Eurycea Cirrigera)

Home , Brook salamander

Genetic Distinctiveness of a Peripheral Illinois Population of the

Southern Two-lined Salamander (Eurycea cirrigera)

Thesis submitted to the Department of Biological Sciences Chicago State University

In partial fulfillment of the requirements for the degree of Master of Science in Biological Sciences

Maria Boyle

Eric L. Peters, Thesis Advisor Timothy J. Bell, Committee member Mark A. Erhart, Committee member

April 14, 2005

Approval Sheet

We have examined this thesis manuscript and we verify that it meets the program and university requirements for the degree of Master of Science in Biological Sciences

Eric L. Peters, Ph.D. (Thesis Advisor) (Date)

Timothy J. Bell, Ph.D. (Date)

Mark A. Erhart, Ph.D. (Date)

Mark A. Erhart, Ph.D. (Graduate Committee Chair, Biological Sciences) (Date)

Floyd W. Banks, Ph.D. (Department Chairperson, Biological Sciences) (Date)

Anitra Ward, Ph.D. (Acting Dean of The Graduate School) (Date)

Table of Contents

Acknowledgements...... 1

Abstract ...... 2

Introduction ...... 3

Molecular evaluation of population differentiation ...... 4

The two-lined salamander species complex ...... 5

Geographic isolation of the southern two-lined salamander in eastern Illinois...... 7

Materials and Methods...... 9

Sampling...... 9

DNA primers ...... 10

DNA Sample processing ...... 11

Statistical Analyses ...... 11

Results ...... 13

Identification and evaluation of primers ...... 13

Differentiation among populations ...... 14

Allelic variability among the KRSP population(s) ...... 15

Discussion ...... 17

Appendix A ...... 22

Literature Cited...... 23 1

Acknowledgements

I must extend my sincerest thanks and appreciation to the members of my thesis committee: my advisor, Dr. Eric Peters for his encouragement, field work assistance, expertise and the crea- tion of an alliance between me and several of his colleagues (Mr. Tony Mills, Dr. Olga Tsyusko,

Dr. Steve Harper) at the University of Georgia’s Savannah River Ecology Laboratory who as- sisted me in sample retrieval, statistical analyses and imaging, Dr. Tim Bell, whose office door was always open to me in Dr. Peters’ absence for questions of all sorts and for accompanying me to my initial visit to Indiana, Dr. Mark Erhart who extended the use of the resources in his lab to facilitate the completion of this project. I am so appreciative of Dr. Joyce Ache Gana’s assistance with this project. She unconditionally gave of her time and lab resources so that I could move forward with the data collection for this project. I was fortunate to have been provided with all the primers used in this project from Dr. Paul Cabe’s lab at Washington and Lee University. His generous donation allowed me to screen all the primers available from his lab’s work on P. cinereus. I would also like to acknowledge the Illinois Department of Natural Resources for granting a Special Use Permit for the collections in Will County, IL and the Indiana Department of

Natural Resources for granting a Scientific Collectors License (No. 2988) for the collections in

Warren County and Lake County, IN. My sincerest appreciation to Dave Mauger (Will County

Forest Preserve District) for assisting me in locating some of the collection sites at KRSP and for providing me with background literature from his personal research on E. cirrigera and related species and Michelle Boyle, Angela Boyle, and Tim Thompson for their field work assistance.

And finally once again to Dr. Peters, in what may have been a moment of altered consciousness, but nonetheless… referred to me as a colleague. I thank you. 2

Abstract

The southern two-lined salamander (Eurycea cirrigera) is a predominantly southeastern

North American species whose peripheral range extends into eastern Illinois. An isolated population inhabits tributaries of the Kankakee River within and near the Kankakee River State Park

(KRSP) in Will County, Illinois: the northwestern-most extent of its known range. Using seven microsatellite loci developed for the red-backed salamander (Plethodon cinereus), the genetic variability of the KRSP population was compared with nearby populations in Warren County,

Indiana (WC) and a distant population in Aiken County, South Carolina (AC). Pair-wise comparisons indicated significant differences at all loci between the KRSP and AC samples and significant differences in six out of seven loci between KRSP and WC population samples. There was a significant correlation (r2 = 0.96) between the mean genetic and geographical distance over all population locations. Differentiation within the KRSP samples demonstrated significant variability at three of the seven loci, with pair-wise comparisons of Fst (correlation measurement of genetic difference between pairs of populations) showing significant genetic differences in eight out of ten population matches, including two of three site comparisons on the north and south sides of the Kankakee River, suggesting that the river forms an isolating boundary between these locations.

