient and cost‐effective tool in population genomic analyses of nonmodel organisms, allowing simultaneous resequencing of many regions of multi‐genomic DNA from multiplexed samples. Here, we detail our synthesis of protocols for targeted resequencing of mitochondrial and nuclear loci by generating indexed genomic libraries for multiplexing up to 100 individuals in a single sequencing pool, and then enriching the pooled library using custom DNA capture arrays. Our use of DNA sequence from one species to capture and enrich the sequencing libraries of another species (i.e. cross‐species DNA capture) indicates that efficient enrichment occurs when sequences are up to about 12% divergent, allowing us to take advantage of genomic information in one species to sequence orthologous regions in related species. In addition to a complete mitochondrial genome on each array, we have included between 43 and 118 nuclear loci for low‐coverage sequencing of between 18 kb and 87 kb of DNA sequence per individual for single nucleotide polymorphisms discovery from 50 to 100 individuals in a single sequencing lane. Using this method, we have generated a total of over 500 whole mitochondrial genomes from seven cetacean species and green sea turtles. The greater variation detected in mitogenomes relative to short mtDNA sequences is helping to resolve genetic structure ranging from geographic to species‐level differences. These NGS and analysis techniques have allowed for simultaneous population genomic studies of mtDNA and nDNA with greater genomic coverage and phylogeographic resolution than has previously been possible in marine mammals and turtles.

Marine turtle mitogenome phylogenetics and evolution.

The sea turtles are a group of cretaceous origin containing seven recognized living species: leatherback, hawksbill, Kemps ridley, olive ridley, loggerhead, green, and flatback. The leatherback is the single member of the Dermochelidae family, whereas all other sea turtles belong in Cheloniidae. Analyses of partial mitochondrial sequences and some nuclear markers have revealed phylogenetic inconsistencies within Cheloniidae, especially regarding the placement of the flatback. Population genetic studies based on D-Loop sequences have shown considerable structuring in species with broad geographic distributions, shedding light on complex migration patterns and possible geographic or climatic events as driving forces of sea-turtle distribution. We have sequenced complete mitogenomes for all sea-turtle species, including samples from their geographic range extremes, and performed phylogenetic analyses to assess sea-turtle evolution with a large molecular dataset. We found variation in the length of the ATP8 gene and a highly variable site in ND4 near a proton translocation channel in the resulting protein. Complete mitogenomes show strong support and resolution for phylogenetic relationships among all sea turtles, and reveal phylogeographic patterns within globally-distributed species. Although there was clear concordance between phylogenies and geographic origin of samples in most taxa, we found evidence of more recent dispersal events in the loggerhead and olive ridley turtles, suggesting more recent migrations (<1 Myr) in these species. Overall, our results demonstrate the complexity of sea-turtle diversity, and indicate the need for further research in phylogeography and molecular evolution.

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).

Characterization of polymorphic microsatellite markers for the green turtle (Chelonia mydas)

We describe primers and polymerase chain reaction conditions to amplify 12 microsatellite loci from the green turtle (Chelonia mydas), including one dinucleotide, four trinucleotide and seven tetranucleotide loci. The primers were tested on 78 individuals from a Pacific population nesting in the Hawaiian Islands. The primer pairs developed in this study yielded an average of 8.33 alleles per locus (range of 3–15 alleles), an average observed heterozygosity of 0.668 (range 0.309–0.910), and an average polymorphic information content of 0.647 (range 0.287–0.894).

Genetic Stock Structure of Green Turtle (Chelonia mydas) Nesting Populations across the Pacific Islands

Abstract: More than two decades have passed since the first studies documenting genetic population structure of green turtles (Chelonia mydas) were published. Since then many more have followed and characterization of the genetic structure of green turtle rookeries now covers most of the global distribution of the species, benefitting conservation of this threatened species worldwide. However, important data gaps still exist across a large part of the western and central Pacific Ocean (WCPO). This large area is made up of hundreds of scattered islands and atolls of Micronesia, Melanesia, and Polynesia, most of which are remote and difficult to access. In this study, we assessed stock structure of green turtles throughout the WCPO using mitochondrial (mt) DNA from 805 turtles sampled across 25 nesting locations. We examined whether sequencing longer fragments (770 bp) of the control region increases resolution of stock structure and used genetic analysis to evaluate level of demographic connectivity among island nesting populations in the WCPO. We identified a total of 25 haplotypes characterized by polymorphism within the 770 bp sequences, including five new variants of haplotypes that were indistinguishable with shorter 384 bp reads from previous studies. Stock structure analysis indicated that rookeries separated by more than 1,000 km were significantly differentiated from each other, but neighboring rookeries within 500 km showed no genetic differentiation. Results presented in this paper establish that sequencing of longer fragments (770 bp) of the control region does in some cases increase resolution and that there are at least seven independent stocks in the region.

