Monday, May 11, 2015

Proposed Research to Further Study Evolution in Box Turtles Compared to Evolution in the Galapagos Giant Tortoise


Project Title: Evolutionary changes in mitochondrial DNA due to ecological and environmental shifts on subsets of populations of the St. Louis Box turtle compared to that of various subspecies of the Galapagos Giant Tortoise

Specific Aims

The Galapagos Giant Tortoise is a model organism that is used to study evolutionary patterns and changes, specifically because evolutionary biologist Charles Darwin discovered the evolutionary differences among these tortoises during his trip to the Galapagos Islands in the 16th century. However, now in the 21st century, research is currently going on in Forest Park in St. Louis, Missouri, that is specifically looking at box turtles, or Terrapene and human impact and influences in the migration patterns of these turtles. I would like to further the research that is currently going on to see if the changes in the migration patterns of these turtles is leading to any evolutionary changes in the mitochondrial DNA of the turtles. Then, I would like to compare any evolutionary changes found in the St. Louis box turtles to those of the Galapagos Islands and note if there are any similarities between change in migration patterns, reduced gene flow, and genomic consequences. The human impact in Forest Park has led to the park resembling an archipelago like that of the Galapagos Islands, so my research will compare and contrast the two geographic environments and note any similarities between the evolutionary genomic consequences between the two organisms, specifically looking at mitochondrial DNA. I hypothesize that human impact will affect the migration patterns of box turtles in Forest Park, and that because of a lack of gene flow due to affected migration patterns, turtles in Forest Park will begin to evolve into separate subpopulations, like what happened to the Galapagos Giant Tortoise.

Specific Aim 1: To see if human impact in areas where box turtles (Terrapene) are common (like in Forest Park, St. Louis) is causing environmental and ecological shifts for the box turtles. Currently, there is a decrease in the number of box turtles in Forest Park, St. Louis, and researchers hypothesize that this decrease is due to turtles migrating to new areas because human impact has changed the geography of the park, leading the park to resemble an archipelago, but instead of being separate land masses separated by water, the park resembles separate land masses that are separated by roads, buildings, and golf courses. My research aims to study and note any environmental and ecological shifts of the box turtles due to this human impact in the park. The migration patterns of the turtles will be tracked to determine any changes in the migration patterns and to see where the turtles have chosen to make their new habitats.

Specific Aim 2: To then look at the population genomic consequences of the St. Louis box turtle due to the environmental and ecological shifts, specifically looking at mitochondrial DNA of the turtles, and then determining any genomic changes in the DNA between various population subsets in the park, and whether or not those DNA differences seem to stem from the environmental and ecological shifts that the turtles have undergone. After looking at changes in the migratory patterns of the Forest Park box turtles due to the environmental and ecological shifts, I will then look at the turtle population in Forest Park as a whole and note any genomic consequences in the population. This will be done by looking at the subsets of populations throughout the park. These subsets will be determined by geographic location. I will take mitochondrial DNA samples of each population subset and look to see if there are any noticeable differences in the DNA samples of the various population subsets. This aims to determine if any noticeable differences in the mitochondrial DNA stem from the environmental and ecological shifts that have been undergone by the turtles in Forest Park.

Specific Aim 3: To compare these differences in mitochondrial DNA between subsets of populations of the St. Louis box turtle due to environmental and ecological shifts to those of various subspecies of the Galapagos Giant Tortoise (if applicable). If there are any noticeable differences in the mitochondrial DNA between subsets of populations of the St. Louis box turtle, then the next aim will be to look at Galapagos Giant Tortoises, take mitochondrial DNA samples of the different populations in each island, and then to compare any DNA differences found to those found in the box turtles. This research aims to hopefully note if geographic restrictions, such as an archipelago-like set up in the environment, lead to reduced gene flow and evolutionary differences of genes, specifically mitochondrial DNA, between populations of organisms that are separated by geographic constraints, such as water, roads, or buildings.