Introduction

Gene flow between members of small subpopulations can homogenize allele frequencies and influence the effects of selection and drift by thwarting the fixation of alleles that may promote genetic differentiation (Balloux 2002). In natural populations, however, population structuring may segregate individuals into small sub-structured, internally-reproducing units (demes) that may promote allelic variation as a consequence of restricted gene flow (Kirkpatrick 2002). As has been shown for simulated populations (Church and Taylor 2002), speciation events may be enhanced in the absence of migration among demes. In natural populations, however, it can be difficult to know where subpopulation boundaries occur. Populations that are reduced in size along with habitat dimensions may be subject to inbreeding effects. Inbreeding can alter geno- typic frequencies (not allele frequencies) by increasing the occurrence of homozygotes within a population. The effective population size (the number of individuals in a population who contribute offspring to the next generation) is an important factor in determining the extent to which individuals are able to overcome the effects of inbreeding.

Gene flow supports the integrity of populations by homogenizing alleles and opposing the influences of differentiation induced by adaptation and genetic drift (Morjan 2004). In an isolated population, the absence of gene flow can have pronounced influences on genetic diversity. The differentiation among alleles subject to the effects of selection will be dependent on a trait’s heri- tability and its ability to express under environmental influence (Garcia-Ramos 1997). Small populations can be particularly susceptible to the detrimental effects of drift, which can fix alleles that may reduce fitness. If random changes in allele frequencies do not lead to adaptive traits in a population, fitness may be further compromised (Freeman 2004).

Populations that develop independently while adapting to their isolated habitats can eventu- ally become subject to speciation events. Reproductive isolation secondary to habitat fragmentation may initiate speciation (Garcia-Ramos1997, Slatkin 1987). Although most populations display genetic differentiation among their members (variations in the gene pool which do not result in divergence), geographically-isolated populations typically demonstrate greater genetic differentiation than is found among populations in close (but barrier-free) proximity (Balloux

2002). Where such reproductively-isolating barriers are present, divergences can develop rapidly in peripheral populations. Under such conditions, evolution of pre- and post-zygotic barriers can inhibit successful reproduction among subpopulations and lead to speciation. Although there is an appreciation of the genetic factors that can result in speciation, these population genetic prin- ciples alone have thus far not been sufficient to completely explain speciation events, given other contributing biogeographical, ecological, and behavioral factors. This may be because the time interval for speciation is generally too long to study directly, and the abundance of evolutionary factors that may interact to influence speciation is difficult to isolate and interpret individually

(Kirkpatrick 2002).

Molecular evaluation of population differentiation

Genetic characterization of species has advanced research of population biology, particularly where populations cannot be distinguished morphologically. The accumulation of genetic differences among individuals within and among populations can be detected and evaluated by current molecular genetic techniques, including: nuclear microsatellite loci, mitochondrial DNA, and single nucleotide polymorphisms (Morin 2004). In natural populations, alterations within a line- age and macroevolutionary processes may be explained by changes in allele frequencies and trait distributions that occur secondarily to gene flow restriction. Several mechanisms (e.g., disruptive

5 selection, isolation, force of selection, mutation) have been proposed in support of speciation events as a consequence of gene flow restriction (Kirkpatrick 2002) and the accumulation of genetic differences (Gavrilets 2000). Molecular markers used to identify these have been instru- mental in canvassing genetic variability within small-scale and large-variant sampling in natural populations. Examining genetic variability among and within populations provides insight to micro-evolutionary changes within species and can also provide information on the migratory patterns between natural populations.

Microsatellites are highly-variable sequence repeats found in coding and non-coding segments of DNA that can be used as genetic markers in population structuring (Slatkin 1995). The discovery of these variable segments of DNA, some of which are flanked by evolutionary conserved regions, has facilitated inter-species genetic comparisons (Zane 2002). Microsatellite alleles that are present in one population may not be present in another population that is reproductively isolated. Though many segments of DNA are conserved among phylogenetically- related species (Voss 2001), differentiation in allele frequency within species can provide insights into molecular diversification and evolution (Camp 2000).

