Allozymic Variation in the Marsh Rice Rat, Oryzomys palustris, and the White-footed Mouse, Peromyscus leucopus, on the Virginia Barrier Islands and Southern Delmarva Peninsula

ACKNOWLEDGEMENTS

Many thanks to my advisor Dr. John Pagels, who provided his time, constructive criticisms and most importantly the occasional, but necessary, 'motivational talks.' I would especially like to thank Dr. Nancy Moncrief; without her help, this project would not have happened. Dr. Moncrief gave of her time, knowledge, and she provided her lab and all of the equipment needed for this study. I would like to thank my other committee members, Dr. Donald Young, Dr. Bonnie Brown, and Dr. Steven Rein, for their time and suggestions, which were helpful in preparing this thesis. In addition, I appreciate their insights into the design and execution of this project. Also, I thank John Anderson, Sharon Moll, and Matt Barnes, who helped me in the field as well as in the lab and Ellen Compton-Gooding for preparing most of the figures. Similarly, thanks to the Long Term Ecological Research Project on the Virginia barrier islands, which aided in the field portion of this research, in particular Randy Carlson, who was invaluable in the field. I would also like to thank Dr. Ray Dueser, who provided suggestions on this manuscript and insight into the project design. From the University of Virginia, I wish to thank Dr. John Porter who not only helped with my field work on the islands but who also aided in preparing maps of the study area, which were necessary for this thesis. Special thanks to E. A. Forys, D. Giesler, L. Laguerra, and T. Asami, who helped in the field with procurement of animals. Thanks to my friend, roommate, and co-graduate student, Sherry Rinehart, who worked with me in the field and helped me maintain my sanity while traveling to and from Martinsville, Virginia. I also appreciate and thank all of the faculty and staff, as well as the graduate students in the Biology Department at Virginia Commonwealth University, who collectively made my years in Richmond both educational and fun. Finally, I thank my family for their support and love, without which I would not be here today. This study was supported by funding from the Virginia Museum of Natural History and by NSF grants BSR-8702333-06, DEB-9211772, and DEB-9411974. I thank the staff of Eastern Shore of Virginia National Wildlife Refuge, Chincoteague National Wildlife Refuge, and the Virginia Coast Reserve of The Nature Conservancy for their assistance and cooperation. I would like to dedicate this thesis to the memory of my grandfather, H. R. Loxterman, he would be very proud.

Table 1. Summary of genetic variability measures including mean heterozygosity (H), number of expected heterozygotes (Hexp; Nei, 1978), and percent polymorphism (P) for Oryzomys palustris. Allelic frequencies for polymorphic loci are indicated in parentheses. See Appendix for locus abbreviations

Table 2. Matrix of Rogers' (1972) genetic distance measures for Oryzomys palustris (above diagonal) and Peromyscus leucopus (below diagonal) from the Virginia barrier islands and southern Delmarva Peninsula. Peromyscus leucopus does not occur on Parramore and Myrtle islands

Table 3. Results of the F-statistics analysis for each variable locus and estimated rate of gene flow (Nm) for Oryzomys palustris from nine populations combined. Significance of FST indicated by the chi-square value

Table 4. Results of the F-statistics analysis for each variable locus and estimated rate of gene flow (Nm) for Oryzomys palustris from populations in three regions of the Virginia barrier islands and southern Delmarva Peninsula (see text). Significance of FST indicated by the chi-square value

Table 5. Summary of genetic variability measures including mean heterozygosity (H), number of expected heterozygotes (Hexp; Nei, 1978), and percent polymorphism (P) for Peromyscus leucopus. Allelic frequencies for polymorphic loci are indicated in parentheses. See Appendix I for locus abbreviations

Table 6. Results of the F-statistics analysis for each variable locus and estimated rate of gene flow (Nm) for Peromyscus leucopus from seven populations combined. Significance of FST indicated by chi-square value

Table 7. Results of the F-statistics analysis for each variable locus and estimated rate of gene flow (Nm) for Peromyscus leucopus from populations in three regions of the Virginia barrier islands and southern Delmarva Peninsula (see text). Significance of FST indicated by the chi-square value

Figure 1. Sample localities for Oryzomys palustris (sites 1-9) and Peromyscus leucopus (sites 1-7) on the Virginia barrier islands and the southern Delmarva Peninsula

Figure 2. Distribution of allelic frequencies at 6PGD in nine samples of Oryzomys palustris. Relative frequencies of alleles are shown as pie diagrams at each locality

Figure 3. Distribution of allelic frequencies at NP in nine samples of Oryzomys palustris. Relative frequencies of alleles are shown as pie diagrams at each locality

Figure 4. Distribution of allelic frequencies at ADA in nine samples of Oryzomys palustris. Relative frequencies of alleles are shown as pie diagrams at each locality

Figure 5. Distribution of allelic frequencies at PGM3 in nine samples of Oryzomys palustris. Relative frequencies of alleles are shown as pie diagrams at each locality

Figure 6. Phenogram based on UPGMA cluster analysis using Rogers' (1972) genetic distance among nine samples of Oryzomys palustris (cophenetic correlation is 0.898)

Figure 7. Distribution of allelic frequencies at NP in seven samples of Peromyscus leucopus. Relative frequencies of alleles are shown as pie diagrams at each locality

Figure 8. Distribution of allelic frequencies at PGM1 in seven samples of Peromyscus leucopus. Relative frequencies of alleles are shown as pie diagrams at each locality

Figure 9. Distribution of allelic frequencies at G6PD in seven samples of Peromyscus leucopus. Relative frequencies of alleles are shown as pie diagrams at each locality

Figure 10. Distribution of allelic frequencies at MPI in seven samples of Peromyscus leucopus. Relative frequencies of alleles are shown as pie diagrams at each locality

Figure 11. Distribution of allelic frequencies at PEPA in seven samples of Peromyscus leucopus. Relative frequencies of alleles are shown as pie diagrams at each locality

Figure 12. Distribution of allelic frequencies at CK2 in seven samples of Peromyscus leucopus. Relative frequencies of alleles are shown as pie diagrams at each locality

Figure 13. Phenogram based on UPGMA cluster analysis using Rogers' (1972) genetic distance among seven samples of Peromyscus leucopus (cophenetic correlation is 0.879)

ALLOZYMIC VARIATION IN THE MARSH RICE RAT, ORYZOMYS PALUSTRIS, AND THE WHITE-FOOTED MOUSE, PEROMYSCUS LEUCOPUS, ON THE VIRGINIA BARRIER ISLANDS AND SOUTHERN DELMARVA PENINSULA

By Janet Lee Loxterman, M. S.

