In the United States there are three distinct geographical groups of Crotalus horridus: the Eastern Timber Rattlesnake, the Western Timber Rattlesnake, and the Canebrake Rattlesnake. Historically, C. horridus has ranged throughout the majority of the eastern part of the United States and Canada, from Ontario and Minnesota to Florida and Texas (Clark et al., 2003). It is believed that the Eastern Timber Rattlesnake originally came from the southern Appalachian Mountains. After the departure of the final glacier about 18,000 years BP, the Eastern Timber Rattlesnake moved out across the majority of the Appalachians all the way out to New Hampshire and southwestern Maine, north on the edges of the Champlain lowland to southwestern Quebec, and in the karst belts and Ohio to the western fringe of Lake Erie and north along the Niagara Escarpment to Georgian Bay.
When the Europeans first settled in the United States, the Eastern Timber Rattlesnake inhabited an almost uninterrupted range from northeastern Georgia to southwestern Massachusetts. There were strewn populations from north to southwestern Maine and north-central New Hampshire and all the way to the Champlain Valley, most likely as far as southwestern Quebec. It is believed that they existed in a couple of areas in Michigan, in southern Ontario near Lake Erie and the Niagara Gorge, and probably by the Niagara Escarpment up to Manitoulin Peninsula and Fitzwilliam Island (reviewed in Martin, 1978).
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Currently, the Eastern Timber Rattlesnake inhabits areas from the mountains of northeastern Georgia above 2000 feet, north through the Appalachians up to Ohio, New England and New York. They have a more patchy distribution in the Piedmont of northern Virginia, Maryland, and Pennsylvania. Overall, the populations of this species are much more fragmented than they were a few hundred years previously (Figures 1A and 1B).
The fragmentation of habitat ranges has been shown to have a negative effect on species in general. For example, a study done by Braschler et.al. (2005), showed the effects of fragmentation on orthopterans such as grasshoppers and bush crickets in grasslands. Dry calcareous grasslands are teaming with species, but the population sizes of these species have been in decline because of modern agricultural practices, like lawn mowing. This has resulted in a high level of fragmentation. The overall conclusion was that even fragmentation on a small level can have an impact on Orthopteran communities, and the effects of the fragmentation increased with time.
Even vegetative species are affected by fragmentation. A study done by O'Connell, et al. (2006), studied the effects of fragmentation on the reproductive success of white spruce trees. As tree populations declined in size as a result of happenings such as forest fires, insects, disease, and human interference, tree populations have become more isolated. It was found that trees that were a part of small stands produced 38% less seeds per cone and trees that were in medium stands produced 30% less seeds per cone. As the size of stands of the white spruce became smaller in size, the number of seeds in every cone goes down. It was discovered that there was a positive correlation between population size and reproductive success of white spruce.
Because of concerns about the effects of fragmentation, the Commonwealth of Pennsylvania listed C. horridus as a candidate species. Being a candidate species means that there is considerable concern for this population because, if the number of individuals drops any further, the species could then become threatened or endangered in the Commonwealth.
One of the main causes of decline in C. horridus populations can be traced to the loss of their habitats and fragmentation; fragmentation is most often a result of human actions such as development, which encroach on the habitat of these snakes (reviewed in Villarreal et. al., 1996). A study that was done by Clark et al., (2009) has shown that snakes in hibernacula which are isolated as a result of roads have a big reduction in their genetic diversity and more genetic differentiation than snakes that do not have their environment interrupted by roads. It was found that the roads interfered with the snakes' seasonal migration. Even roads that were just newly constructed had a major impact on C. horridus. Because C. horridus have a long life span, but also a low rate of reproduction, they are especially vulnerable to environmental disturbances (reviewed in Clark et. al., 2003).