The two-lined salamander species complex

The southern two-lined salamander (Eurycea cirrigera) is a member of the Family Pletho- dontidae (ITIS 2004), the largest and most widely distributed family of salamanders inhabiting

North America (Petranka 1998). Sub-classed as a brook salamander, E. cirrigera is a forest- dependent species, requiring rocky spring outlets, streams or creeks for reproductive and population viability. Courtship usually occurs in the fall, with eggs laid in late fall and winter. Larvae usually emerge in the spring. Plethodontids have been studied in detail and, while many cryptic species are continually emerging (Conant 1998), genetic investigations have led to the division

6 of numerous morphologically conserved taxa into defined species (Camp 2000). Because of the high phenotypic conservatism of plethodontids, evaluations of molecular differences are neces- sary to assess population dynamics and the potential evolutionary significance of these populations.

A study of 22 species of salamanders (21 of which were plethodontids) was conducted to determine the levels and rates of gene flow among populations (Larson 1984). Using an average allele frequency (average frequency of a protein variant) and the fraction of the total population in which it occurred, the authors compared observed patterns of allelic frequencies to those expected at various levels of gene flow. They concluded that populations of plethodontids are generally not connected by gene flow, as appears true of amphibians in general (Newman 2001).

Furthermore, the high morphologic concordance among populations isolated from gene flow over extensive geographic distances, may be consistent with their life KRSP WC history mode over millions of years

(Larson 1984).

Initially, three subspecies of a single AC species of two-lined salamanders were Eurycea cirrigera Eurycea wilderae recognized (Kozak 2001), ranging across Eurycea bislineata the U.S. east of the Mississippi River from Figure 1. Distribution of E. cirrigera (southern two-lined Louisiana to southern Ontario (Figure 1): salamander) as well as E. wilderae and E. bislineata in North America (redrawn from Conant 1998). The map Eurycea b. bislineata (the northern two- shows the approximate locations of the Kankakee River State Park, Illinois (KRSP), Warren County, Indiana (WC), and Aiken County, South Carolina locations (AC) sampled in this study.

a 7 lined salamander, a northeastern form), E. b. wilderae (the Blue Ridge two-lined salamander, found in the Appalachian Mountains from southwest Virginia to northern Georgia), and E. b. cirrigera (the southern two-lined salamander, a lowland form of the Atlantic and Gulf Coastal

Plains. The debate over whether the members of this “Eurycea bislineata” (two-lined salamanders) complex represent subspecies or full species was addressed by Petranka (1998), who did not in fact recognize E. cirrigera as a separate species, based on allozyme studies that did not examine areas of contact zones or assess the degree of inbreeding between populations in close proximity. Two subsequent studies (Kozak and Montanucci 2001, Camp et al. 2000) disagreed with this more conservative view, finding significant genetic differences (inferred from allozyme frequencies) between cirrigera and wilderae. Both groups are, in the absence of any geographical barrier, occupying contact zones (i.e., both “species” occupy the same niche), however there is little or no gene exchange, nor any evidence of hybridization (Kozak and Montanucci 2001,

Camp et al. 2000). Based on these results and on morphological analyses, the authors agreed that

E. cirrigera and E. wilderae, should be regarded as separate species. The salamanders used in this study will be referenced as E. cirrigera, a distinct species, as suggested by Kozak and Mon- tanucci 2001 and Camp et al. 2000.

Geographic isolation of the southern two-lined salamander in eastern Illinois

Within the Kankakee River State Park (KRSP, located at the boundary of Will and Kankakee

Counties, Illinois), E. cirrigera is found in rocky streams and above-ground seeps that occur on both sides of the Kankakee River. The KRSP is located within a sparsely-forested area (a likely remnant of a more extensively wooded area) that was formerly surrounded by prairie (Mierzwa

1989) and is currently subject to the influences of agriculture and human inhabitance. The geographic isolation of the KRSP population is likely due to their requirements for cool water and

8 shaded forest; as such habitats are uncommon in northeastern Illinois. E. cirrigera residing in this location represent a population (or populations) of the species isolated from the main body of its range by a distance of more than 100 km (Mierzwa

1989, see Figure 2). Geological evidence suggests that the isolation of the KRSP population from other nearby populations likely dates from Wisconsin glacial events Figure 2. Distribution of E. cirrigera in Illinois and Indi- occurring approximately 14,000 years ago ana, based on locations plotted by Mierzwa (1989) and county records as of 1990 (Illinois Natural History Survey (Mierzwa 1989). Reproductive isolation 2004). Arrows approximate the KRSP (Illinois) and WC (Indiana) locations. may have also occurred subsequently, however, due to the geographical barrier of the Kankakee River. It appears unlikely that populations on the north and south sides would be able to interbreed freely, as individuals would have to traverse the swift current of the Kankakee River and elude its predatory fishes. However, mat- ing among members of neighboring tributaries on the same side of the river might occur. If this is true, then differences in allelic frequencies should be greater between the populations on the north and south sides of the river than among sub-populations on each side of the river.

