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 By 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. cin- ereus. I would also like to acknowledge the Illinois Department of Natural Resources for grant- ing 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 popula- tion 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 com- parisons indicated significant differences at all loci between the KRSP and AC samples and sig- nificant 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. 3 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 con- tribute 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 in- fluences 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 al- leles 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). 4 Populations that develop independently while adapting to their isolated habitats can eventu- ally become subject to speciation events. Reproductive isolation secondary to habitat fragmenta- tion 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