Population structure and phylogeography reveal pathways of colonization by a migratory marine reptile (Chelonia mydas) in the central and eastern Pacific.

Climate, behavior, ecology, and oceanography shape patterns of biodiversity in marine faunas in the absence of obvious geographic barriers. Marine turtles are an example of highly migratory creatures with deep evolutionary lineages and complex life histories that span both terrestrial and marine environments. Previous studies have focused on the deep isolation of evolutionary lineages (>3 mya) through vicariance; however, little attention has been given to the pathways of colonization of the eastern Pacific and the processes that have shaped diversity within the most recent evolutionary time. We sequenced 770 bp of the mtDNA control region to examine the stock structure and phylogeography of 545 green turtles from eight different rookeries in the central and eastern Pacific. We found significant differentiation between the geographically separated nesting populations and identified five distinct stocks (FST = 0.08–0.44, P < 0.005). Central and eastern Pacific Chelonia mydas form a monophyletic group containing 3 subclades, with Hawaii more closely related to the eastern Pacific than western Pacific populations. The split between sampled central/eastern and western Pacific haplotypes was estimated at around 0.34 mya, suggesting that the Pacific region west of Hawaii has been a more formidable barrier to gene flow in C. mydas than the East Pacific Barrier. Our results suggest that the eastern Pacific was colonized from the western Pacific via the Central North Pacific and that the Revillagigedos Islands provided a stepping-stone for radiation of green turtles from the Hawaiian Archipelago to the eastern Pacific. Our results fit with a broader paradigm that has been described for marine biodiversity, where oceanic islands, such as Hawaii and Revillagigedo, rather than being peripheral evolutionary “graveyards”, serve as sources and recipients of diversity and provide a mechanism for further radiation.

Hawksbill turtle terra incognita: conservation genetics of eastern Pacific rookeries

Abstract Prior to 2008 and the discovery of several important hawksbill turtle (Eretmochelys imbricata) nesting colonies in the EP (Eastern Pacific), the species was considered virtually absent from the region. Research since that time has yielded new insights into EP hawksbills, salient among them being the use of mangrove estuaries for nesting. These recent revelations have raised interest in the genetic characterization of hawksbills in the EP, studies of which have remained lacking to date. Between 2008 and 2014, we collected tissue samples from 269 nesting hawksbills at nine rookeries across the EP and used mitochondrial DNA sequences (766 bp) to generate the first genetic characterization of rookeries in the region. Our results inform genetic diversity, population differentiation, and phylogeography of the species. Hawksbills in the EP demonstrate low genetic diversity: We identified a total of only seven haplotypes across the region, including five new and two previously identified nesting haplotypes (pooled frequencies of 58.4% and 41.6%, respectively), the former only evident in Central American rookeries. Despite low genetic diversity, we found strong stock structure between the four principal rookeries, suggesting the existence of multiple populations and warranting their recognition as distinct management units. Furthermore, haplotypes EiIP106 and EiIP108 are unique to hawksbills that nest in mangrove estuaries, a behavior found only in hawksbills along Pacific Central America. The detected genetic differentiation supports the existence of a novel mangrove estuary “reproductive ecotype” that may warrant additional conservation attention. From a phylogeographic perspective, our research indicates hawksbills colonized the EP via the Indo‐Pacific, and do not represent relict populations isolated from the Atlantic by the rising of the Panama Isthmus. Low overall genetic diversity in the EP is likely the combined result of few rookeries, extremely small reproductive populations and evolutionarily recent colonization events. Additional research with larger sample sizes and variable markers will help further genetic understanding of hawksbill turtles in the EP.

Evaluating Buccal and Cloacal Swabs for Ease of Collection and Use in Genetic Analyses of Marine Turtles

Abstract 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.

