spatial and temporal variability of microgeographic genetic structure in ...

SPATIAL AND TEMPORAL VARIABILITY OF MICROGEOGRAPHIC GENETIC STRUCTURE IN WHITE-TAILED DEER KIM

T.

SCRIBNER, MICHAEL

H. SMITH, AND RONALD K. CHESSER

Department of Zoology, University of Georgia, Athens, GA 30602, and Savannah River Ecology Laboratory, Drawer E, Aiken, SC 29802 (KTS) Department of Genetics, Institute of Ecology, School of Forest Resources, University of Georgia, Athens, GA 30602, and Savannah River Ecology Laboratory, Drawer E. Aiken, SC 29802 (MRS) Department of Genetics and Institute of Ecology, University of Georgia, Athens, GA 30602, and Savannah River Ecology Laboratory, Drawer E. Aiken, SC 29802 (RKC) Present address of KTS: Alaska Science Center. Biological Resources Division. United States Geological Survey, 1011 East Tudor Road, Anchorage. AK 99503

Techniques are described that define contiguous genetic subpopulations of white-tailed deer (Odocoileus virginianus) based on the spatial dispersion of 4,749 individuals that possessed discrete character values (alleles or genotypes)'during each of6 years (1974-1979). Whitetailed deer were not uniformly distributed in space, but exhibited considerable spatial genetic structuring. Significant non-random clusters of individuals were documented during each year based on specific alleles and genotypes at the Sdh locus. Considerable temporal variation was observed in the position and genetic composition of specific clusters, which reflected changes in allele frequency in small geographic areas. The position of clusters did not consistently correspond with traditional management boundaries based on major discontinuities in habitat (swamp versus upland) and hunt compartments that were defined by roads and streams. Spatio-temporal stability of observed genetic contiguous clusters was interpreted relative to method and intensity of harvest, movements, and breeding ecology. Key words: Odocoileus virginianus, white-tailed deer, genetics, management boundaries, population ecology, spatial subdivision Populations of most manunalian species are not randomly distributed in space but exhibit considerable geographic structuring. The degree of structuring varies from species to species relative to life-history characteristics, ecological requirements, vagility and degree of habitat fragmentation, mating behavior, and degree of sociality. Large species that are highly mobile. such as ungulates, would be expected to show little spatial structuring at microgeographic scales due to their potential to disperse, large home ranges, and in certain instances, seasonal patterns of migration. Yet many species of ungulates exhibit considerable site fidelity and philopatry (Garrott et aI .• 1987; Mathews and Porter, 1993; Nelson and Mech. 1987). Journal of Mamma/olm 78(3):744-755, 1997

Over large geographic areas, recognition of distinct subspecies, races, or geographic populations implies some degree of reproductive isolation. Direct techniques such as resightings of collared animals (Bartman and Steinert, 1981) and telemetry (Garrott et aI., 1987; Nelson and Mech, 1987) have revealed much of the current information on movements and geographic structuring of populations of ungulates. Unfortunately, most of these studies simply documented the presence or absence of interpopulational movements of individuals without actually determining if animals contributed reproductively to another population. In the absence of readily identifiable traits, or adequate data on movements, assessment of the degree of population structuring becomes 744

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problematic. relying on perceived barriers to dispersal (i.e., water courses or topographic relieO or simply the relative proximity of populations. One augmentative approach to defining population structuring involves the collection of genetic data. Geographic variation in the frequency of heritable genetic markers may provide an indirect measure of gene flow and, thus, the degree of spatial structuring. Many species of ungulates.exhibit spatial genetic structuring (e.g., white-tailed deer, Odocoileus virginianus-Chesser et aI., 1982b; Mathews and Porter, 1993; Sheffield et aI., 1985; red deer, Cervus elephus--Gyllensten et al., 1980; reindeer, Rangifer tarandus-Roed, 1985; moose, Alces alces-Chesser et aI., 1982a; Ryman et aI., 1977, 1980), despite their capacity to disperse over large distances. Most genetic studies of ungulates have focused on macrogeographic variation by comparing popUlations that were separated by distances beyond the dispersal capabilities of the species. Little attention has been focused on the degree to which populations are structured over short distances (i.e., within single-generation dispersal distances), the stability of this structure over time, or the degree of concordance between spatial dispersion of genetically distinguishable subdivisions and boundaries of a popUlation that have been defined based on direct observation or other traditional criteria. Our null hypothesis was that white-tailed deer are panmictic or randomly distributed in space with regard to genotype. Under this scenario, we would not expect to observe evidence for significant microgeographic genetic structuring. Previous studies of the species' behavioral ecology (Hawkins and Klimstra, 1970; Hirth, 1977) and patterns of movement (Mathews and Porter, 1993; Nelson, 1993; Nelson and Mech, 1987) suggest a series of working hypotheses on how populations of whitetailed deer may be spatially structured and the resiliency of this structure over time. Using a novel analytical approach that