A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science at Virginia Commonwealth University

Virginia Commonwealth University, 1995

Thesis Director: Dr. John F. Pagels, Professor, Department of Biology

Allozymic variability was examined within and among nine populations of Oryzomys palustris and seven populations of Peromyscus leucopus from the Virginia barrier islands and southern Delmarva Peninsula. Oryzomys palustris is an effective disperser over water and is present on 21 of the 24 islands. In contrast, P. leucopus is a less effective disperser over water and occurs on only four of 24 islands. Of 31 loci, four were variable in O. palustris and six were variable in P. leucopus. For both species, average heterozygosity was lower within the island populations. The nine populations of O. palustris had average heterozygosity of 2.4% and percent polymorphic loci was 6.7%. For seven populations of P. leucopus, the average heterozygosity was 3.6% and percent polymorphic loci was 12.3%. Both species had lower levels of variation among the mainland populations than among the island populations. Populations of P. leucopus exhibited considerable genetic differentiation (FST=0.180) and lower levels of gene flow (Nm=1.14) among populations; whereas O. palustris had moderate levels of differentiation (FST=0.135) and higher levels of gene flow (Nm=1.60) among populations. The potential levels of gene flow and founder effects appear to be the primary evolutionary factors influencing the observed patterns of variation in these species. In addition, the observed levels of gene flow and genetic differentiation among populations of the rice rat and the white-footed mouse appear to be related to their respective abilities to disperse over water.

Since MacArthur and Wilson (1967) formulated the theory of island biogeography, genetic studies of island-dwelling organisms have provided much information pertinent to conservation biology and evolutionary theory. As levels of human-induced habitat fragmentation increase, research into the impact of insularization on the genetic composition of populations is becoming more important for conservation purposes. Much conservation-related research of island populations is concerned with the assertion that levels of genetic variability determine a population's long-term growth rate and success in adapting to changing environmental conditions (Leberg, 1993). Research on island populations also provides insights into evolutionary patterns and processes. Population divergence as a result of geographic isolation on islands is a topic of interest to evolutionary biologists (Wayne et al., 1991). Populations on islands provide a natural model of genetic change in continental populations because island populations are frequently isolated, often smaller in population size than mainland populations, and their geological histories are usually known (Wayne et al., 1991).

Studies involving island populations of mammals have focused largely on population dynamics, often evaluating the patterns and effects of colonization and extinction (e.g., Forys, 1990; Crowell, 1983; Hanski, 1986; Heaney, 1986; Lomolino, 1986), and community structure (e.g., Adler and Wilson, 1985; Lawlor, 1986; Sullivan et al., 1990). To a lesser extent, genetic variation and biogeography have been studied in island populations of mammals (Ashley and Wills, 1987, 1989; Avise et al., 1974; Browne, 1977; Calhoun and Greenbaum, 1991; Aquadro and Kilpatrick, 1981; Selander et al., 1971; Wayne et al., 1991).

The genetic variability of insular populations is affected by several evolutionary forces including genetic drift, inbreeding, selection, mutation, and gene flow (Kilpatrick, 1981). The level of genetic variation (as measured by mean heterozygosity and allelic diversity) in island populations of mammals is usually expected to be lower than it is in mainland populations of similar size (Berry, 1986; Browne, 1977; Kilpatrick, 1981; Wayne et al., 1991). With isolation and increasing geographic distance among populations, there is often a corresponding decrease in gene flow and an increase in the levels of inbreeding and drift (Ashley and Wills, 1987; Wayne et al., 1991). Not surprisingly, species with greater vagility (i.e. ability to disperse) are less affected genetically by the degree of isolation and increasing distance than are less vagile species (Kilpatrick, 1981; Lomolino, 1986). Thus, the degree of subdivision, as a function of dispersal ability, can affect local population dynamics and genetic diversity (Hastings and Harrison, 1994; Weishampel, 1990).

For this study, two small mammal species with potentially different dispersal abilities were compared and contrasted to investigate the evolutionary forces influencing their population genetics on the southern Delmarva Peninsula and Virginia barrier islands. The 24 Virginia barrier islands (Fig. 1) extend 150 kilometers (km) along the seaward margin of the Delmarva Peninsula and are separated by various inlets, marsh islands, tidal flats, and salt marshes (Dueser, 1990). Islands range in size (Appendix I) from 23 ha, Crescent Island (ID) to 7029 ha, Assateague ID, and distance from the mainland (Fig. 1) ranges from less than 1 km (Raccoon ID and Fowling Point) to about 12 km (Cobb ID). The primary vegetation on these islands includes a variety of marsh and dune grasses (Spartina spp.) and shrub thickets; only a few islands support tree species or maritime forests (McCaffrey and Dueser, 1990).

The Virginia barrier island system is subject to frequent storms and islands are often inundated, thus continuously changing the local topology. The dynamic and diverse nature of these islands has prompted considerable research on their flora and fauna. Recent studies have been conducted on the vegetation (McCaffrey and Dueser, 1990; Levy, 1990), reptiles and amphibians (Conant et al., 1990), mammals (Cranford and Maly, 1990; Forys, 1990; Forys and Dueser, 1993; Forys and Moncrief, 1994; Keiper, 1990; Kirkland and Fleming, 1990; Krim et al., 1990), and birds (Beck et al., 1990; Byrd, 1990; Patterson et al., 1990).

Although nine species of non-volant small mammals are known from the Virginia barrier islands, at the time of this study, only two, the marsh rice rat, Oryzomys palustris, and the white-footed mouse, Peromyscus leucopus, were present on enough islands, occurred in sufficient numbers, or were otherwise suitable for broad scale genetic studies. Therefore, only these two species were included in the present analysis.