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Harassment from humans, such as snake roundups, is another cause for decline in Pennsylvania (reviewed in Reinert, 1990). Hunting rattlesnakes for recreational purposes in Pennsylvania has occurred for many years. A study done by Reinert (1990) showed that the hunts injured snakes. A total of 28.8% of the snakes at snake hunts displayed injuries. Most of the injuries were premature tears in their epidermis, meaning that these tears occurred before their natural molting period, but one had a badly injured eye and lacerations to the neck. A few C. horridus had nasal bleeding which is indicative of serious internal injury. Also, no care was taken to release the snakes where they were found and as a result snakes were likely to become confused, display abnormal movement, and even die. Moving the snakes from one population to another could have a negative effect on the population at the site where the "foreign" snake is released. A high proportion of the females that were captured were found to be gravid. Gravid females that are handled could be injured as well as the offspring that she is carrying; this of course could have a negative impact on the population level. Male C. horridus were caught in disproportionately higher levels than females in Pennsylvania. The most logical reason for this is that males are on average, longer than the females and prizes are given to those with longest snakes.
The preservation of C. horridus is important because it is an important part of the forest ecosystems in the northeastern United States. The decrease in the populations of C. horridus can have effects on many levels, which will not only impact the forest ecosystems, but also humans. Rodents are common in the diet of C. horridus (Reinert et. al., 1984). In fact, C. horridus feed more or less completely on rodents. A single C. horridus eats 15 to 20 rodents in a year (reviewed in Reber and Reber, 1994). Feeding upon rodents plays a big role in keeping the rodent population down. If the population of snakes is reduced, the rodent population will increase, as rodents are prolific breeder. As of late, there has been an increase in the rodent population and with this, in North America there has been an increase in Hantavirus, a life threatening disease carried by rodents (reviewed in Mushinsky et. al., 2006).
Another benefit that snakes have to humans is their venom. Snake venom has many medical benefits. It has been used in the treatment of strokes and cancerous tumors. There are many more possible medical uses for snake venom that are still in the process of being discovered (reviewed in Mushinsky et al., 2006).
Importantly, factors that are negatively impacting populations of C. horridus are most likely having a negative effect on other plants and animals in the ecosystem. C. horridus, particularly the young ones, are in turn part of the diet of other animals such as crows, ravens, hawks, and other carnivorous mammals (Urban, 2004). So rattlesnakes are important predators and prey, and their reduction or extinction will dramatically affect other species of animals (reviewed in Reber and Reber, 1994).
Much ecological information has been obtained to date. A study done by Reinert (1984) revealed that physiological conditions played an important role when it came to ascertaining the organization of populations of snakes living in temperate environments. Snakes that were close to giving birth preferred basking sites, open areas with little vegetation, or rocky regions. These gravid female snakes also had a preference for higher body temperature than snakes that were not gravid.
Another study done by Reinert et al. (1988) showed that neonatal C. horridus have the same winter refugia as the adults of this species. It was shown that it was necessary for baby snakes to find an appropriate winter refugium soon after birth in order to survive in areas that have severe weather and although visual signals and celestial positioning are possibly important for the fully grown snakes for movement to and from hibernaculum (places where animals go to hibernate), it is unlikely that these mechanisms are used by neonatal snakes. By marking snakes, it was found that neonates accomplished this task by locating and following adult snakes. This enabled them to find suitable areas in which to hibernate.
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A better understanding of the genetics of C. horridus is important for the development of effective management plans. Gene flow is important when it comes to lasting viability of populations. The reason for this is that gene flow maintains genetic variability. Gene flow also prevents the effects of inbreeding in populations small in size (reviewed in Clark et al., 2008). For a lot of species that are philopatric (which means that individuals stay in or return to the place of birth) gene flow and spreading out is done not through actual migration to new populations, but by mating with individuals from different populations. These individuals would come into contact with those from other populations, mate, and then each goes back to their own original populations. Understanding the genetic make-up in populations having indirect gene flow is critical because population connectivity might not only be determined by the surroundings or landscape, but also by behavioral and social aspects (Bushar et al., 1998; Clark et al., 2008).