Significance and Impact

This research is significant because it builds off of projects that are already going on, but just takes these projects a step further by looking at the genes of box turtles in Forest Park. Currently, the Galapagos Tortoise Programme and the St. Louis Box Turtle Project are tracking both Galapagos Giant Tortoises and St. Louis box turtles, and are determining migratory patterns and changes due to geographic and environmental constraints. My research is significant because it takes this research and continues it another step further by looking at how the changes in the migratory patterns of these organisms can cause reduced gene flow due to separation of population subsets, and how this reduced gene flow could lead to evolutionary changes between the population subsets. This is important because the Galapagos tortoise is a model organism used for studying evolution, but many subspecies of the organism have either gone extinct or are in grave danger. The population of box turtles seems to be steadily declining, so it is important to note if reduction in gene flow and evolutionary changes may be leading to decreased fitness among the turtles, and as a result, less box turtles surviving and reproducing. If any significant changes are found in the mitochondrial DNA sequences of that of the box turtle, and these evolutionary changes resemble those of the Galapagos tortoise, then maybe humans can take precautionary measures to reduce human impact in Forest Park and other areas where box turtles are common, can work to promote gene flow between the subsets of populations, and hopefully increase fitness, reproduction, and survival rate in the box turtles so that they do not end up becoming endangered or extinct like many of the species of the Galapagos Giant Tortoise.

