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Article Excerpt
ABSTRACT
Detailed molecular studies of portions of the cytochrome b and 12S rRNA genes in three species of common West Michigan frogs, Rana clamitans, R. pipiens, and R. sylvatica, reveal different patterns of genetic variation and different recent evolutionary histories for the three species. These patterns are correlated with habitat requirements throughout the life cycle of each species. Green frogs are dependent on permanent water throughout the year and their dispersal appears to be constrained by rapid human development and, when dispersal does occur into newly constructed wetlands, it may be accompanied by a loss of genetic variation. Leopard frogs are associated with water throughout their life cycle but live in more open areas than green frogs. West Michigan leopard frogs are severely genetically impoverished in the populations studied, possibly due to their pronounced sensitivity to chemical pollution in water. Wood frogs are the least aquatic/most forest dependent of the species studied and their populations have considerable genetic variability associated with a recent history of relatively stable populations that have had time to accumulate mutations. Tracking frog abundance in Michigan by calling surveys alone may be inadequate to identify species at risk from factors that erode their genetic variability and their subsequent ability to evolve in response to environmental pressures.
INTRODUCTION
Molecular data are being used increasingly to study metapopulation dynamics (the ability of organisms to move--or not move--between subpopulations), phylogeography (genetic history of the subpopulations tied to historic and geologic phenomena), phylogeny (the evolutionary history--selection, mutation, and genetic drift--within and between species), and many other aspects of the biology of organisms. These types of studies are important because much of modern biology is becoming a study of the decline and extinction of species as well as a study of the most effective methods to preserve biodiversity.
Among species groups, amphibians (and especially frogs) are in serious decline (Stuart et al. 2004) and have been subjected to intense natural history and genetic scrutiny as a result. For example, metapopulation studies indicate that land use changes associated with human development create barriers that reduce the genetic variability of populations (Knutson et al. 1999; Marsh and Trenham 2001; Guerry and Hunter 2002; Semlitsch and Bodie 2003), a feature important to their ability to recolonize suitable habitats where they have been extirpated (Reh and Seitz 1990; Hitchings and Beebee 1997). Other examples include phylogeographic studies that look at intraspecific or interspecific genetic variation patterns to discern cryptic species, genetically diverse population units that require separate conservation efforts, and recent versus ancient events that have impacted the genetic structure of the species of interest (Nielson et al. 2001; Carolina and Carnaval 2002; Hoffman and Blouin 2004; Vieites et al. 2006). Lastly, in frogs the evolutionary history of whole families is being elucidated by studies of the growing data base of molecular information available online (NCBI, GenBank) and through new DNA sequencing efforts (Hillis and Wilcox 2005; Bossuyt et al. 2006).
Molecular-based studies of species must balance the ability to sample sufficiently to obtain statistically reliable data with the cost of sampling (Lowe et al. 2004). In the case of molecular sampling, the cost is exceptionally high so there is a strong tendency for small sample sizes at localities separated by large geographic distances. For example, typical studies have 3-4 samples at each locality and have localities separated by tens to hundreds of kilometers (e.g., Tanaka-Ueno et al. 1999; Nielson et al. 2001; Jaeger et al. 2001; Carolina and Carnaval 2002; Vieites et al. 2006; Lee-Yaw et al. 2008). Another difficulty is sampling to accurately represent the true ecology of the species (Lowe et al. 2004; Waples and Gaggiotti 2006). With frogs, this is particularly difficult because their distribution is patchy (Marsh and Trenham 2001), varies with the time of year and the sex of the frog (e.g., Newman and Squire 2001; Regosin et al. 2003), and they may be locally extirpated periodically by events like droughts (Marsh and Trenham 2001).
The DNA samples for this study were collected as a byproduct of a larger, comprehensive survey of frogs on the GVSU Campus and of a GIS habitat study of the Bass River area. The purpose of this study was to capture a snapshot of genetic information in three frog species in a very restricted geographic region to gain insight into their population dynamics, phyiogeography, and fine-scale phytogeny. We have found no other studies that have attempted to gain this close of a view within local populations of frogs or, for that matter, other species. The species chosen--green frogs, leopard frogs, and wood frogs--are all common but differ in their physiological, ecological, and life cycle sensitivities. Of the three species, green frogs are most dependent on permanent water sources (Conant and Collins 1991; Knutson et al. 1999), leopard frogs are the most sensitive to environmental pollutants (Brodkin et al. 2003; Simon et al. 2002; Vatnick et al. 1999), and wood frogs are the least dependent on water and have the longest dispersal distances (Berven and Grudzien 1990). Because of these life history differences, we expect modern genetic patterns within each species to reflect different responses to recent pressures. We also expect differences in their genetic patterns based on their postglacial dispersal into the region. For example, wood frogs apparently entered the lower peninsula of Michigan from the north (Lee-Yaw et al. 2008) whereas leopard frogs and many other amphibians apparently entered from the south (Hoffman and Blouin 2004; Lee-Yaw et al. 2008).
MATERIALS AND METHODS
Mitochondrial DNA (mtDNA) was used as our molecular tool because it is relatively easy to collect without harming the frog, its mutation rate is very fast compared to other sources of DNA, it is phylogenetically interpretable, maternally inherited, shows strong differentiation, and is very useful in assessing diversity and bottlenecks (Lowe et al. 2004). Molecular genetics is used most frequently to determine the phylogenetic relationships among higher taxa (e.g., Garcia-Paris et al. 2000; Reeder and Montanucci 2001; Garcia-Paris and Wake 2000; Sumida et al. 2000), but fast evolving mtDNA genes are also useful to determine relationships within species (e.g., Hidetoshi et al. 1998; Sumida and Ogata 1998; Sumida et al. 2002). Short sequences of the cytochrome b gene contain phylogenetic information extending from the intraspecific to the inter-generic level (Kocher et al. 1989) and, together with the 12S rRNA gene, which evolves more slowly than the cytochrome b gene (e.g., Sumida et al. 1998; Garcia-Paris and Wake 2000), they provide a powerful time-sensitive indicator of frog dispersal or lack of it (Sumida et al. 2000) as well as an indicator of genetic variability.
From 2002 to 2003, we studied three common species of Ranid frogs--Rana clamitans, R. pipiens, and R. sylvatica (green frogs, leopard frogs, and wood frogs, respectively)--in the Lower Grand River Drainage Basin in Ottawa County, Michigan, with intensive but unequal sampling of frogs from the Bass River State Recreation Area and the Grand Valley State University (GVSU) Allendale Campus. Some frogs were also collected from the Pigeon Creek area, a small watershed south of the Grand River. After observing the results of the 2002-2003 genetic studies, in 2004 we added small samples of each of the species from the major watersheds north (Muskegon River, Muskegon County) and south (Pigeon Creek, Ottawa County; Kalamazoo River, Allegan County) of the Grand River Drainage (Figure 1).
[FIGURE 1 OMITTED]
The GVSU Campus encompasses 450 hectares that include areas of intensive development as well as a relatively undisturbed eastern ravine system that borders the Grand River. Much...
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