The rice rat occurs in much of the southeastern United States from Maryland to Florida and west to Texas and Mexico (Wolfe, 1982). The Delmarva Peninsula and Virginia barrier islands represent the northern limit of its distribution (Hall, 1981). Despite its widespread distribution throughout the coastal plain of southeastern North America, the marsh rice rat has been the subject of only two studies examining genetic variation within and among populations (Forys and Moncrief, 1994; Schmidt and Engstrom, 1994). The rice rat inhabits grasslands and wetlands, which are present on all of the Virginia barrier islands, and this small mammal is typically closely associated with water (Forys, 1990; Forys and Dueser, 1993; Wolfe, 1982). Oryzomys palustris has been documented on 21 Virginia barrier and marsh islands: Assateague, Cedar, Chimney Pole, Cobb, Crescent, Fishermans, Fowling Point, Hog, Metompkin, Mink, Mockhorn, Myrtle, Parramore, Raccoon, Revel, Rogue, Ship Shoal, Skidmore, Smith, Wallops and Wreck (Dueser, 1979, 1990, pers. comm.). This semi-aquatic species is a competent swimmer and has been observed crossing up to a 300 m expanse of water, making water an ineffective barrier to dispersal (Esher et al., 1978; Forys and Dueser, 1993). In a study examining swimming behavior in the rice rat, Esher et al. (1978) found that rice rats readily entered water and were observed to swim underwater up to 10 m. In addition, the fur of rice rats is water repellant and traps air, which increases their buoyancy and decreases their heat loss in water (Esher et al., 1978; Wolfe, 1982).

Peromyscus leucopus is not necessarily associated with water and is typically found in wooded areas. At the time of this study, eight of the Virginia barrier islands (Assateague, Cedar, Fishermans, Hog, Mockhorn, Parramore, Smith, and Wallops) supported tree species (McCaffrey and Dueser, 1990); however, P. leucopus has been documented on only four islands: Assateague, Cedar, Fishermans, and Wallops (Dueser, 1990, pers. comm.). Presumably, populations of P. leucopus surrounded by water are isolated from other populations. Savidge (1973) examined homing behavior in P. leucopus and found that mice removed from their capture site had difficulty homing and migrating across a stream. Savidge (1973) concluded that even a small stream (3 m to 4 m) can be an effective natural barrier to homing and dispersal in white-footed mice. The white-footed mouse is widely distributed in the eastern and southern United States, as well as Mexico (Hall, 1981). In contrast to O. palustris, genetic variation within and among populations of P. leucopus has been more extensively examined (Baccus and Wolff, 1989; Browne, 1977; Krohne and Baccus, 1985; Nelson et al., 1987; Robbins et al., 1985; Schnake-Greene et al., 1990).

The purpose of the present study was to characterize allozymic variation within and among populations of O. palustris and P. leucopus from the Virginia barrier islands and the southern Delmarva Peninsula. The primary objectives were to assess relative levels of gene flow and genetic subdivision among subpopulations and to gain insight into evolutionary factors affecting genetic variation in these two species, which apparently have different dispersal abilities.

A total of 118 Oryzomys palustris from nine sites (Fig. 1; sites 1-9) and 96 Peromyscus leucopus from seven sites (Fig. 1; sites 1-7) was live trapped on the southern Delmarva Peninsula and associated Virginia barrier islands in 1989, 1990 and 1993. Sample sizes for each species at each site are given in Appendix I. Representative pairs of island and mainland sites were used to assess the potential relationship between genetic distance and geographic proximity. Also, sampling sites were divided into groups to represent northern, middle, and southern portions of the mainland and adjacent barrier islands to allow analysis of allelic frequency data by region.

Heart, kidney, skeletal muscle, and liver, when available, from each specimen were assayed using horizontal starch gel electrophoresis. Methods for tissue preparation and staining followed those described by Harris and Hopkinson (1976) and Murphy et al. (1990).

Each individual was analyzed for 23 enzyme systems encoded by 31 presumptive gene loci (Appendix II). Numerous side-by-side comparisons of electromorphs were made to confirm relative mobilities. Representatives from several sites were analyzed on a single gel to ensure appropriate assignment of alleles across all samples. For confirmation, individuals from all combinations of sites were repeated on subsequent gels. In addition, each gel contained two repeated individuals, which served as internal controls.

For enzymes with more than one interpretable gene locus the most anodal migrant was designated as locus 1. Alphabetic assignments were made for loci with multiple alleles, with the most common allele usually designated as A. Buffer systems used for each enzyme are listed in Appendix II.

The BIOSYS-1 program (Selander and Swofford, 1981) was used to describe and statistically analyze the results. To ensure that the samples of each species collected in different years (1989, 1990, and 1993) could be pooled for each site in subsequent analyses, cluster analysis was performed using the unweighted pair group method with arithmetic averaging (UPGMA; Sneath and Sokal, 1973) using Rogers' (1972) genetic distance. For each species, individuals from 1989, 1990, and 1993 were designated by sample site and by year as separate operational taxonomic units (OTU's), resulting in a total of fourteen OTU's for O. palustris and twelve OTU's for P. leucopus. For each species, all samples from the same site, regardless of year, clustered together, indicating that the year of capture was not a segregating factor. Therefore, samples of O. palustris and samples of P. leucopus, respectively, were pooled across years for each site to conduct subsequent analyses.

The BIOSYS-1 program was used to compute several

within-population parameters including average heterozygosity (H) estimates using the 'direct count' method, number of expected heterozygotes (Hexp; Nei, 1978), and percent polymorphic loci (P) where the frequency of the most common allele is <95%. In addition, deviation from Hardy-Weinberg equilibrium was calculated using chi-square goodness of fit with Levene's (1949) formula for small samples.

Several among-population measurements were also computed, including Rogers' (1972) genetic distances and F-statistics (FST's).

F-statistics include three measurements: FIS, FIT, and FST, where FIS measures inbreeding in the individuals (I) relative to the subpopulation (S) to which they belong; FIT measures inbreeding in individuals (I) relative to the total population (T); and FST measures inbreeding in subpopulations (S) relative to the total population (T) of which they are a part (Hartl and Clark, 1989). Additionally, a UPGMA cluster analysis (Sneath and Sokal, 1973), using Rogers' (1972) genetic distance, was performed for each species. The relationship between genetic distance and linear, geographic distance (in km) was tested for each species using Mantel's (1967) general regression test.

For calculation of one series of F-statistics, the samples were divided into regions based on geography. For P. leucopus, the North Region included Wattsville and Assateague ID, the Middle Region included Quinby, Cedar ID, and Nassawadox, and the South Region included ESVNWR and Fishermans ID (Fig. 1). The regions for O. palustris were the same as P. leucopus except that the Middle Region also included Parramore ID, and the South Region also included Myrtle ID (Fig. 1). For each species, a total of four series of F-statistics were calculated including: within each region, among all regions, among island samples only, and among mainland samples only, to ascertain the degree of genetic differentiation among subpopulations in each of these groupings (Hartl, 1988). The estimated level of gene flow (Nm) was calculated within each region, among all samples, and among the mainland samples only. In addition, chi-square contingency tests were performed to examine homogeneity of allele frequencies within each region and among all samples.