According to Wright's "shifting balance theory," gene flow can allow for the spread of well-adapted combinations of genes to additional populations (reviewed in Slakin, 1985). The "shifting balance theory" hypothesizes that infrequent dispersal can result in well-adapted combinations of genes becoming established in a population. But if the gene flow is frequent, well-adapted combinations of genes cannot get a foothold in any population. On the other hand, if the gene flow is low, the combinations can get established in a single population, but cannot be dispersed to other populations (reviewed in Slakin, 1985).
Gene flow plays an important role in creating genetic diversity and genetic diversity, in turn, plays a major role in facilitating adaptation, and therefore survival, of populations in changing environments. Lower levels of genetic diversity can limit a population's ability to adapt to changes in the environment and can lower the fitness of a population because of inbreeding depression (reviewed in Vali et al., 2008). An example of a population that has suffered from, dwindling health, and reduced survivability as a result of inbreeding depression would be the Florida panther (reviewed in Land & Lacy, 2000). Another species that has been affected by the detrimental effects of low genetic diversity would be the Cheetah, which is suffering from a bottleneck effect. In 1981, up to 70% of the male cheetahs in the National Zoo in Washington D.C were found to have abnormal sperm, which would lower fitness (reproduction). DNA samples taken from Cheetahs have often shown them to be the same genetically (reviewed in Cohn, 1986).
Populations, such as those of C. horridus that have gone through a reduction in demographic size often display a reduction in genetic diversity. A number of threatened or endangered species and populations have been shown to possess a low amount of genetic variation. Whenever low genetic variation is found in a population, it is often assumed that the population went through a bottleneck, but not all populations that have been reduced to small numbers have noticeably low levels of genetic diversity (reviewed in Spencer et al., 2000). Consequently, assessing the genetic diversity of a population is common in conservation genetics.
There are a number of different markers than have been used to assess genetic diversity in populations. Usually, genetic diversity at loci with functional significance, like protein coding, RNA coding, or regulatory sequences, is what has an effect on a population's capability to act in response to selection. But measuring genetic variability across all or a large portion of these functionally significant loci is currently unfeasible because of their vulnerability to selection. Because they are so vulnerable to selection, coding (functionally significant) loci show little variation. Selection generally favors the best allele, the more "fit" allele, so then any mutation from the ideal form will be less common in the population.
On the other hand, non-coding alleles are not affected by selection and so they can mutate without selection affecting their frequencies in the population, resulting in the buildup of more variation. It is because of this that non-coding alleles are often a more ideal choice for assessing genetic variation in populations. Genetic diversity at neutral and functional loci should be correlated because they are both reliant on effective population size, which means the portion of the population that is breeding, as well as other demographic factors (reviewed in Vali et al., 2008).
There are several techniques that are used to assess genetic variation at coding regions. Two that will be discussed here are allozymes and mitochondrial DNA. Allozymes are alternate forms of a protein that are distinguishable by electrophoresis. Allozymes were one of the first molecular markers used to characterize the genetic makeup of populations. The drawback of using allozymes is that they are not representative of a random sample of genomes and so as a result of sample size effect, the data could be skewed. Also allozymes might be selectively controlled in ways not seen in non-coding regions. One example of selection that controls allozymes is balancing selection, which means that many alleles are actively conserved in the genomes that make up a population., and it is because of this that allelic likenesses among populations are overestimated a lot more than they are at neutral loci (reviewed in Aagaard et. al, 1998). Allozymes are simple and low-cost to use but it is harder to spot differences among different populations on a small-scale level. They work better when it comes to detecting variation on a large scale (reviewed in Urbanelli et al., 2007). In addition, there are some species that give little or no variation at allozyme loci and as a result of this, estimating inter-population diversity is hard to do (reviewed in Seeb et al., 1998).