Specific Aims

The specific aims of this study were to evaluate the genetic difference of the KRSP Illinois populations of E. cirrigera using microsatellite loci, relative to less-isolated populations and to evaluate subpopulations within the KRSP as to whether genetic differentiation could be detected

9 among population sites (including those divided by a geographical barrier) that may in fact represent demes.

Materials and Methods

Sampling

Tissue samples from Eurycea cirrigera were collected within the borders of the KRSP, along tributaries of Big Pine Creek in Warren County, Indiana (WC), and in southern Aiken County

(AC), South Carolina (Figures 1 and 2) in accordance with Chicago State University’s Institu- tional Animal Care and Use Committee protocol (approved July 2003). Adult E. cirrigera were hand-collected from under rocks, logs and leaf debris at the KRSP sites, in July and August

2003. The (5 to 8 mm) tail tip was collected from each individual, and animal was then immedi- ately released at the capture site. The samples were stored in 2.0-ml polypropylene cryogenic vials and kept frozen for DNA analyses.

Salamanders were collected from the KRSP at five locations (Figure 3). The sampling site designations followed the protocol of

Mauger et al. (2000), and reflect the proximity of Illinois State Routes 102 and

113, which parallel the Kankakee River and the KRSP. These sites included localities previously identified (Mierzwa

1989, Mauger et al. 2000), and a new site Figure 3. Map of sampling locations within the Kankakee River State Park (KRSP), Will County IL (the park is bor- (102-5). The Indiana samples were dered on the north and south by Illinois State Routes 102 and 113, respectively). The Park is bisected by the Kankakee collected from three locations (Figure 2) in River. The five locations (also see Mauger et al. 2000) lie along small, unmapped springs and seeps that are tributaries of the river.

June and July of 2004, using the same sampling protocol as the Illinois sites. Except for a single specimen collected from Upper Three Runs Creek near the Savannah River Ecology Laboratory

Conference Center east of New Ellenton, all AC salamanders were collected on a rainy night in

September 2004 on a road adjacent to the Savannah River floodplain near Jackson, SC. The samples were frozen after collection, and the AC samples placed in 95% ethanol for shipment to

Chicago State University. For the purpose of DNA analyses, all of the AC salamanders were considered to represent a single population.

Tail clips of two Plethodon cinereus collected at the Indiana Dunes National Lakeshore

(Lake County, IN) were used as a control in the initial screening of the supplied primer pairs.

DNA primers

Thirteen dinucleotide microsatellite loci primer pairs (Connors and Cabe 2003) developed from the genome of Plethodon cinereus (a closely-related plethodontid), were used to test the E. cirrigera samples. These primers were developed by random-cloned procedures, so there was no information available as to where these segments might bind in either the P. cinereus or E. cirrigera genomes. Success in applying the same primers to different taxa depends on the presence of conserved loci. Based on morphologic divergences and evolutionary traits among plethodontids and on phylogenetic relationships between salamander tribes, it is estimated that P. cinereus and E. cirrigera diverged from a common ancestor >40 Ma ago (Highton 1995, Ruben 1993,

Lombard 1986). Other than in mammalian studies, there have generally not been inter-species analyses of microsatellite loci. However, conserved microsatellite polymorphic loci were docu- mented between two families of marine turtles with an estimated time since divergence of approximately 300 Ma. The ability to amplify microsatellite loci across such distantly-related

11 families of marine turtles implies an evolutionary conservation of these segments (FitzSimmons et al. 1995).

DNA Sample processing

DNA was extracted from tail tissue samples using a DNeasy kit (Qiagen) following the manufacturer’s instructions. Each of the samples was quantified using Genequant (Pharmacia), to determine the concentration of DNA (µg/ml). After DNA isolation, thirteen primer pairs were screened with a control (P. cinereus) and a random sampling of E. cirrigera subjects. Each PCR reaction (20 µl) contained approximately 250 ng of genomic DNA, 0.054 µM of MgCl2, 0.09

µM of 10X buffer, 0.02 µM of dNTP, 0.02 µM of specified primer pair (25 pmol/µl), and 0.5 units of Taq polymerase (Promega). Deionized H2O was added as needed to achieve the required volume. Reactions were run in a Master-gradient thermo-cycler (Eppendorf). PCR products were run on a 3% Amresco (Solon, OH) agarose gel using 1X TBE buffer. Each lane was loaded with

15 µl of sample. A DNA marker of 50-1000 bp (Cambrex) was loaded in one or two lanes per gel as a reference. Gels were run at 100 V for 60 min. The gels were then placed into a staining tray containing an ethidium bromide (0.5µg/ml) solution and soaked with slight agitation for 30 min. The gels were then rinsed, viewed and imaged on a Fluor–S (Biorad) imager.