Background and Significance

The box turtle, or Terrapene, is currently threatened and is a high priority to many conservationists. A common place to find box turtles, and a place where box turtles are currently being tracked, monitored, and studied, is Forest Park in St. Louis, Missouri. Box turtles are declining in health and in numbers, and conservationists have reason to believe that this decline is due to human impact, which may have caused environmental and ecological shifts for the turtles. These environmental and ecological shifts may be causing population genomic changes for the box turtle. A model example of an organism’s population genomics being affected by environmental and ecological shifts is the Galapagos giant tortoise. The Galapagos giant tortoise has weakened genetic diversity due to environmental factors, human impact, and to loss of genetic drift (6) because the Galapagos Islands are an archipelago, and the tortoises are unable to migrate from island to island, so they have evolved into various subpopulations. Because box turtles in Forest Park of St. Louis, Missouri are unable to migrate throughout the entire park, this same phenomenon of evolving into various subpopulations in the park may happen to the box turtles. Galapagos tortoises are commonly studied when looking at evolutionary genetics because they are a model organism and a prime example for evolution, as can be seen in their differences in their carapaces from island to island (3). But, little is known about the evolutionary genetics of the box turtle, so more research must be done on these reptiles in order to determine if they are evolving like the Galapagos tortoises once did.
            This experiment aims to see if human impact in areas where box turtles, or Terrapene, are common is causing ecological and environmental shifts for the turtles, and for the sake of this experiment, I will be particularly focusing on box turtles in Forest Park in St. Louis, Missouri. It also aims to look at population genomic consequences of the St. Louis box turtle due to ecological and environmental shifts and human impact in Forest Park. And, finally, this experiment aims to compare the population genomics of box turtles to those of the Galapagos giant tortoise and see if there are any similarities between the changes in population genomics in the box turtles and in the Galapagos tortoise. In 2002, researchers at Yale University mapped the phylogeography of 161 Galapagos tortoises, specifically looking at their mitochondrial DNA and maternal lineage, so much is currently known about the genomics of the Galapagos tortoise. The research showed that Galapagos tortoises found on the same island and in the same population had mitochondrial DNA that showed that these tortoises were from the same maternal lineage (2).
            In an article entitled “Population genomics of the endangered Galapagos tortoise,” researchers look at population genomics of the Galapagos tortoise, and studied how changes in environmental conditions and ecological shifts affected the population genomics of the Galapagos tortoise. Researchers looked at Galapagos tortoises because the tortoises colonized the Galapagos Islands archipelago and have been facing environmental hardships and limited resource availability since then. Based on the researchers’ analyses of the mitochondrial genes of the tortoises, researchers concluded that there is a reduced amount of genetic diversity amongst these species, and there is weakened selection, most likely due to environmental factors and the restricted geography of the islands. Molecular evolution in the Galapagos is greatly influenced by genetic drift, which in turn weakens the efficiency of natural selection and creates a heightened mutation load. Molecular evolution of an endangered taxon in a stressed environment, such as environmental hardships due to human impact or climate change, is helpful in studying deleterious effects on genome evolution and reduced effective population sizes (8). This is an example of the population genomics of Galapagos tortoises already being studied and recorded in scientific literature, and hopefully this study can be further extended to box turtles in Forest Park and to their population genomics, too.
Mitochondrial DNA is commonly looked at when doing DNA extractions. In this research, I will be taking caudal blood samples of box turtles and extracting mitochondrial DNA. There are many cases in which mitochondrial DNA has been looked at in reptiles, such as in loggerhead turtles. Researchers took blood samples of loggerhead turtles and then used PCR amplifications to further study mitochondrial DNA, which will be the same method used in this experiment. PCR amplifications were used to amplify the mitochondrial DNA that was taken from caudal blood samples of loggerhead turtles (9).
In another article, entitled “Short-Term Forest Management Effects on a Long-Lived Ectotherm,” researchers look at how timber harvesting affects the eastern box turtle. Timber harvesting can be both beneficial and harmful to forest dwelling organisms. In conclusion, researchers discovered that timber harvesting, or things like deforestation in general, have negative effects on the thermal ecology of ectotherms. Researchers found that in relatively contiguous forested landscape, timber harvests have little effect on the short-term annual behavior of box turtles. However, they did detect a behavioral effect at the local scale where available microenvironmental temperatures were altered. Researchers concluded that there is much variation in the annual behavior and home ranges of T. c. carolina that should be considered when establishing management strategies for forests and this species (4). This article is specific to the aim of looking at how human impact can affect box turtles, and maybe even affect their environment, migratory patterns, and population genomics in the long run. In comparison, the populations of Galapagos Giant Tortoises have greatly depleted. Just like how humans creating deforestation in the habitats of box turtles and negatively affecting the box turtles, many researchers in the Galapagos Islands believe the depletion in the numbers of Galapagos tortoises greatly results from human impact having negative consequences for the tortoises in the Galapagos.
Deforestation seems to negatively impact the box turtles, but it is important to also discuss how road mortality has caused depletion in the numbers of painted turtles in Eastern Ontario. Researchers found a significant level of road mortality rate of these turtles (5). This research is relevant to the research done in this project because one of the main factors believed to be affecting the migratory patterns of the box turtles in Forest Park is the separation of land in the park because of roads running through the park. Because of these roads, box turtles have had to change their migratory patterns though the park to avoid being killed by cars and other vehicles, but have also become separated into various subsets of populations because the turtles cannot migrate throughout the park easily due to the obstacle of the roads.
Another article, entitled “Giant tortoises are not so slow: Rapid diversification and biogeographic consensus in the Galapagos,” discusses how evolutionary events cause associations between genetic variation and geography in archipelago radiations. In this experiment, researchers specifically studied the Galapagos Islands because molecular studies done there revealed conflicting biogeographic patterns, so it made sense to study Galapagos tortoises for the purpose of this experiment. Based on analysis of mtDNA of the tortoises, researchers discovered that their studies indicated an intimate association of temporal patterns of genetic variation with geophysical aspects of the environment (1). This article provides a closer look at the aim of how Galapagos tortoises have evolved due to their geography and environments, and hopefully how these evolutionary changes due to geography and environment can also be applied to the St. Louis box turtles of Forest Park. A common method to study migratory patterns of organisms, in this case Galapagos tortoises and box turtles, is through the use of GPA trackers. An example of this is the use of GPA trackers to study movement patterns and distribution of East Pacific green turtles. By tagging the turtles with GPA trackers, researchers were able to study the migration patterns of the East Pacific green turtles (7), and this same method is being used to study the movement of Galapagos tortoises and box turtles in Forest Park of St. Louis, Missouri.
My research will differ from research and investigations already conducted in known literature because it specifically focuses on box turtles in Forest Park of St. Louis, Missouri. Also, it looks at evolutionary changes, population genomics, and ecological and environmental shifts of the Galapagos tortoise and compares these to see if there are any similarities that can be applied to the St. Louis box turtle. Currently, there are research projects going on that are looking into the migratory patterns and the health of the St. Louis box turtle, but my research will go further into investigating if the box turtles are in danger of weakening their genetic diversity due to being unable to migrate throughout the entire park, just like what happened to the Galapagos giant tortoise when it was unable to migrate from island to island.
            Knowledge of how geography, human impact, and environmental and ecological shifts affects the migratory patterns and the population genomics of the box turtle of Forest Park, St. Louis will be gained from this experiment. This will impact known literature because many facts are known about box turtles on their own and many facts are known about the Galapagos giant tortoise on its own, but the two are rarely compared. Forest Park may just be a small-scale set up of the Galapagos Islands, functioning like an archipelago and preventing mixing of genes among box turtles like in the Galapagos Islands, so this research will impact known literature by drawing similarities and conclusions between the two organisms and their environments.