Inter-nesting movements and habitat-use of adult female Kemp’s ridley turtles in the Gulf of Mexico

Species vulnerability is increased when individuals congregate in restricted areas for breeding; yet, breeding habitats are not well defined for many marine species. Identification and quantification of these breeding habitats are essential to effective conservation. Satellite telemetry and switching state-space modeling (SSM) were used to define inter-nesting habitat of endangered Kemp’s ridley turtles (Lepidochelys kempii) in the Gulf of Mexico. Turtles were outfitted with satellite transmitters after nesting at Padre Island National Seashore, Texas, USA, from 1998 through 2013 (n = 60); Rancho Nuevo, Tamaulipas, Mexico, during 2010 and 2011 (n = 11); and Tecolutla, Veracruz, Mexico, during 2012 and 2013 (n = 11). These sites span the range of nearly all nesting by this species. Inter-nesting habitat lies in a narrow band of nearshore western Gulf of Mexico waters in the USA and Mexico, with mean water depth of 14 to 19 m within a mean distance to shore of 6 to 11 km as estimated by 50% kernel density estimate, α-Hull, and minimum convex polygon methodologies. Turtles tracked during the inter-nesting period moved, on average, 17.5 km/day and a mean total distance of 398 km. Mean home ranges occupied were 725 to 2948 km2. Our results indicate that these nearshore western Gulf waters represent critical inter-nesting habitat for this species, where threats such as shrimp trawling and oil and gas platforms also occur. Up to half of all adult female Kemp’s ridleys occupy this habitat for weeks to months during each nesting season. Because inter-nesting habitat for this species is concentrated in nearshore waters of the western Gulf of Mexico in both Mexico and the USA, international collaboration is needed to protect this essential habitat and the turtles occurring within it.

Proceedings of the Thirty-fourth Annual Symposium on Sea Turtle Biology and Conservation : 2014 International Sea Turtle Symposium, 14 to 17 April, 2014, New Orleans, Louisiana, USA