To determine the sizes of the bands imaged from the electrophoretic gels, exponential regression analyses were performed using the size (bp) of the migrating bands as a function of the distance (mm) traveled. Each band was assigned a calculated size (bp) in a three-digit format from the results of the regression analysis.

Statistical Analyses

Deviations from Hardy-Weinberg equilibrium (HWE) were tested using χ2 analyses across all loci within each state population and at each locus and subpopulation using GENEPOP ver-

12 sion 3.4 (Raymond and Rousset 1995), estimating P-values by the Markov chain method (default parameters) testing the null hypothesis that the observed allele frequencies are not significantly different from predicted frequencies for a population in equilibrium. In addition, an evaluation of heterozygosity for each locus at each population using Fis (correlation of alleles within individuals; within a population) as in Weir and Cockerham (1984) and Robertson and Hill (1984) was conducted.

Tests for differences in allelic distribution among populations were conducted using

GENEPOP 3.4, under the null hypothesis of no variability among alleles for pair-wise and overall population comparisons. In addition, analyses for the variability among the distribution of alleles were conducted with all seven loci for pair-wise and overall populations within the KRSP and WC sites. A Markov Chain method (batches = 1000, iterations per batch = 1000) was used to determine the unbiased P-value for every population pair at each of the seven loci. The Fst, a correlation measurement of the genetic difference between pairs of populations (Weir and Cocker- ham 1984, Michalakis 1996), was computed to determine the overall population differentiation at each of the loci among the three state populations as well as within the KRSP and WC populations.

The Mantel correlation coefficient (Mantel 1967) was calculated using the Mantel version

2.0 (Liedloff 1999) nonparametric test calculator (1000 iterations) to test for a correlation between Fst (genetic difference) and geographic distance. Relationships between the measures of

Fst/(1 – Fst) and geographical distance at different scales were evaluated by logarithmic regres- sions (Rousset 1997).

Table 1. Microsatellite primers developed for P. cinereus Results (Connors and Cabe 2003), annealing temperatures used in this study and allele size ranges obtained in this study.

Identification and evaluation of primers Locus Annealing Allele Size (GenBank Accession Temperature Range Seven of the 13 microsatellite primers No.) (°C) (bp) PcXD23 (AY151376) 50.0 62-171 developed for P. cinereus were used for PcCCO4 (AY151380) 58.9 59-146 genetic comparisons (Table 1). PCR PcI16 (AY151379) 61.8 52-61 PcFX08 (AY151377) 55.2 56-72 products produced from 0-5 bands per lane. PcII14 (AY151372) 57.2 60-171

Approximately 17% of the samples revealed PcKX02-2R (AY151373) 58.9 252-519 PcLX23 (AY151374) 61.8 61-74 more than two bands in any given lane. This result was predominantly seen at two loci (PcXD23 and PcII14). Bands that were eliminated from the data were >>100 bp longer than the “expected” size fragments. In all cases, questionable bands were compared with other subjects in the same sub-population and similarly-sized bands were chosen to represent the allele(s).

Overall, both the KRSP (χ2 >> 25, df = 10, P < 0.0001) and WC populations (χ2 = 25.40, df = 8, P = 0.0013) showed departures from HWE, but the AC population was not significantly out of equilibrium (χ2 = 9.982, df =6, P = 0.1254). The results at each locus however, showed that only one locus (PcXD23) did not conform to HWE. Four loci displayed a single allele in one to five subpopulations (Table 2). It is unclear whether the single-allele results would represent a null allele, which could be the subject of bias in genetic differentiation testing (Newman 2001).

Two of the loci (PcI16 and PcPX23) did not produce any usable data for pair-wise tests or Fst determination. The other two loci that displayed the single alleles (PcC04 and PcFx08) were util- ized in the analyses with single allele data omitted.

Table 2. Genetic variation (displayed as variable loci) and heterozygosity frequencies for E. cirrigera.