Research Designs and Methods

Specific Aim 1: To see if human impact in areas where box turtles (Terrapene) are common (like in Forest Park, St. Louis) is causing environmental and ecological shifts for the box turtles.
First, I will survey the land of Forest Park and observe and note any and all types of human impact in the park, such as roads, buildings, golf courses, hiking trails, bikes, domesticated pets, litter, and more. After looking at what types of human impact currently exist in Forest Park, I will begin to research more closely how this human impact could be causing environmental and ecological shifts for the box turtles in Forest Park.
In order to study the environmental and ecological shifts, I will look at movement/migration patterns and distribution of the box turtles throughout the park. I will use a sample size of fifty box turtles for my research, and I will determine which turtles I use based on their location. I will survey the park and pick five different areas of the park to draw my samples from. I will use five samples, each sample containing ten turtles. These five samples will serve as representations of five different geographic locations in the park and will be representative of five different populations of box turtles. Individual turtles will be researched and studied, but population samples will also be researched and studied, too, so it is important to draw samples from different geographic locations in the park.
Every time a turtle is found that will be used for a sample, the turtle’s carapace will be attached with fitted radio tags, or VHF, which emit radio frequencies so that the turtles can be tracked. These radio tags can be obtained from the Tyson Research Center in St. Louis, Missouri. Research will take place over the course of a year, starting in the summer month of May and continuing into the following May, that way the migration patterns can be tracked and studied throughout all of the various seasons of the turtles, even when the turtles are hibernating. It is important to look at patterns of movement of the box turtles in all seasons in order to note any deviations from the normal movement pattern of turtles during migratory months and during hibernation months, and to compare if turtles change their migratory patterns or to note any differences in migratory patterns between the five different turtle populations. Migratory patterns for each turtle population will be analyzed, and then human impact surrounding each population of turtles will be looked at to determine if certain populations of turtles seemed to be affected by humans in a more serious or direct way, and if so, if more direct human impact seemed to affect the movement patterns of the box turtles in each population.
Researchers will follow the migration patterns of the turtles by using the radio tags and the emitted frequencies. Researchers will map data points of migratory patterns for each individual turtle, and will also map data points of migratory patterns for each population of turtles as a whole. Mapping data points of migration patterns will take place on a weekly basis.
Limitations of this research is that most of it is subjective, so researchers are held accountable to note what they believe counts as human impact in the park. Also, ideally, box turtle samples will be representative of different geographical locations, but a turtle could be tagged in a geographic location and it could be assumed that that specific turtle is an inhabitant of that geographic location, but the turtle could actually just be migrating between locations. This could be problematic for research because it does not provide an accurate representation of samples from different geographic locations. However, hopefully having at least five samples will eliminate sampling errors because researchers will have enough samples to look at and to compare.

Specific Aim 2: To then look at the population genomic consequences of the St. Louis box turtle due to the environmental and ecological shifts, specifically looking at mitochondrial DNA of the turtles, and then determining any genomic changes in the DNA between various population subsets in the park, and whether or not those DNA differences seem to stem from the environmental and ecological shifts that the turtles have undergone.
            Mitochondrial DNA samples of the fifty box turtles will be obtained via caudal blood samples of the box turtles. At the end of the year, after the migration patterns have been tracked and studied, researchers will take a caudal blood sample of each turtle in order to obtain mitochondrial DNA. Unlike humans, turtles have nuclei in their red blood cells, so red blood cell samples will give researchers a sufficient amount of mitochondrial DNA needed to perform the research. The sampling will not be invasive because all it entails is to turn each turtle over onto its back, locate the caudal vein on the ventral side of the turtle, and to extract a blood sample.
Once researchers have taken blood samples of each of the fifty turtles, researchers will then use PCR to amplify the mitochondrial DNA obtained from the blood samples. Then, single stranded conformation polymorphism analysis will be used to screen for sequence variants in the samples.
Controls for comparison will be made by sequencing multiple individuals from each population sample group that exhibit the same single stranded conformation polymorphism gel band for their sample group. If turtles differ from the control from their sample group in their single stranded conformation polymorphism gel band, then further research will go into comparing the differences that were observed. Differences will be analyzed, compared between individuals, and on a larger scale, compared between population sample groups that were determined based on geographic locations. The gel bands of the control turtles will be compared between the five different population sample groups in order to note any similarities or differences in the mitochondrial DNA between the different groups. Then, the gel bands of the individuals in each group will be compared from group to group to notice any differences or similarities in the mtDNA of the five different groups of turtles. Differences and similarities between population samples will be analyzed to note if there are any indications of mitochondrial DNA differing between populations as a result of each population evolving into its own subspecies of box turtles due to the environmental and ecological shifts in Forest Park.
Each turtle and its mitochondrial DNA will be analyzed on an individual level. However, the turtles will also be looked at in their population samples. Each of the five population samples will be analyzed and compared in order to look at changes in mitochondrial DNA on a population genomic level, not just on an individual level.
Limitations of this research are that even though there might be similarities among turtles based on analysis of their mitochondrial DNA, it cannot be assumed that these similarities are strictly due to evolution into a new subpopulation or even a subspecies of box turtles. Also, mutations in mitochondrial DNA could disrupt data because it could lead researchers to believe that turtles were not from the same lineage or the same population, but in reality, differences in mitochondrial DNA could just be a result of a spontaneous mutation occurring in that turtle’s mtDNA. Other limitations are that it could be hard to determine which turtles will be the ones to serve as the control for their population sample.