Because mitochondrial DNA (mtDNA) is highly variable, it has also been implemented for genetic studies (reviewed in Bos et al., 2008). Mitochondrial DNA is very sensitive when it comes to identifying the genetic composition of a population. Because mtDNA is passed down from the maternal side and is non-recombinant, the genetic relationships between individuals can be studied more easily than with nuclear loci. Also, because mtDNA only has one copy of each gene, gene flow is less likely to result in the homogenization of the genetic material. It is because of all these features that mtDNA can pick up on differences that allozymes do not (reviewed in Hale and Singh, 1987). Another feature that makes mtDNA favorable for genetic studies is the fact that it has a mutation rate that is 5 to 10 times more rapid than nuclear DNA. This rapid mutation rate is because of the lack of repair mechanisms for mtDNA that exist for nuclear DNA. As a result of this, mtDNA is highly variable between individuals (reviewed in Castro et al., 1998). Unfortunately, for unknown reasons, mtDNA does not seem to be very variable in C. horridus.
There are a number of techniques used to assess genetic variation that rely on the analysis of non-coding DNA. Techniques that will be discussed here include different "fingerprinting" techniques. DNA fingerprinting can be used as a way to distinguish individuals or populations from each other or to identify similarities between them. Fingerprinting can be accomplished using markers such as random amplified polymorphic DNA (RAPD), amplified fragment length polymorphism (AFLP), restriction fragment length polymorphism (RFLP), minisatellites, and microsatellites. All four techniques will be discussed in the following paragraphs.
Random amplified polymorphic DNA (RAPD) markers are easily procured and are simple. They are non-specific markers, so no genome information is needed before using RAPDs. In comparison to allozymes, RAPD markers are more polymorphic and are less influenced by selection (reviewed in Jenczewski et al., 1999). But although there are a plethora of RAPD markers, the success of reproducing them is low (reviewed in Urbanelli et al., 2007).
Amplified fragment length polymorphisms (AFLP) are reproducible when it comes to identifying polymorphisms among individuals in a population, populations, and lineages that are autonomously evolving, meaning that the lineages are unrelated and separate even though they may have evolved similar features (reviewed in Urbanelli et al., 2007). The polymorphism of the AFLP fragments is mainly produced through variation in restriction enzymes sites and polymerase chain reaction (PCR) facilitates rapid and proficient marker generation.
The restriction fragment length polymorphism (RFLP) technique is a DNA to DNA hybridization method that includes cutting genomic DNA with restriction endonuclease(s) and then Southern hybridization with labeled specific probes. This results in highly reproducible fingerprint patterns, valuable markers for studying genetic diversity among species.
Minisatellites are a form of RFLP that are made up of 6 to 100 nucleotide repeats, arranged one after the other, these sequences can be as long as 100 base pairs to many kilobases (Vergnaud and Denoeud, 2000). Minisatellites are markers that are very polymorphic and therefore are good to use in genetic studies (Bally et al., 2010). Also, the fact that they are able to cross-hybridize with many other comparable loci in the genome also makes them versatile for genetic studies (reviewed in Vergnaud and Denoeud, 2000).
A marker that has proven especially useful for population genetic studies is the microsatellite. Microsatellites are short tandem repeat sequences that are typically less than 6 base pairs in length. Dinucleotide, trinucleotide and tetranucleotide repeats are the type of microsatellites usually used in genetic studies. The majority of microsatellites found in most species are of the dinucleotide variety. The repeat sequences of microsatellites mutate often by means of slippage during DNA replication (which often happens when there are repetitive sequences, which results in deletions and insertions of nucleotides) and proofreading errors (reviewed in Selkoe and Toonen, 2006).
Microsatellites have many features that make them favorable tools for population genetic studies of organisms. The high rate of mutation in microsatellites makes them especially useful for estimating the levels of genomic diversity in populations (Vali et al., 2008). Eukaryotic genomes usually have a high amount of extremely polymorphic microsatellite loci. It is because of their abundance and polymorphism that they are such useful tools for population genetic studies (Bruford and Wayne, 1993). Microsatellites are unique to a species; therefore cross contamination is not as big of a problem as it is in other methods that use universal primers like AFLP (Selkoe and Toonen, 2006). Also because microsatellites are so short, even in situations were there is some DNA degradation, they can usually still be amplified (Selkoe and Wayne, 1993). These microsatellites can be amplified by PCR (reviewed in Villarreal et al., 1998). Each microsatellite can then be identified and characterized for the species or population being studied (Ashley and Dow, 1994).