Number of Alleles (Observed/Expected Heterozygosity Frequencies) Location PcXD23 PcCC04 PcI16 PcFX08 PcII14 PcKX02-2R PcLX23

102-2 3 (0.25/0.25) 2 (0.50/0.33) 2 (–) 3 (0.25/0.25) 2 (0.50/0.33) 2 (0.25/0.25) 1 (–) 102-3 4 (1/0.70) 3 (0.50/0.30) 2 (–) 3 (0.33/0.33) 3 (0.33/0.28) 4 (0.67/0.52) 2 (–) 102-5 2 (0.75/0.45) 2 (0.50/0.27) 2 (–) 3 (0.75/0.55) 2 (1/0.57) 2 (–) – (–) 113-2 2 (0.60/0.36) 1 (–) 1 (–) 4 (0.40/0.40) 2 (0.60/0.36) 2 (0.20/0.20) 1 (–) 113-4 2 (0.60/0.36) 2 (–) 1 (–) 3 (0.20/0.20) 2 (1/0.56) 2 (0.20/0.20) 2 (–) WC-1 2 (0.71/0.40) 1 (–) 1 (–) 2 (0.33/0.22) 2 (0.67/0.38) 4 (0.50/0.43) 1 (–) WC-2 2 (0.75/0.45) 2 (0.25/0.25) 2 (–) 3 (0.50/0.42) 2 (0.75/0.45) 2 (0.50/0.33) 1 (–) WC-3 2 (1/0.67) 1 (–) – (–) 1 (–) 2 (1/0.67) 2 (1/0.67) 2 (–) AC 4 (0.88/0.59) 3 (0.38/0.28) 1 (–) – (–) 3 (0.25/0.17) 2 (0.13/0.13) 1 (–) Total 11 9 7 10 8 12 6

Mean 2.56 (0.72/0.47) 1.89 (0.43/0.29) 1.5 (–) 2.75 (0.39/0.34) 2.22 (0.68/0.42) 2.44 (.43/.34) 1.38 (–)

The microsatellites used in this study displayed multiple alleles at each locus. Computed Fis values were consistently < 0, which implies that there is no evidence of a deficiency of heterozy- gotes in any of the subpopulations. These results are consistent with the deviation from HWE

Table 3. Estimates of genetic difference (Fst) and (seen in the KRSP populations and WC allelic variability among KRSP, WC and AC populations of E. cirrigera at each of the seven loci used populations) that is observed in subpopulations in this study.

Locus Populations Fst P displaying heterozygote excess (Rowe et al. PcXD23 KRSP & WC 0.3186 <0.00001 KRSP & AC 0.3156 <0.00001 2000). AC & WC 0.4366 <0.00001 Differentiation among populations PcCC04 KRSP & WC 0.2051 0.02618 KRSP & AC 0.2870 <0.00001 In pair-wise comparisons, 18 of 19 probability WC & AC 0.3333 0.00002 PcI16 KRSP & WC NA 0.01371 tests (locus PcFX08 produced results for only one KRSP & AC NA 0.00013 WC & AC NA 0.00484 of three comparisons) led to a rejection of the null PcFX08 KRSP & WC 0.2015 <0.00001 hypothesis of no difference in allelic variability PcII14 KRSP & WC 0.2426 <0.00001 KRSP & AC 0.3014 <0.00001 ability among the three state-wide populations WC & AC 0.5000 <0.00001 PcKX02-2R KRSP & WC 0.2264 0.00041 (Table 3). Only one locus (PcLX23) displayed a KRSP & AC 0.1905 0.00021 WC & AC 0.3571 0.00005 P > 0.05 between the KRSP and AC populations. PcLX23 KRSP & WC NA 0.00235 KRSP & AC NA 0.07287 WC & AC NA 0.03728

At each locus, there was significant allelic variability among all three of the state populations.

Differentiation over all three state populations was also significantly different (P < 0.0001) among all seven loci.

The Fst value of each state-wide population pair was significantly greater than zero, suggesting that to varying extents, the populations are genetically different from each other. Among the nine subpopulations, there were 36 pair-wise Fst estimates. Four of 36 values were negative, and were treated as zero (Jaramillo et al. 2001). Pair-wise comparisons of all subpopulations within the three states, showed that eight of 36 values did not show significance differences in genetic variability, with only two of these values Table 4. Fst values and probability of no differences in allelic frequencies among 7 loci tested between populations of E. cirrigera on the north and south representing intra-state population pairs (IL and sides of the Kankakee River.