Specific Aim 3: To compare these differences in mitochondrial DNA between subsets of populations of the St. Louis box turtle due to environmental and ecological shifts to those of various subspecies of the Galapagos Giant Tortoise (if applicable).
Once mitochondrial DNA of box turtles has been extracted, amplified, and gone through single stranded conformation polymorphism, the next step is to compare any differences in the mitochondrial DNA gel bands between the five populations of box turtles to research already done on the mitochondrial DNA of the Galapagos Giant Tortoise. In 2002, researchers in the Department of Ecology and Evolutionary Biology at Yale University mapped the phylogeography and the history of 161 individual Galapagos tortoises from 21 different sample sites in the Galapagos Islands, specifically looking at mitochondrial DNA (2). In order to not have to perform unnecessary blood tests on the Galapagos tortoises, researchers will use these findings, which have been published and made public by the researchers at Yale University. Researchers will compare my data of the extraction of mitochondrial DNA in the box turtles to that of the Galapagos tortoise, and then look for any similarities and differences between the two groups of reptiles. Researchers will look to see if differences and similarities in the mitochondrial DNA between different geographic populations of box turtles mirrored those in the mitochondrial DNA between different geographic populations of Galapagos Giant Tortoises.
Any significant findings that lead researchers to believe that there are differences in the population genomics of the box turtles will be recorded and compared to the population genomics of the Galapagos Giant Tortoise. Researchers will note how and if mitochondrial DNA of the box turtles yielded significant changes based on geographic location of the turtle, and researchers will also note if mitochondrial DNA had significant changes based on geographic locations of the Galapagos tortoises. If there are significant differences in mitochondrial DNA from population to population in both the box turtles and the Galapagos tortoises, then the next step is to look at the causes of these differences. Researchers will look for similarities in the mitochondrial DNA between individual box turtles within the same population and determine if the similarities are due to the same maternal lineages in the population of turtles. Same maternal lineages will mean that turtles within the same population are breeding with one another, and offspring are coming from the same lineage in the population, meaning that there is reduced genetic diversity in that population because genes are staying within the same population. Researchers will refer back to the migratory patterns of the populations of the turtles and determine if human impact seemed to restrict migratory patterns, resulting in reduced gene flow between the populations and an increase in inbreeding in populations of box turtles. The next step for future research is to look at reduced gene flow in the populations of the box turtles and determine if reduced gene flow is resulting in, or will eventually result in, the different populations of box turtles evolving into their own subspecies of turtles, just like what happened to the Galapagos Giant Tortoise. Different subspecies of Galapagos tortoises can be traced back through the same maternal lineage by using mitochondrial DNA samples, so researchers will study mitochondrial DNA samples of box turtles and determine if populations are coming from the same maternal lineage or not.
            Limitations of this research are that although researchers may find significant similarities and differences in the mitochondrial DNA between different populations of box turtles, these similarities and differences may be difficult to compare to those of the Galapagos tortoise because these are two different species of turtles and they live in completely different parts of the world.
Alternative approaches to test all three specific aims are to physically put trackers on Galapagos tortoises, like what was done to the box turtles, and to follow the migration patterns of five populations of Galapagos tortoises. Then, researchers can take caudal blood samples and look at mitochondrial DNA of these five groups of tortoises so that research of the Galapagos tortoises better mirrors the research done in the box turtles. However, these alternative approaches require more time and money because of traveling to the Galapagos, as well as also being in St. Louis to look at the box turtles. This is why this an alternative approach to the one already stated. Other alternative approaches are to not use blood samples of box turtles to obtain mtDNA, but to look at other DNA instead of mtDNA. Researchers can focus on DNA that codes for specific genes of interest in the box turtles, not just mtDNA.