Oryzomys palustris.-- For the rice rats sampled, 24 of 31 loci were monomorphic for the same allele in all nine samples: ACN2, AK, CK1, CK2, GDA, G6PD, GLUD, GOT1, GOT2, IDH1, IDH2, LDH1, LDH2, MDH1, MDH2, ME, MPI, PEPA, PEPB, PEPD, PEPS, PGI, PGM1, and PGM2 (See Appendix II for enzyme names and Enzyme Commission Numbers). Three loci (ACN1, G3PD, and SOD) were not consistently interpretable, and four loci were polymorphic: PGM3, 6PGD, NP and ADA (Table 1). The mean number of alleles per locus was 1.0 in one sample (Fishermans ID), 1.1 in seven samples (Assateague ID, Cedar ID, ESVNWR, Nassawadox, Parramore ID, Quinby, and Wattsville), and 1.2 in one sample (Myrtle ID), with an average of 1.1 alleles per locus for all nine samples. Percent polymorphic loci (P) for all nine samples ranged from 0.0 to 10.7% and averaged 6.7% (Table 1). Mean heterozygosity (H) averaged 2.4% among all nine samples and ranged from 0.0 to 3.6%. For the five island samples, H and P averaged 2.0% and 5.7%, respectively. For the four mainland samples, H and P averaged 3.0% and 8.0%, respectively.

Chi-square analysis for conformance to Hardy-Weinberg equilibrium using Levene's (1949) correction for small samples (data not shown) revealed that three of nine samples (Assateague ID, Cedar ID, and Quinby) deviated significantly from Hardy-Weinberg expectations at one or more loci. Assateague and Cedar islands were heterozygote deficient at the following loci: 6PGD (Assateague ID), NP (Assateague ID and Cedar ID), and PGM3 (Cedar ID). In contrast, an excess of heterozygotes was observed at ADA in the Quinby sample.

All four variable loci (6PGD, NP, ADA, and PGM3) had noteworthy allelic frequency distributions. At 6PGD (Fig. 2), the B allele was present in the two northern samples, two of the three southern samples (ESVNWR and Myrtle ID), and one of the four middle samples (Quinby). However, the frequency of the 6PGDB allele at Assateague ID was much higher (50%) relative to the other four samples exhibiting the B allele (Table 1). The NPB allele (Fig. 3) was detected in all four mainland samples and in three of five island samples (Assateague ID, Cedar ID, and Myrtle ID). In general, the frequencies of the NPB allele were higher in the four mainland samples than the frequencies of this allele in the island samples (Table 1). Three alleles were present at the ADA locus (Fig. 4); but neither the B nor the C allele was present at Fishermans ID. The ADAB allele was present in both of the northern samples and all four of the middle samples, but occurred in only one of the three southern samples (Myrtle ID). In addition, the frequency of the ADAC allele was about 30% in five samples (ESVNWR, Myrtle ID, Nassawadox, Parramore ID, and Wattsville; Table 1). At PGM3 (Fig. 5), the PGM3B allele occurred only in two island samples (Cedar ID and Myrtle ID) and was present in relatively low frequencies (Table 1).

Rogers' (1972) genetic distance measures ranged from 0.006 (between Myrtle ID and Wattsville and also between Myrtle ID and Parramore ID) to 0.039 (between Assateague ID and Fishermans ID), and averaged 0.016 for all nine samples (Table 2). The phenogram based on UPGMA cluster analysis of Rogers' (1972) genetic distance (Fig. 6) indicated that the northernmost island (Assateague ID), and the southernmost island (Fishermans ID), were the most genetically divergent of the rice rat samples. Also, a mainland sample (Quinby) was grouped together with the three most similar samples (Myrtle ID, Parramore ID, and Wattsville). Nassawadox and ESVNWR, on the mainland, were grouped with a middle island sample (Cedar ID). A Mantel (1967) test using Rogers' (1972) genetic distance (Table 2) indicated that genetic distance and geographic distance (in km) were significantly associated in these samples of rice rats (r=0.39, p=0.018).

Analysis of Rogers' (1972) genetic distance measures indicated less average genetic distance among the four mainland samples (0.010) relative to average distance among the five island samples (0.021). The average genetic distance between pairs of mainland and island samples was 0.015, which was intermediate between the mainland-to-mainland average and the island-to-island average (Table 2).

F-statistics revealed significant allele frequency differences for rice rats at 6PGD and ADA when all nine samples were combined (Table 3), at 6PGD in the North Region (Table 4), and at ADA in the South Region (Table 4). In addition, differences averaged for all variable loci were significant in the North Region (Table 4), the South Region (Table 4), and when all samples were combined (Table 3). Heterogeneity of allele frequencies was not significant for any variable locus in the Middle Region (Table 4).

Furthermore, results of the F-statistic analyses of all nine samples combined indicated that only 13.5% (FST= 0.135) of the total variance of allele frequencies was due to the genetic differences among samples (Table 3). Thus, approximately 87% of the total genetic diversity in these samples of rice rats is found within any one sample, indicating moderate genetic differentiation and moderate to high levels of gene flow among samples (Chesser, 1983; Wayne et al., 1991). In addition, the average FST (0.054) calculated among the four mainland samples only was much lower than the average FST (0.187) calculated among the five island samples only (data not shown).

The high positive mean values for FIT suggested a deficiency of heterozygous individuals among all nine samples combined (Table 3), as well as in the North and Middle regions (Table 4). This is in agreement with the chi-square analysis for the North Region. Although there was an excess of heterozygotes at the ADA locus in one Middle Region sample (Quinby), Cedar ID (Middle Region) was heterozygote deficient at two loci (NP and PGM3), which may explain the high FIT for the Middle Region (Table 4). The high positive mean FIS values in the North Region indicated a deficiency of heterozygotes within the two samples in this region (Table 4), which was also reflected in the chi-square analysis. However, the overall average FIS (0.082) indicated only a slight deviation from random mating (FIS=0.0) for all nine populations combined (Table 3).

In rice rats, Nm among all nine samples (Table 3) was 1.60 migrants per generation, indicating moderate to high levels of gene flow. The values for Nm computed within each region were also greater than 1.0, suggesting high levels of gene flow within each of the three regions. In fact, the Middle Region had an Nm of 4.30 (Table 4), and Nm calculated among the four mainland samples only was 4.40 migrants per generation (data not shown). In general, values of Nm greater than 1.0 indicate that levels of gene flow are sufficient enough to offset random genetic drift, which often leads to population divergence (Wright, 1931; Slatkin, 1985), and values of Nm>4.0 are indicative of panmixia (Wright, 1967).