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makes no a priori assumptions of how individuals are spatially dispersed, we examine microgeographic genetic structuring of white-tailed deer. Our specific objectives were to 1) identify non-random genetic aggregations of individuals that were spatially contiguous, 2) determine degree of stability of observed genetic associations over time in terms of location and genetic composition, 3) assess concordance between the geographic position of genetic associations and traditional boundaries that were established based on major discontinuities in habitat or on the road network in the study area, and 4) relate location and stability of genetic associations to harvest conditions, movements, and breeding ecology. STUDY AREA

Our study was conducted on the United States Department of Energy's Savannah River Site (ca. 800 lcm2 ) in westcentral South Carolina, bordering the Savannah River (33°17'N, 81°40'W). Vegetation varied based on soil type and topographic relief. Plantations of pines (Pinus) and oaks (Quercus) dominated well-drained upland areas. Swamp and bottomland hardwoods fonned principal overs tory vegetation along riparian corridors and the Savannah River. The Savannah River Site supported a large population of white-tailed deer that has been harvested annually since 1965 and studied intensively since 1971 (Novak et aI., 1991; Scribner et aI., 1985; Urbston, 1967). Pre-hunt size of the population varied greatly from year to year (3, 621-5, 368 or 4.7-6.7Ikm' over 1965-1985-Novak et al., 1991) as a function of annual variation in hunter effort. Hunts were highly controlled by personnel from the United States Forest Service and provided large samples of white-tailed deer (ca. 600-1,200) annually to assess population structuring and stability of that structure over time. The Savannah River Site traditionally has been divided into distinct units based on several criteria and has been subdivided coarsely into swamp and upland areas

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FIG. I.-Diagrammatic presentation of three methods by which the Savannah River Site (SRS) has been subdivided for analysis and management: a) swamp and upland subdivisions based on dominant overstory vegetation; b) hunt compartments defined based on the road system and riparian corridors; and c) 4-Jan2 cells.

based primarily on vegetation characteristics (Fig. 1). The site also has been divided into 50 hunt compartments (Fig. 1) whose boundaries are defined based on the existing road system and riparian conidors. METHODS AND MATERIALS

Collection of samples.-Since 1965. two methods of harvest for white-tailed deer have been employed on the Savannah River Site (Scribner et aI., 1985). Hunting with dogs was used in most compartments from 1965 to the

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present. Still hunting was pemtitted in certain compartments from 1969 to 1980. On the stillhunted area. 200-400 hunters usually were allowed on each day of hunting, and hunters were allowed to hunt anywhere in designated areas and harvest as many deer of any sex and age as desired. Hunts in the dog-hunt area consisted of an average of 90 hunters assigned to permanent stands located ca. 100 m apart. Forty packs of dogs, each having two handlers and a minimum of six dogs, would drive deer through each hunt compartment. Hunters and handlers were allowed to harvest as many deer of any age or sex as desired. The hunting season began in most years in October and ended in December. During each year, all deer killed were brought to check stations. Individuals were sexed, and age was determined based on patterns of tooth eruption and wear (Severinghaus, 1949). The precise location of the kill (based on pennanent hunter-stand locations within each hunt compartment) was determined for each deer using an x-y cell system. A contiguous matrix of 4_km2 cells that encompassed the entire Savannah River Site was constructed (Fig. 1), and all deer were assigned a specific cell. Spatial dispersion of all deer killed each year fonned the basis of subsequent analyses. Cells of this size were chosen so that samples for each cell were available during each year of the study period (1974-1979). Given the young age structure of the kill (typically >65% of the animals were :-;; 1.5 years old--Scribner et al., 1985) and the small size of home ranges (ca. 58 ha) of deer in this region (Jeter and Marchington. 1967), it was likely that individuals were killed in or near the cell in which they were born, which could provide some indication of breeding structure. Genetic ana/ysis.-Blood and tissue samples were taken from each individual and used for electrophoretic analysis. Samples were kept on wet ice in the field. Blood was separated into fractions of hemolysate and plasma in the lab. All tissue and blood samples were subsequently frozen at -70°C. Preparation of samples and electrophoretic buffers and staining followed Manlove et al. (1975). Each of the 4,749 individuals killed over the period 1974-1979 was assigned a genotype at the sorbitol dehydrogenase (Sdh; E.C. 1.1.1.14) locus. Sdh is one of several loci routinely assayed (Breshears et al .•