Use of Animal Subjects

This study will use fifty box turtles from St. Louis Forest Park as subjects. Turtles will not be intentionally harmed in the study, but will have VHF radio trackers attached to their carapaces for the period of one year. At the end of the year, each turtle will have a caudal blood sample taken from it in order to allow researchers to study the mitochondrial DNA of the turtles. In order to use animal subjects, I, and other researchers, must follow Guidelines for Ethical Conduct in the Care and Use of Nonhuman Animals in Research, as stated by the American Psychological Association. Violations of these guidelines must be reported to an APA member immediately to ensure that nonhuman subjects, in this case box turtles, are not treated unethically throughout the course of this research project. However, this research project already uses data collected for the Galapagos Giant Tortoise, so no Galapagos tortoises will be used as subjects in this experiment.

Literature Cited

(1) Beheregaray, L. B., Gibbs, J. P., Havill, N., Fritts, T. H., Powell, J. R., & Caccone, A. (2004). Giant tortoises are not so slow: Rapid diversification and biogeographic consensus in the Galápagos. Proceedings of the National Academy of Sciences of the United States of America, 101(17), 6514–6519. doi:10.1073/pnas.0400393101
 (2) Caccone A, Gentile G, Gibbs JP, Frirts TH, Snell HL, Betts J, Powell JR. Phylogeography and history of giant Galápagos tortoises. Evolution. 2002 Oct;56(10):2052-66. PubMed PMID: 12449492.
(3) Caccone, A., Gibbs, J. P., Ketmaier, V., Suatoni, E., & Powell, J. R. (1999). Origin and evolutionary relationships of giant Galápagos tortoises. Proceedings of the National Academy of Sciences of the United States of America, 96(23), 13223–13228.
(4) Currylow, A. F., MacGowan, B. J., & Williams, R. N. (2012). Short-Term Forest Management Effects on a Long-Lived Ectotherm. PLoS ONE, 7(7), e40473. doi:10.1371/journal.pone.0040473
(5) Dorland, A., Rytwinski, T., & Fahrig, L. (2014). Do Roads Reduce Painted Turtle (Chrysemys picta) Populations? PLoS ONE, 9(5), e98414. doi:10.1371/journal.pone.0098414
(6) Froyd, C. A., Coffey, E. E. D., Knaap, W. O., Leeuwen, J. F. N., Tye, A., Willis, K. J., & Sax, D. (2014). The ecological consequences of megafaunal loss: giant tortoises and wetland biodiversity. Ecology Letters, 17(2), 144–154. doi:10.1111/ele.12203
(7) Hart, C. E., Blanco, G. S., Coyne, M. S., Delgado-Trejo, C., Godley, B. J., Jones, T. T., … Nichols, W. J. (2015). Multinational Tagging Efforts Illustrate Regional Scale of Distribution and Threats for East Pacific Green Turtles (Chelonia mydas agassizii). PLoS ONE, 10(2), e0116225. doi:10.1371/journal.pone.0116225
(8) Loire, E., Chiari, Y., Bernard, A., Cahais, V., Romiguier, J., Nabholz, B., … Galtier, N. (2013). Population genomics of the endangered giant Galápagos tortoise. Genome Biology, 14(12), R136. doi:10.1186/gb-2013-14-12-r136
(9) Shamblin, B. M., Bolten, A. B., Abreu-Grobois, F. A., Bjorndal, K. A., Cardona, L., Carreras, C., … Dutton, P. H. (2014). Geographic Patterns of Genetic Variation in a Broadly Distributed Marine Vertebrate: New Insights into Loggerhead Turtle Stock Structure from Expanded Mitochondrial DNA Sequences. PLoS ONE, 9(1), e85956. doi:10.1371/journal.pone.0085956