Peromyscus leucopus.-- White-footed mice exhibited variation at six of 31 loci: G6PD, PGM1, PEPA, MPI, NP, and CK2 (Table 5). Twenty-three were monomorphic for the same allele among all seven samples: ACN1, ACN2, ADA, AK, CK1, GDA, GLUD, GOT1, GOT2, IDH1, IDH2, LDH1, LDH2, MDH1, MDH2, ME, PEPB, PEPD, PEPS, 6PGD, PGI, PGM2, and PGM3. G3PD and SOD were not consistently interpretable. The mean number of alleles per locus was 1.1 in three samples (Assateague ID, ESVNWR, and Fishermans ID) and 1.2 in four samples (Cedar ID, Nassawadox, Quinby, and Wattsville), with an average 1.2 alleles per locus for all seven samples. Percent polymorphic loci (P) ranged from 6.9 to 20.7% and averaged 12.3% (Table 5). Mean heterozygosity (H) ranged from 0.9 to 5.7% and averaged 3.6% for all seven samples (Table 5). For the three island samples, H and P averaged 2.7% and 12.6%, respectively. The four mainland samples averaged 4.2% and 12.0% for H and P, respectively.

Chi-square analysis for conformance to Hardy-Weinberg equilibrium using Levene's (1949) correction for small samples (data not shown) revealed that five of seven samples deviated significantly from Hardy-Weinberg expectations at one or more loci: Cedar ID, ESVNWR, Fishermans ID, Nassawadox, and Wattsville. These samples were heterozygote deficient at the following loci: PGM1 (Cedar ID, ESVNWR, Nassawadox, and Wattsville) and PEPA (Cedar ID and Fishermans ID).

In white-footed mice, all six variable loci had noteworthy patterns of variation in allelic frequencies. At the NP locus (Fig. 7), the B allele was present in all samples, however, the frequency of the B allele was much lower at Fishermans ID (4%; Table 5). The NPC allele was present only on the mainland in three of the four samples (Nassawadox, Quinby, and Wattsville). At PGM1 (Fig. 8), the PGM1B allele was present only in two island samples (Assateague ID and Cedar ID); in contrast, the PGM1C allele was present in three of four mainland samples (ESVNWR, Nassawadox, and Wattsville). The G6PDB allele (Fig. 9) was present in the Middle Region only, with a higher frequency on Cedar Island, relative to the two mainland samples (Nassawadox and Quinby; Table 5). At the MPI locus (Fig. 10), four alleles were present. The MPID allele was only found in two mainland samples, ESVNWR and Nassawadox. The MPIB allele occurred in five of seven samples; it was absent at Assateague ID and Fishermans ID. The MPIC allele was present in two mainland samples (Nassawadox and Wattsville) and two island samples (Assateague ID and Fishermans ID). At the PEPA locus (Fig. 11), the B allele occurred in all samples except one southern mainland sample (ESVNWR) and one middle mainland sample (Nassawadox). At the CK2 locus (Fig. 12), only three samples, two mainland (Quinby and Wattsville) and one island (Cedar ID), exhibited the B allele.

Rogers' (1972) genetic distance measures ranged from 0.015 (between Nassawadox and Wattsville) to 0.065 (between Cedar ID and Fishermans ID) with an average genetic distance of 0.035 (Table 2). The phenogram based on UPGMA cluster analysis of Rogers' (1972) genetic distance (Fig. 13) indicated that Cedar ID was the most genetically divergent sample of P. leucopus. In addition, the most genetically similar samples in white-footed mice were two mainland samples (Nassawadox and Wattsville). In contrast to O. palustris, the Mantel (1967) regression test using Rogers' (1972) genetic distance and geographic distance (in km) indicated no significant association between the two parameters in P. leucopus (r=-0.08, p=0.719).

The average genetic distance between the four mainland samples was 0.028, while the average among the three island samples was 0.045 (Table 2). As in rice rats, the average genetic distance between pairs of mainland and island samples (0.036) was intermediate to averages of mainland-to-mainland pairs of samples and island-to-island pairs of samples (Table 2).

F-statistics revealed significant differences in allele frequencies at all variable loci, except CK2, when the seven samples were combined (Table 6). In the analysis by region, the differentiation of allele frequencies was significant at MPI in the North Region; G6PD, PGM1, PEPA, MPI, and NP in the Middle Region; and PGM1 and NP in the South Region (Table 7). In addition, differences in allele frequencies were significant when all loci were combined in all three regions (Table 7), as well as when all samples were combined (Table 6).

The F-statistic analyses (FST's) for all seven samples of P. leucopus (Table 6) indicated that 18.0% (FST= 0.180) of the total variance of allelic frequencies was due to genetic differences among samples. Therefore, 82% of the total genetic diversity can be found within any one sample of white-footed mice, indicating considerable genetic differentiation and low levels of gene flow (Wright, 1978). The FST value for the four mainland samples only averaged 0.106, which was half of the average 0.220 for the three island samples only (data not shown).

The high positive mean values of FIT in the Middle and South regions (Table 7), and when all samples were combined (Table 6), indicated a deficiency in heterozygous individuals among all of the samples combined. These FIT values were in accordance with the chi-square analysis, in which five of the seven samples deviated significantly from Hardy-Weinberg expectations. Similarly, the mean values of FIS in the Middle and South regions suggested a heterozygote deficiency within the samples in these regions (Table 7). This level of differentiation was also apparent in the

chi-square analysis to test the deviation from Hardy-Weinberg expectations.

In white-footed mice, the values of Nm in the North and Middle regions were greater than 1.0 (Table 7), suggesting moderate levels of gene flow within these regions (Wright, 1978). However, Nm was 0.06 in the South Region (Table 7), indicating lower levels of gene flow in the South Region, relative to the North and Middle regions. Among the four mainland samples only, the average level of gene flow was 2.11 migrants per generation (data not shown). Overall, Nm was 1.14 migrants per generation (Table 6), indicating low to moderate levels of gene flow among all seven samples (Wright, 1978).

Oryzomys palustris.-- Average heterozygosity of O.palustris is lower (2.4%; Table 1) than both Schmidt and Engstrom's (1994) value of 4.1% and Forys and Moncrief's (1994) value of 5.0%. In the present study, values for H and P, when calculated separately for the island samples (5.7% and 2.0%, respectively) and mainland samples (8.0% and 3.0%, respectively), indicated higher levels of heterozygosity and polymorphism among the mainland samples. The different values reported by Forys and Moncrief (1994) may be explained in part by the number of loci examined. Their estimate was based on fewer loci (they analyzed only blood) than were analyzed in this study. In Schmidt and Engstrom's (1994) study, the area they examined, which does not include islands, may explain differences in average heterozygosity. Schmidt and Engstrom (1994) examined 23 populations of O. palustris, some of which were separated by more than 900 km.

