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.
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