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1988) and was chosen for detailed analysis to illustrate microgeographic spatial structuring. Spatial analysis.-Counts of the number of individuals possessing a given Sdh genotype were made for each cel1 for each of the 6 years. From these genotypic counts. the number of alleles in each cell also was calculated. Allelic and genotypic counts for each cell were used to identify significant contiguous clusters of cells from deer of similar genetic characteristics. Identification of contiguous clusters of alleles or genotypes was derived from hypergeometric sampling probabilities (Chesser and Van Den Bussche. 1988; Scribner and Chesser. 1993). Clusters were based on the probability of observing contiguous cells that contained individuals with the same genotypes or alleles. Parameters necessary for the calculation of hypergeometric probabilities included: 1) size of the total sample during each year (N); 2) numbers of individuals in the sample that possessed a specific allele or genotype (A); 3) numbers of individuals in the sample that possessed a genotype or allele other than the one considered in the cluster analysis (B; B = N - A); 4) numbers of individuals in the cells of a potential cluster (n); 5) numbers of individuals in the cells of a potential cluster that possessed the specific genotype or allele (a); 6) numbers of individuals in the cells of a potential cluster that possessed alleles or genotypes other than the one considered (b; b = n - a). The probability (Pc) that a potential cluster of cells containing n individuals included a individuals that possessed the specific allele or genotypes was estimated as:

Pr (a, b I A, B)

~

(~) -

(~)

(:)

Each cell was in tum used as a reference point. Another cell that contained individuals of the same genotype or allele was chosen. and an ellipse was constructed encompassing cells that were being considered as part of a cluster. We considered individuals to be from the same cluster (i.e.• were contiguous) if they feU within an ellipse with foci (fl and f 2); foci were the midpoints of the most distant grids within a potential cluster (Chesser and Van Den Bussche. 1988). Elliptical eccentricity was determined from the distance between mid-points of the two refer-

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ence grids relative to the maximum distance between mid-points of grids that contained individuals of the same genotype or alleles over the entire Savannah River Site. Numbers of like and unlike genotypes or alleles in the ellipse (containing all individuals within the enclosed cells) were calculated, and the probability of observing such a cluster was estimated using hypergeometric sampling probabilities. Tests for each genotype or allele were conducted. A matrix that summarized the pair-wise probabilities of grids co-occurring in a contiguous cluster was constructed for each of the 6 years. Because pairs of grids could have been tested as members of several potential clusters. only their lowest pairwise probability was retained. Only clusters significant at the P < 0.005 level were used because of multiple testing and probability of Type J errors. Criteria for cluster fonnation were similar to those used for conditional clustering (Lefkovitch. 1982) and were completely independent of knowledge of the ecology of white-tailed deer or perceived environmental constraints to movements. Analyses may have been affected by the spatial distribution of deer within the harvest. However, all compartments were hunted during each year of the study. and samples were obtained from all cells during each year. Additional analyses were necessary to determine if spatial clusters of grids with deer of similar genetic characteristics were coincident with earlier conclusions of spatial structuring based on compartments or areas of swamp and upland. Binary matrices were constructed to summarize all pairwise relationships between cells based on their co-occurrence or lack of co-occurrence in areas of swamp versus upland and within hunt compartments. Mantel analyses (Mantel. 1967; Smouse et al.. 1986) were conducted to test if co-occurrence of cells within the major regions or within compartments was correlated significantly to co-occurrence of cells within a cluster. Matrices of the probabilities of co-occurrence of inter-cell clusters were further used to test for concordance of the positions of clusters between years (e.g., 1974 versus 1975. 1975 versus 1976). Analyses tested if the probability of the ith and jth grid being in the same cluster in year x was predictive of their co-occurrence within a cluster in year x+ 1. The degree of yearly variation in Sdh allele frequency was evaluated using chi-square anal-