Forys and Moncrief (1994) reported a mean FST of 0.082 in O.

palustris populations from the Virginia barrier islands and southern Delmarva Peninsula. This value (0.082) is of the same order of magnitude as the value of 0.055 for populations examined from the same general area (Middle Region) in this study (Table 4). Schmidt and Engstrom (1994) reported a much higher FST (0.24) for O. palustris from three regions in the southern United States and Mexico: they studied three populations from Georgia (region 1), thirteen populations from eastern Texas (region 2), and seven populations from southeastern Texas and northeastern Mexico (region 3). They found that populations in region 3 were linearly restricted to available coastal habitat and had an FST of 0.370, which subsequently inflated their overall average. Populations restricted to one-dimensional habitats would be expected to undergo greater divergence than populations in two-dimensional habitats (Hartl and Clark, 1989). Rice rats in Schmidt and Engstrom's (1994) region 2 were less subdivided by habitat, resulting in an average FST of 0.166, a value slightly higher than the average 0.135 (Table 3) reported in this study for nine populations of rice rats from the Virginia barrier islands and southern Delmarva Peninsula.

In Schmidt and Engstrom's study (1994), recalculation of estimated gene flow using only the thirteen populations of rice rats from region 2 (data not shown) yielded a value greater than 1.0 (Nm=1.26). Forys and Moncrief (1994) also reported an estimated rate of gene flow greater than 1.0 (Nm=2.8). Similarly, the estimated rate of gene flow for O. palustris in the present study was 1.60 migrants per generation, indicating that the rate of gene flow among populations of rice rats from the Virginia barrier islands and southern Delmarva Peninsula is high enough to offset the potential effects of random genetic drift. Moreover, among the four mainland samples only (Nm=4.40) and within the Middle Region (Nm=4.30), the level of gene flow is indicative of panmixia (Hartl, 1988). The Nm found in the Middle Region of the present study is higher than that (Nm=2.8) of Forys and Moncrief (1994) from the same general area.

Results from the Mantel (1967) test indicated a significant association between genetic and geographic distances. This was also reflected in the phenogram (Fig. 6), in which the two most genetically divergent samples (Assateague ID and Fishermans ID) were also the most geographically distant sites (Fig. 1). The average genetic distance for O. palustris from the Virginia barrier islands and southern Delmarva Peninsula (0.016; Table 2) was lower than the average distance for populations of O. palustris from Texas and Mexico (0.054; Schmidt and Engstrom, 1994). These higher average genetic distance values documented among populations in Schmidt and Engstrom's (1994) study are not unexpected considering the large geographic extent (>900 km) of their study area.

Peromyscus leucopus.-- Average heterozygosity (3.6%; Table 5) was lower than values reported for white-footed mice from southwestern Virginia, 15.8% (Baccus and Wolff, 1989), central Oklahoma, 7.5% (Nelson et al., 1987), the eastern United States and Mexico, 9.1% (Robbins et al., 1985), and southwestern Missouri, 7.8% (Schnake-Greene et al., 1990). Like O. palustris, the degree of geographic isolation among P. leucopus populations in the present study relative to previous studies may explain differences in average heterozygosity.

When examined separately, average H and P for the island samples (2.7% and 12.6%, respectively) and mainland samples (4.2% and 12.0%, respectively) resulted in a pattern similar to that reported in Browne's (1977) study of island and mainland populations of P. leucopus from Ohio and Canada. In that study, the mean heterozygosity and percent polymorphic loci were 7.1% and 21.4%, respectively, for the island populations, and 8.0% and 32.1%, respectively, for the mainland populations. Both this study and Browne's (1977) had higher H estimates in the mainland samples than in the island samples. However, the P value in this study was slightly higher for the island samples; whereas Browne (1977) found a higher P in his mainland samples. Browne's (1977) consideration that a locus is polymorphic if any variation is detected yielded a less conservative calculation of polymorphism than that used in this study. Island studies of other Peromyscus species including P. maniculatus (Aquadro and Kilpatrick, 1981), P. eremicus (Avise et al., 1974), P. gossypinus (Boone et al., 1993), and P. polionotus (Selander et al., 1971) have also reported lower heterozygosity in island populations than in mainland populations. In addition, a mtDNA study of P. maniculatus on the California Channel Islands concluded that heterogeneity was much lower in the island populations than in the mainland populations (Ashley and Wills, 1987, 1989).

Values for FST (0.180) in this study indicated that considerable population divergence has occurred among the seven P. leucopus populations from the Virginia barrier islands and southern Delmarva Peninsula (Table 6). A similar value was reported for four populations of P. leucopus from southwestern Missouri (0.143, Schnake-Greene et al., 1990). Although Schnake-Greene et al. (1990) offer no causative explanation, they suggest that genetic drift may be prompting differentiation in white-footed mice from southwestern Missouri. Krohne and Baccus (1985) reported a slightly lower FST value for populations from Indiana (0.094) than the overall FST calculated in the present study. Krohne and Baccus (1985) examined three populations of P. leucopus from contiguous habitat in the center of their study area and two populations that were separated from the other three, one by a 90 m gorge and one by a temporal stream. The FST for the three contiguous samples was much lower (0.021) than the average for all five samples (0.094, Krohne and Baccus, 1985). They concluded that the overall degree of genetic differentiation they observed was the result of the two separated samples (Krohne and Baccus, 1985).

Values of FST comparable to estimates in this study have also been reported for island populations of P. maniculatus (0.158, Avise et al., 1979; 0.156, Calhoun and Greenbaum, 1991) and P. gossypinus (0.219; Boone et al., 1993). Calhoun and Greenbaum (1991) reported a substantially lower FST in mainland populations of P. maniculatus (0.048) versus island populations (0.200) and concluded that the potential level of gene flow (Nm=0.05) was the major factor influencing their results. The values in this study indicate a similar trend with a mainland FST of 0.106 versus a much higher island FST of 0.220. As in the P. maniculatus populations examined by Calhoun and Greenbaum (1991), high levels of genetic drift are likely due to low levels of dispersal and hence lower levels of gene flow among P. leucopus populations in this study.