IN). Locus Fst P PcXD23 0.1090 0.0072 Allelic variability among the KRSP population(s) PcCC04 NA 0.0007 Comparisons of the allelic differentiation PcI16 NA 0.1656 PcFX08 0.0323 0.1091 among the seven loci tested for the sampled popu- PcII14 0.1930 0.0005 lations on the north and south sides of the PcKX02-2R 0.0000 0.1841 PcLX23 0.0763 0.5243 Kankakee River demonstrated that three of the seven loci (PcXD23, PcCC04, PcII14) were significantly different (Table 4). Among the three subpopulations sampled on the north side of the Kankakee River, the distances between sites

102-2 and 102-3, and between 102-3 and 102-5 were 0.571 and 2.41 km, respectively. The distance between the two sites on the south side of the Kankakee River was 3.7 km. Of the ten combinations of population pairs compared, only two (102-2 & 102-3, and 102-3 & 113-2) did not significantly differ. It is interesting to note that while 102-2 and 102-3 were the closest adjacent sites on the north side of the river, 102-3 and 113-2 are located on opposite sides of the

16 river. Further examination of the banding patterns for sites 102-3 & 113-2 revealed that at three of the seven loci (PcXD23, PcCC04,

PcII14), over 50% of the samples shared indistinguishable fragments, and these were presumed to be the same alleles. Even at fine- scale distances (<10 km), there is a significant Figure 4. Logarithmic regression of genetic difference regressed on the geographic distance between KRSP and relationship between genetic differentiation WC population pairs. among the subpopulations that is correlated with geographic distance at KRSP, but not at

WC (Figure 4).

There was a significant correlation (Man- tel’s, r = 0.261, P = 0.002) between genetic difference (Fst) and geographical distance

ranging over 1000 km among all the sampled Figure 5. Logarithmic regression of genetic difference regressed on the geographic distance between all popula- subpopulations in this study (Figure 5). The tion pairs. relationship between mean genetic differences and mean geographical distances of the populations separated by geographical barriers was highly correlated (Figure 6), with the populations on the north and south sides of the

Figure 6. Logarithmic regression of mean genetic difference across all loci regressed on the mean geographic distance for populations separated by geographic barriers.

Kankakee River (KRSP N & S) showing a significant amount of genetic difference.

Discussion

Peripheral populations exemplify a challenge in estimating the effect of gene flow disruption when evaluating genetic variability among populations. Populations at the margins of a species’ range may be subject to decreased genetic variability as a consequence of reduced population size, decreased sources of immigrants and potential population bottlenecks (Garner 2004). Alter- natively, changes in allele frequencies can occur when an isolated population evolves in response to adaptation to a favorable environmental situation (Garcia-Ramos 1997).

The genetic differences among the three state-wide populations as well as most of the subpopulations in this study were highly correlated with the geographic distance between them. The level of genetic differentiation measured by Fst, indicated that in general, populations in closest geographic proximity to one another displayed the greatest amount of genetic similarity. This may imply that gene exchange may be facilitated by the ability of breeding individuals to freely migrate to adjacent breeding areas or that subpopulations in close proximity would have had less time to “diverge”, assuming that they once represented a more contiguous population.

The geographic proximity of populations does not always present a clear picture of habitat parameters. In the case of the KRSP subpopulations, some of the areas between and possibly within adjacent breeding sites have been compromised to accommodate human activities at the park. Roads, designated hunting sites, campgrounds and picnic areas are located within the

KRSP boundaries and may have influenced population structuring of E. cirrigera. In addition, natural habitat stresses may also challenge optimum niches for this species. For example, a site identified by Mauger et al. (2000) and annually visited by classes from Chicago State University

18 was flooded in 2002 by a beaver dam, and was inaccessible during the collection period in 2003.

It is unclear as to whether any E. cirrigera are still maintaining a population in that location.

There were also signs of habitat stresses in what appeared to be a slightly less corrupted envi- ronment in Warren County, Indiana. On the first visit to Big Pine Creek (WC), two sampling locations were found. On a return visit to the same locations, the residual mud that adhered to the streamside flora of the WC-2 location suggested that it had been dammed to allow installation of a culvert under the road, and no salamanders were found during an extensive search of the area.

It is uncertain as to whether this population was eradicated or if it successfully migrated to avoid the temporary flooding. Although plethodontids are typically phylopatric, these types of disrup- tions could compromise the success of a stream-associated salamander in migrating to a neighboring breeding site or influence the perpetual existence of subpopulations.