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yses (Sokal and Rohlf, 1981). Allele frequencies were calculated for each of the 6 years for the entire Savannah River Site. To assess temporal variation on a microgeographic scale, allelic frequencies were estimated from two 20_km2 contiguous areas of swamp and upland (indicated by X in Fig. la). Detailed descriptions of allele frequencies at Sdh and other loci were presented elsewhere (Breshears et al., 1988; Smith et aI., 1990). RESULTS

Presence of genetic clusters.--GeneticaIly, white-tailed deer were not distributed unifonnly over the Savannah River Site, but showed considerable non-random, spatial-genetic structuring. Significant (P < 0.005) contiguous clusters of each of the three alleles (defined as M. F, S) were documented at the Sdh locus during each of the 6 years (Fig. 2). Areas encompassed by each cluster generally were large, but size and location of clusters varied among years. Clusters during each year were not discrete, but overlapped frequently. Significant non-random clusters were documented based on the non-random dispersion of deer that possessed specific genotypes (e.g., MS, FM), which further indicated microgeographic genetic structuring in the Savannah River Site (Fig. 3). Locations of these clusters generally were concordant with clusters based on alleles. Clusters of cells with predominantly heterozygous genotypes (e.g., FM, FS, MS) generally were observed in areas of overlap between clusters based on counts of alleles (Figs. 2 and 3), which suggested that clusters based on genotypes may confer some information on localized breeding structure. Temporal variation.-Annual changes in the allele that characterized clusters in a specific area were observed. Cells that were observed jointly in a cluster in one year were likely to co-occur in subsequent years (Table I) and suggested some degree of temporal stability. However, the predominant allele that characterized a cluster within a specific area often changed from year to year. This was particularly evident in

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central areas of the Savannah River Site where alleles that defined clusters changed from F to S over time (Fig. 2). Clusters tended to occur in the same areas in different years, but they often were based on different alleles, which implied that there was continuity in biological structure within localized areas, but deer varied temporally in their genetic characteristics. Two contiguous areas of 20-lan2 on the Savannah River Site were chosen for analysis to assess the magnitude of changes in allelic frequency in localized areas over the 6-year study. When individuals from across the entire Savannah River Site were combined, annual changes in allelic frequency were minimal (range of common allelic frequency ~ 0.76 - 0.78; Ii' ~ 1.71, d,f. ~ 10, P > 0.05). In contrast, when allelic frequencies from the smaller areas were compared across years, a greater range in allelic frequency was observed within each area (swamp ~ 0.70 - 0.84, Ii' ~ 17.57, d,f. ~ 10,0.10 > P > 0.05; upland ~ 0.73 - 0.87, Ii' ~ 9.84, d,f. ~ 10, P > 0.05) than was documented across the Savannah River Site, although the differences were not significant. Predictability of structural boundaries.-Mantel matrix-regression analyses revealed that there was little concordance between geographic locations of clusters based on genetic characteristics and those based on major habitat types (swamp versus upland) or hunt compartments (Table 2). Locations of clusters based on alleles did not correspond to the location of boundaries of either habitats or hunt compartments (P > 0.05 for each year). In contrast, there was a significant correspondence between the location of clusters based on genotypes and the location of boundaries of management units during most years. However, the predictability (r-values) was generally low or relationships were negative, which suggested that structural boundaries established based on habitat differences or roads have little relevance to the genetic structure of

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SCRIBNER £1' AL.--UENETIC SUBPOPULAII0NS Or DbbR

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FIG. 2.-Contiguous cluster analysis based on the dispersion of alleles at the Sdh locus revealing the extent of spatial genetic structure for white-tailed deer killed during each of 6 years on the Savannah River Site in westcentral South Carolina.

the population of white-tailed deer on the Savannah River Site. DISCUSSION

The population of white-tailed deer on the Savannah River Site was genetically structured spatially. Significant contiguous clusters were observed during each of the 6 years of study based on the non-random

dispersion of deer with specific alleles and genotypes. Clusters were documented consistently in the same geographic locales in consecutive years, implying some degree of maintenance of biological structure. However, the allele (or genotype) on which these localized clusters were based varied annually due to temporal variation in genetic characteristics in limited areas. When the

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