The overall level of gene flow (Nm) for white-footed mice in this study (1.14; Table 6) was lower than Nm=1.50, the value calculated for four populations of P. leucopus from southwestern Missouri (Schnake-Greene et al., 1990). In addition, the value for white-footed mice from the Virginia barrier islands and southern Delmarva Peninsula was also lower than the estimated rate of gene flow for five populations of white-footed mice sampled in Indiana (2.41; Krohne and Baccus, 1985). Calculating the rate of gene flow for the three contiguous populations in Krohne and Baccus's (1985) study yields a much higher Nm of 11.65. However, the value of 2.41 (Krohne and Baccus, 1985) is comparable to the average rate of gene flow calculated for only the mainland populations of P. leucopus in this study (Nm=2.11).

The Mantel (1967) test indicated no association between genetic and geographic distance, which is also reflected in the cluster analysis (Fig. 13). The phenogram did not exhibit a clear pattern between genetic distance and geographic proximity. The average genetic distance for populations of P. leucopus from the Virginia barrier islands and southern Delmarva Peninsula (0.035; Table 2) is lower than reported averages for mainland populations of P. leucopus (>0.10, Robbins et al., 1985; 0.06, Schnake-Greene et al., 1990). Lower average genetic distance reported in this study corresponds to the smaller study area examined relative to the area examined by Robbins et al. (>1000 km; 1985) and Schnake-Greene et al. (> 125 km; 1990).

Comparison of O. palustris and P. leucopus.-- Populations of P. leucopus from the Virginia barrier islands and southern Delmarva Peninsula are more genetically differentiated from one another than are populations of O. palustris. Average Rogers' (1972) genetic distance for P. leucopus was higher than the average genetic distance for O. palustris (Table 2). Furthermore, for both species the average distance was lowest between mainland-mainland samples and highest between island-island samples.

In a review of the genetics of insular populations of mammals, Kilpatrick (1981) stated that percent polymorphic loci and heterozygosity should increase with increasing island size and with decreasing distance to the mainland. Contrary to findings reported by Kilpatrick (1981), no relationship was detected in this study between island size (Appendix I), distance to mainland, and percent polymorphism. For example, in O. palustris, the population from the smallest island (Myrtle ID) had levels of heterozygosity comparable to those on the other islands. Similar levels of heterozygosity among all five island populations indicates that gene flow is occurring among island populations of the rice rat, thereby maintaining heterozygotes in these populations. In P. leucopus, the highest levels of heterozygosity and polymorphism were found in white-footed mice from Cedar ID, which was not the largest island sampled. This may be explained by the dynamic nature of this island. Cedar Island is frequently overwashed and the white-footed mouse population there may reflect recent recolonizing events, whereas the other island populations of the white-footed mouse have likely been isolated for a longer period of time. In both species, the populations from the island closest to the mainland (Fishermans ID) had the lowest levels of polymorphism (Tables 1 and 5). However, actual distance to the mainland does not take into account the nature of the area surrounding the islands. Although Fishermans ID is closest to the mainland, the area surrounding many of the other islands is quite marshy, thus providing stepping stones for animals dispersing to the islands. Therefore, actual distance to the mainland is probably not as important as characteristics of the area separating the islands (i.e. open, deep water versus shallow marshes) from the mainland.

The indicators that provide the most insight into the genetic structure of these populations are the F-statistics, which facilitate estimation of gene flow (Nm). In general, an FST value that falls between 0.05 and 0.15 implies moderate genetic differentiation, whereas a value between 0.15 and 0.25 implies considerable genetic differentiation among subpopulations (Wright, 1978). Results of the overall FST analysis in both white-footed mice and rice rats suggest that genetic divergence has occurred among populations of both species (Wright, 1978). For rice rats, the FST (0.135) value indicates moderate levels of differentiation among populations (Wright, 1978). In contrast, the FST (0.180) for the white-footed mice populations suggests considerable genetic differentiation among populations (Wright, 1978). Again, these values support the contention that the rice rat populations are less genetically divergent than are populations of the white-footed mouse.

The values of FST for both species in this study were lower than those reported for other island populations of mammals, including the masked shrew (Sorex cinereus) from Atlantic Canada (24%; Stewart and Baker, 1992) and the island fox (Urocyon littoralis) from California (56%; Wayne et al., 1991). These authors attributed the levels of genetic differentiation observed in their studies primarily to genetic drift due to small population size (Stewart and Baker, 1992; Wayne et al., 1991), founder effects (Wayne et al., 1991), and reduced levels of gene flow (Stewart and Baker, 1992; Wayne et al., 1991). For populations of O. palustris and P. leucopus from the Virginia barrier islands and southern Delmarva Peninsula, founder effects and the level of gene flow are likely the major forces influencing the observed patterns of genetic variation.

Kilpatrick (1981) noted that founder effects are extremely important in determining the genetic characteristics of insular populations. Considering the average heterozygosity levels observed in both rice rats and white-footed mice (Tables 1 and 5), inbreeding is probably not an important factor in the genetic structuring of these populations. The moderate (O. palustris) to considerable (P. leucopus) degree of differentiation among populations is evidence for founder effect (Kilpatrick, 1981; Stewart and Baker, 1992). With increased incidence of founder effects, corresponding increases in the level of differentiation occur, as observed in white-footed mice (Stewart and Baker, 1992).

Comparisons of island and mainland FST's for rice rats and

white-footed mice indicate higher levels of genetic differentiation among the island populations of both species. The average FST of 0.220 for island populations of P. leucopus was higher than the average of 0.187 for island populations O. palustris. Moreover, the mainland FST in P.leucopus (0.106) was also higher than the mainland value for O. palustris (0.054). Increased genetic divergence and reduced gene flow among mainland populations of white-footed mice may be a result of long-term agricultural development on the southern Delmarva Peninsula, which has fragmented and decreased the available woodland habitat (Brown and Craver, 1985).

In the Middle and South regions, levels of gene flow (Nm) for rice rats (Table 4) were higher than those for white-footed mice (Table 7). The overall estimated rate of gene flow was also higher for O. palustris (Table 3) than for P. leucopus (Table 6). In addition, in the Middle Region and among the mainland populations only the levels of gene flow among rice rat populations are indicative of panmixia. The higher rate of gene flow among rice rat populations supports the hypothesis that rice rats are able to disperse over water more effectively than white-footed mice, which subsequently results in increased homogeneity among rice rat populations when compared with white-footed mouse populations.