Opening Remarks and Keynotes..........................................................................................1 Anatomy, Physiology and Health..........................................................................................4 Collaborative Fisheries Research and Mitigating Marine Turtle Bycatch: Special Session.....................40 Conservation, Management and Policy.................................................................................43 Education, Outreach and Advocacy.....................................................................................77 Fisheries and Threats.......................................................................................................91 Genetics and Population Biology......................................................................................117 In-Water Biology........................................................................................................144 Nesting Biology..........................................................................................................189 Research Highlights from Southeast U.S.A..........................................................................221 Sea Turtle Biology and Conservation in the Gulf of Mexico: Special Session..................................230 Social, Economic and Cultural Studies................................................................................241 Video Presentations.....................................................................................................250 Author Index.............................................................................................................253 Abstract titles marked with an * at the end of the title denote an Oral Presentation.titles marked with an * at the end of the title denote an Oral Presentation. April 2014 New Orleans, Louisiana, USA iv PRESIDENT’S REPORT 34rd Annual Symposium on Sea Turtle Biology and Conservation 10-17 April 2014, New Orleans, Louisiana, USA Roldán A. Valverde, President, ISTS A total of 785 people from 73 countries registered for the Symposium. The venue for the symposium was the Marriott Hotel on Canal Street, New Orleans. A total of 176 oral papers and 273 posters were originally submitted to organizers. These original submissions included the highest number of oral presentations on inwater biology science ever submitted to a symposium, with a total of 35, or approximately 20% of all oral presentations originally submitted. Due to normal attrition associated with every symposium, in the end the symposium included a total of 158 oral presentations in general sessions and a total of 235 posters. Of the oral presentations, 32 (20%) corresponded to in-water biology research, more than any other category presented in this symposium. Travel grants: A total of 119 registrants received a travel grant to the New Orleans symposium (12 from Africa, 13 from US/Canada, 5 from English Caribbean, 4 from South Asia, 8 from Asia Pacific, 16 from South America, 6 from Europe, 23 from Mexico-Central America, 32 others). This level of travel grant awards represents about 15% of the total registered participants. Travel grants took the form of room grants, which was highly advantageous for the awardees and for the ISTSociety. Room awards contributed a total of about 561 room nights, which made a significant contribution to our hotel’s room block. Because rooms were awarded to a group and not to individuals the organizing committee was able to serve more people in a more effective way. Also, this strategy saved our Treasurer the time and effort to write checks and keep track of the awards, and gave the ISTSociety better control over how the grants are assigned and used, thus increasing efficiency and effectiveness of the awards. Pre-symposium Meetings Workshops: A total of eight Special Interest and Regional meetings were held prior to the main symposium. These were the Terrapin, Tortoise & Freshwater Meeting, the RETOMALA, the Africa Regional, the Mediterranean Regional, the East Asia Regional, Indian Ocean South East Asia Regional, the Pacific Islands Region-Oceania, Eastern Pacific Hawksbill Initiative and the Marine Turtle Specialist Group. These meetings were successful and contributed to bring attendees early to the symposium. A total of five workshops were offered before the symposium. These were the Sea Turtle Rehabilitation and Health Workshop (with a total of 237 registered participants), the Educators Outreach Workshop (with 18 local, national and international participants), the Digital Marketing Workshop (with 83 participants), the GIS Workshop with 197 participants, and the Temperature-dependent Sex Determination Workshop (with 151 participants). Key Note Speakers: Three Key Note speakers delivered three 30 minutes addresses to symposium participants. Jack Frazier’s presentation gave the audience a comprehensive overview of the topic Sea Turtles and Cultures, which nicely served to frame the theme of the symposium. Duncan MacKenzie immediately Proceedings of the 34th Annual Symposium on Sea Turtle Biology and Conservation v followed Jack’s presentation, speaking about the pros and cons of using sea turtles as animal models to conduct physiological studies. Lastly, David Owens delivered an enthusiastic, informative and entertaining speech about the history of sea turtle research and the historical involvement of women in this research. All three speeches were excellent and very well received by the audience. Symposium Sessions: Two special sessions were held during the symposium: Biology and Conservation of the Sea Turtles of the Gulf of Mexico and Collaborative Fisheries Research. The first session focused on work conducted in the Gulf of Mexico and was held the first day of the symposium. This session included papers from the entire Gulf (Mexican and US waters), and offered an emphasis on in-water work. The second special session on collaborative fisheries focused on work being done by scientists in collaboration with fishermen to collect fisheries-specific information and promote effective conservation and management practices among fishermen. Business Meeting: Very important issues were addressed during the plenary business meeting conducted the last day of the New Orleans symposium. One of the most important issues was the approval of the overhauled Constitution and Bylaws of the Society. Other issues discussed were the travel committee report, the Treasurer’s report and the Resolutions submitted, among others. Social Events: Welcome Social, Live and Silent Auctions, Farewell party, Student Awards were some of the social events held during the symposium. Among those events, a Speed Chatting with Experts event was held the night of the first day of the meeting. Of all these vents, the Welcome Social held the night before the first day of the symposium was arguably the most popular. This included a surprise Mardi Gras-style parade with a second line Jazz band guiding symposium attendees over the streets of New Orleans. Resolutions: A very important component of the every symposium is the issuing of Resolutions, documents that allow the ISTSociety at large to pronounce itself with regard to issues pertaining to sea turtle conservation around the world. Two very important resolutions were passed during the New Orleans symposium: The first resolution was relayed to the Australian Minister of the Environment, Hon Greg Hunt regarding the protection of sea turtle populations in the Great Barrier Reef region. The second resolution was sent to the President of Mexico Enrique Peña Nieto and pertained to the protection of loggerheads in Baja California, Mexico. Receipt of letters was acknowledged and press notices regarding these letters appeared in Australian and Mexican newspapers. Finances: Society’s finances were a major concern going into the New Orleans symposium. Indeed, during the plenary business meeting we learnt that the Society’s finances were in worse shape than originally thought. Fortunately, thoughtful planning by the Society’s Board and effective execution by the organizing committee resulted in a very successful symposium, both scientifically and financially. After paying for all our obligations incurred prior and during the symposium, our revenues were sufficient to overcome past debts and leave us in a solvent situation. Hopefully, the model developed for the New Orleans symposium will be adopted for future symposia. The financial success achieved in New Orleans was due to a series of measures taken. For instance, historical but expensive items, such as simultaneous translation and exceedingly high travel grant levels were significantly reduced or eliminated. Also, the New Orleans symposium was held as a joint meeting with the Southeast Regional Sea Turtle Network, which eliminated any competition for resources. Additionally, important sponsorship was secured from major donors, such as Shell and the National Federation of Wildlife and Fisheries, as well as the Marine Turtle Conservation Act of the USFWS, and the National Atmospheric Aeronautic Administration. Also importantly, organizers were able to secure sponsorship from the Virginia Institute of Marine Science to cover all meeting expenses for one entire day of the Symposium (CFR session), which significantly reduced our costs. Significantly, the organizing committee was able to partner with Southeastern Louisiana University, which allowed us to receive tax-exempt status in Louisiana, among other measures. Finally, significant assistance from The Zenith Group, our contracted meeting provider, made it possible for us to realize significant savings on hotel expenses. April 2014 New Orleans, Louisiana, USA vi EXECUTIVE COMMITTEE President Roldán Valverde Secretary Manjula Tiwari Treasurer Terry Meyer President-Elect Yakup Kaska Past President Ray Carthy ORGANIZING COMMITTEE Activity Coordinator Emma Harrison A