Varied degrees of differentiation have been reported in studies of other amphibian species utiliz- ing microsatellites as a genetic determinant (Table 6). These studies found a direct relationship between genetic variability and distance between populations. The lowest levels of genetic differentiation were observed in the R. sylvatica study, which made fine-scale population comparisons ranging from 50 m to 5.5 km distant. Similar results were obtained for the subpopulations at

WC, however KRSP subpopulation’s overall Fst values ranged from 0.03 to 0.24 on the north side and was 0.32 for the two populations on the south side of the river. The results of this study suggest greater genetic differences among E. cirrigera populations across smaller geographic distances, as compared with Ambystoma maculatum (spotted salamander). The overall genetic difference (as measured by Fst) between the KRSP populations on the north and south sides of the river ranged from 0.13 to 0.42, rivaling estimates for other small-scale amphibianpopulation

19 studies accounting for greater differences between populations and effectively emphasizing the influence of the Kankakee River as a potential inhibitor to gene flow in this species.

Table 6. Population genetic studies of amphibians using Fst as a measure of genetic difference.

Species Distance Fst No. of loci Source

Ambystoma maculatum 10–650 km 0.08-0.14 15 Julian et al. (2003) (spotted salamander) Rana luteiventris Montana/Idaho 0.008-0.508 6 Funk et al. (2005) (Columbia spotted frog) Rana sylvatica 0.05–5.5 km 0-0.059 6 Newman and Squire (2001) (wood frog) Litoria aurea 1–1000 km 0.034-0.454 4 Burns et al. (2004) (green and golden bell frog) Eurycea cirrigera 1–1032 km 0- 0.500 5 This study (southern two-lined salamander) The population of E. cirrigera located in the forested areas in and near the KRSP represents an isolated population of this species at the extreme margin of its known range. The KRSP population appears to be maintaining its genetic integrity despite barriers to gene flow and habitat challenges. This implies that the dispersal capacity of this species may be adequate at present to avert a population catastrophe secondary to bottlenecks or inbreeding depression and to maintain biodiversity among this species. A study investigating the genetic variability of peripheral populations of Rana latastei (Italian agile frog) as compared to those within the central range did not demonstrate a correlation between genetic variation and population location along the range periphery (Garner 2004). Significant variation was seen among populations at one end of the species range suggesting that the variability may be a consequence of dispersal patterns of the species. However, a similar study on a peripheral (disjunct) population of Emydoidea blandingii

(Blanding’s turtle), a North American fresh water turtle, showed that this population displayed significant variability and may contribute to the overall diversity within the species (Mockford

1999). These studies do not support the theory that in natural populations, species’ diversity

20 should decrease as the range periphery is approached. Instead they offer encouraging information for the proliferation of marginal populations.

The genetic differences observed among the three state populations in this study may be influenced by habitat variations as well as limited gene flow. Subtle differences in the availability of food sources and resources that support biological system functions may influence selection for alleles that will increase fitness within a specific habitat parameter. As additional information is made available about the genomes of amphibian species, examination of specific sites (e.g., areas of the genome that flank genes or proteins with known functions) might provide insights into selection processes at work among populations inhabiting different ecological niches. As- sessing genetic differences among natural populations gives insight to population structuring and species’ ability to adapt environmental changes or stresses.

In light of the results presented here, the stability and persistence of the KRSP subpopulations should be evaluated. Future studies should address the limits of species’ persistence to restricted ecological settings at the extreme margins of their range and the overall role peripheral populations play in species diversity. Populations of amphibians that are subject to variations within their environments and discontinuity within their ranges present conditions in which re- searchers may address the contention between genetic differentiation and species conservatism.

Research efforts aimed at detecting decreases in amphibian population numbers secondary to habitat restrictions will assist in securing the continued proliferation of these species. Manage- ment of the population dynamics of E. cirrigera near and within the KRSP is essential to insure the integrity and conservatism of this peripheral population. Additional genetic studies should explore the influence of gene flow, habitat fragmentation and selection within various habitat parameters experienced by this morphologically-conserved species.

Appendix A

PCR products imaged on gel for locus (primer pair) PcII14 (Connors and Cabe 2003). Lanes 2-6 are samples from KRSP at the 113-4 sampling site, 7-12 are samples from WC-1, 13-16 from WC-2 and 17-18 from WC-3.

2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

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