The ubiquity of the rice rat on the islands is related not only to its ability to disperse, but also to its abundance on the mainland. Oryzomys palustris is the most common small mammal species in mainland salt marshes (Dueser pers. comm.). This fact, coupled with their natural history and behavior, probably results in relatively frequent passive transport of rice rats to nearby islands, due to tidal fluctuations and flooding. The natural history and habitat requirements of white-footed mice makes passive transport, as a result of tidal fluctuations and flooding, much less likely.

Additionally, the Mantel (1967) test indicated a significant association between genetic distance and geographic distance in populations of O. palustris. In contrast, there was not a significant relationship between these two variables in populations of P. leucopus. The significant relationship between geographic distance and genetic distance for rice rats reflects high levels of gene flow among rice rat populations. For white-footed mouse populations, reduced gene flow and increased differentiation among populations corresponds to a lack of association between geographic and genetic distances. In conjunction with the FST and Nm results, the results of the Mantel (1967) test provide strong evidence for increased genetic differentiation within and among populations of P. leucopus and provides evidence supporting the contention that water is a barrier to dispersal in this species. Conversely, relatively higher homogeneity within and among populations of O. palustris as indicated by a significant Mantel test and high Nm in the present study, indicates that water is not a significant barrier to dispersal in rice rats.

In summary, the data for P. leucopus in this study support observations that species with reduced ability to disperse undergo greater genetic differentiation among subpopulations, as a result of population subdivision. In contrast, species such as O. palustris, with greater ability to disperse and, hence, higher levels of gene flow, are less affected by population subdivision and undergo less genetic differentiation among subpopulations. By comparing and contrasting two species with differing abilities to disperse, this study provides evidence that gene flow is an important evolutionary force that counteracts the genetic effects of population subdivision.

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Numerals in parentheses following each locality indicate the sample number (underlined) and the number of specimens examined, respectively. A brief description of the trapping site follows each locality. Area measured in hectares, of islands are as follows: Assateague ID, 7029; Cedar ID, 868; Parramore ID, 2197; Myrtle ID, 407; and Fishermans ID, 453.

Oryzomys palustris

VIRGINIA: Accomack Co.:

Assateague Island, Chincoteague National Wildlife Refuge, 5 km NE Chincoteague National Wildlife Refuge Office (6, 10);

-- southwestern portion of the island; mesic grassland with some brush

1.5 km S, 2 km E Wattsville (5, 13);

-- open grass area, interspersed with brambles and poke weed

Cedar Island, (4, 12);

-- northwest portion of island; bayside salt marsh

5 km N Quinby (1, 12);

-- mainland salt marsh; bayside

Parramore Island, (8, 13);

-- northern portion of island; bayside salt marsh and inland to brackish marsh

VIRGINIA: Northampton Co.:

3.3 km E Nassawadox (7, 10);

-- salt marsh along hammocks

Myrtle Island, (9, 25);

-- northeast portion of island; along Ship Shoal Inlet; foredune grassland and over dune ridge into bayside salt marsh

Eastern Shore of Virginia National Wildlife Refuge (2, 10);

-- mainland salt marsh; bayside

Fishermans Island National Wildlife Refuge, (3, 13);

-- northeast portion of island; bayside salt marsh

Peromyscus leucopus

VIRGINIA: Accomack Co.:

Assateague Island, Chincoteague National Wildlife Refuge, 5.2 km NE Chincoteague National Wildlife Refuge Office (6, 13);

-- southwestern portion of island; along edge of thickets;

pine-hardwood complex

1.5 km S, 2 km E Wattsville (5, 9);

-- hardwood dominated woodland; along dense edge habitat

Cedar Island, (4, 11);

-- northeast portion of island along Metompkin Inlet; tall thicket with juniper, cherry and loblolly pine

5 km N Quinby (1, 13);

-- mixed pine and hardwood woodland; xeric edge of brushy field

VIRGINIA: Northampton Co.:

3.3 km E Nassawadox (7, 24);

-- edge along farm road and fence row; dense grass and shrubs with sparse hardwoods; along the edge of hardwood hammocks

Eastern Shore of Virginia National Wildlife Refuge (2, 15);

-- hardwood woodland with shrubby understory; dense edge of woodland

Fishermans Island National Wildlife Refuge, (3, 11);

-- northwestern portion of island; scrub thicket along jeep trail

Buffer Systems and Enzymes

Buffer systems and enzymes used to analyze Oryzomys palustris and Peromyscus leucopus were as follows: tris-citrate, pH 8.0 (TC8) for peptidase A (valyl-leucine used as substrate; PEPA, Enzyme Commission number 3.4.11), peptidase B (leucyl-glycyl-glycine used as substrate; PEPB, 3.4.11), peptidase D (phenylalanyl-proline used as substrate; PEPD, 3.4.13.9), peptidase S (valyl-leucine or leucyl-glycyl-glycine used as substrate; PEPS, 3.4.11), adenylate kinase (AK, 2.7.4.3), creatine kinase (CK, 2.7.3.2), glutamate dehydrogenase (GLUD, 1.4.1.3), adenosine deaminase (ADA, 3.5.4.4), guanine deaminase (GDA, 3.5.4.3), glycerol-3-phosphate dehydrogenase (G3PD, 1.1.1.8); tris-citrate, pH 7.0 (TC7) for malic enzyme (ME, 1.1.1.40), malate dehydrogenase (MDH-1, -2, 1.1.1.37), phosphoglucose isomerase (PGI, 5.3.1.9), nucleoside phosphorylase (NP, 2.4.2.1), glutamate oxaloacetate transaminase (GOT-1, -2, 2.6.1.1), lactate dehydrogenase (LDH-1, -2, 1.1.1.27), aconitase (ACN-1, 4.2.1.3), glucose-6-phosphate dehydrogenase (G6PD, 1.1.1.49), phosphoglucomutase (PGM-1, -2, -3, 2.7.5.1), 6-phosphogluconate dehydrogenase (6PGD, 1.1.1.44), isocitrate dehydrogenase (IDH-1, -2, 1.1.1.42), and mannose phosphate isomerase (MPI, 5.3.1.8), superoxide dismutase (SOD-1, -2, 1.15.1.1).

Janet Lee Loxterman was born on May 28, 1970 in Rochester, New York. She graduated in 1988 from Seneca Valley High School, Harmony, Pennsylvania. In 1992, she received her Bachelor of Science in Behavioral Neuroscience from Lehigh University, Bethlehem, Pennsylvania. Upon completion of her undergraduate education, she entered Virginia Commonwealth University to earn her Master of Science in Biology. She hopes to eventually return to school for her Doctorate.