It was in the human genome where microsatellites were first identified (Litt and Luty 1989; Weber and May, 1989). Despite the many advantages in using microsatellites, there are some pitfalls. Although the high mutation rates of microsatellites produce the large amounts of variety found in alleles needed for genetic studies, the mutational process of these regions can be irregular and unbalanced (Selkoe and Toonen, 2006). The complex mutation of the microsatellite can be a problem when a researcher uses statistical tests like Fst and Rst which calculate allelic frequencies because they solely depend on a mutational model for their calculations (Selkoe and Toonen, 2006). Fst depends on a stepwise mutational model of the alleles while Rst assumes that all alleles are equal.
Since the emergence of microsatellites as a tool in population genetics, they have become popular for use in a wide range of genetic studies such as a study done by Anderson (2006) which showed that microsatellite DNA primers made for the Massasauga Rattlesnake (Sistrurus catenatus) can be used on C. horridus as well. These primers resulted in visible microsatellite DNA fragments for C. horridus when the PCR annealing temperature was lowered and the buffer was optimized at every locus.
Using microsatellites can reveal a lot of information about populations of C. horridus. A study done by Villarreal et al. (1996) showed that six polymorphic microsatellite loci were identified in 32 C. horridus that were unrelated. These snakes came from eastern Pennsylvania, southern New Jersey, North Carolina, South Carolina, and Alabama. Blood and shed skin from the snakes were used to obtain genetic material. It was shown that all of the six loci were polymorphic with anywhere from two to eight alleles and had heterozygote frequencies ranging from 01. to 0.69. These results showed that allelic frequencies differed among geographically divided populations.
The six snakes that came from Ocean County, New Jersey had lower heterozygosity than the snakes from Berks County, Pennsylvania in three out of the four loci that were analyzed. The snakes from New Jersey were all homozygous for the same allele, while the snakes from Pennsylvania were heterozygous. This homozygosity could mean that a bottleneck happened to the New Jersey population of snakes.
When the loci of a mother and her neonates were analyzed, it was found that the loci have a Mendelian pattern of inheritance. Each of the neonates had at least one of its mother's two alleles. Although the father wasn't available for analysis, it was easy to determine, based on the neonates' and the mother's genotypes, that the father had to have been heterozygous at locus 5A. There were no signs of multiple paternity found in C. horridus.
Another study was done by Bushar et al. (1998) in which the researchers used 4 C. horridus microsatellite genetic markers to characterize 32 snakes from five local populations utilizing different hibernacula within a 6000-ha area located in southeastern Pennsylvania. Many alleles were found to be specific to either one or two of the local populations that were studied. Some hibernacula had a small population of snakes, but ironically, the more rare alleles were exclusive to these smaller local populations.
It was discovered that the snakes from the five different local populations were divided into two different genetic groups, snakes from two hibernacula made up one genetic group, while snakes from the three other hibernacula made up the other genetic group. The snakes that were in the same genetic group had considerably high gene flow between them, but the level of gene flow was quite low between snakes from the different genetic groups.
There have been few studies of the population genetic structure of the C. horridus and none that attempt to look at genetic relationships among populations across a large geographic area. My research is part of a larger project that was designed to assess the genetic variation and relationships within and among C. horridus populations across the state of Pennsylvania. Research on populations in various regions of Pennsylvania will add to the genetic profile that we are attempting to build on C. horridus in Pennsylvania. Up to this point, there has been no population genetic analysis carried out on C. horridus populations located in Union County. Our purpose is to determine the level of genetic variability and population genetic structure of populations of C. horridus from Raymond B. Winter State Park in Union County, Central Pennsylvania; Hells Creek in Carbon County, Pennsylvania; Slate Run in Lycoming County, Pennsylvania; Cove Mountain in Perry County, Pennsylvania; and High Knob in Pike County, Pennsylvania. These data will be used to formulate a successful conservation plan for this species in the state of Pennsylvania.