Gustine & Sanderson: Temporal Changes in White Clover Population Structure

and even under grazing produces significant levels of viable seed that end up in the soil (Chapman and AnderWhite clover (Trifolium repens L.) populations persist for years in son, 1987; Charlton, 1977). grazing lands primarily through clonal growth, yet retain high genetic variability. This study was conducted to determine how clone structure In spite of the presence of viable white clover seed dynamics affected intraspecific genetic variation of white clover at in the soil (Tracy and Sanderson, 2000), few of these three pasture sites. Up to 37 trifoliate leaf samples were taken monthly seeds germinate and even fewer seedlings become estabby resampling specific points in four 1.2 x 1.2 m area quadrats from lished in grasslands (Barratt and Silander, 1992; Brink April to September for 2 yr; random amplified polymorphic DNA et al., 1999; Chapman and Anderson, 1987; Fothergill (RAPD) profiles of 1160 and 973 samples, in 1997 and 1998, respecet al., 1997; Grime et al., 1988). However, naturalized tively, were analyzed. Significantly more clones were sampled in 1997 white clover populations persist for many decades in (162) than in 1998 (58) (P 0.0001). The majority of clones were grazed swards at northern midlatitudes. White clover not detected more than once during each year. The soil water content persistence in pastures where commercial seed is not was significantly lower in 1998 than in 1997 (P 0.0001). The number applied is due to two primary mechanisms: (i) New of sampled clonal members in quadrats ranged from 0.5 to 12.8 across both years on the three pastures. Within-population analysis of molecplants (genotypes made up of one or more rooted stoular variances (AMOVA) by date for the three pastures ranged from lons and trifoliates) can be established through rare 15 to 74% and 46 to 80% in 1997 and 1998, respectively, indicating germination and subsequent rare seedling recruitment low to medium genetic diversity in the populations. The fraction of in the spring (Chapman, 1983; Fothergill et al., 1997; clonal samples relative to the total number of samples ranged from Grime et al., 1988); and (ii) plants can increase in size by 0.03 to 0.78 in 1997 and 0.04 to 0.33 in 1998. Higher numbers of clonal clonal propagation via stolon nodes (Chapman, 1983). members appeared to reduce genetic diversity; however, this was When nodes within a stolon die, resulting fragments offset by rapid turnover of clones. We conclude that genetic variability become separate plants of the same genotype. Because of white clover is dynamic at the local scale, which contributes to its white clover spreads primarily by vegetative propagalong-term persistence in grazing lands. tion and because plants are thought to continuously propagate, one might expect many clonal patches and therefore low genetic variability within pastures. AlW clover is an important functional component though a white clover genotype could potentially domiof temperate grazed ecosystems because of symbinate a grassland by fragmenting into many clonal plants otic nitrogen fixation and its high nutritional quality (Cahn and Harper, 1976), the largest diameter clone (Caradus et al., 1996) as an animal feed. White clover reported was less than 6 m (Harberd, 1963). is a stoloniferous, obligately outcrossing, tetraploid speClonal patches generally range in size from 1 to 5 cies. It flowers prolifically during the growing season, Abbreviations: AMOVA, analysis of molecular variance; HU, HunUSDA-ARS, Pasture Systems and Watershed Management Research tingdon County; JU, Juniata County; MI, Mifflin County; RAPD, Unit, Curtin Road, Building 3702, University Park, PA 16802. Rerandom amplified polymorphic DNA; st, correlation of random genoceived 24 Feb. 2000. *Corresponding author ([email protected]). types within populations relative to that of random pairs of genotypes drawn from the whole species. Published in Crop Sci. 41:1143–1149 (2001). 1144 CROP SCIENCE, VOL. 41, JULY–AUGUST 2001 distribution of white clover genotypes was studied in four m2, and patch size is limited by site-specific conditions permanent 2.8-m quadrats. Quadrats 3 and 4 were in a flat, (Cahn and Harper, 1976; Harberd, 1963). Genetic variwell-drained paddock about 2 m above and 100 m away from ability of white clover can be high at the local (Burdon, quadrats 1 and 2. Each of the pair of locations was separated 1980) and regional (Gustine and Huff, 1999) scales. It by 10 to 30 m. The locations were laid out in the spring before is unclear how white clover maintains high intraspecific growth of white clover plants was initiated. genetic diversity in the face of partial dominance by a The mean floristic composition and bare ground areas single successful genotype. Vague explanations such as (sward gap area) were determined from estimates made in 10 “undefined selective or biotic forces” frequently are subquadrats that had not been sampled for white clover. The cited along with infrequent seed germination and seedextent of vegetative cover (composed of white clover, grasses, and other species) and the sward gap area was estimated ling recruitment (Burdon, 1980; Cahn and Harper, 1976). visually at each sampling date. The weed cover in the quadrats The purpose of our study was to investigate how clone on the three pastures ranged from 0 to 50% of the sward. structure dynamics of white clover populations affected Soil water data were recorded for each sampling date with intraspecific genetic variation during two growing seaa Trase time domain reflectometer (Soilmoisture Equipment sons in three rotationally stocked swards in the northCorp., Santa Barbara, CA) using 15 cm waveguides. The mean eastern USA. Random amplified polymorphic DNA volumetric soil water was determined from readings taken at profiles have been used to characterize genetic variabileight points near the perimeter of the quadrat and at two ity, clonal structure, and population structure in sevunsampled subquadrats within the quadrat. The field capacieral plant species (Buso et al., 1998; Gustine and Huff, ties for Hagerstown, Edom, and Clarksburg soils were 0.16 to 1999; Huff et al., 1998; Palacios and Gonzales-Cande0.24, 0.12 to 0.16, and 0.16 to 0.2 m m 3 of water in the surface 15 cm, respectively (USDA, 1981). The total precipitation las, 1997; Sydes and Peakall, 1998). Random amplified from January through August, 1997, was 606 mm at the HU polymorphic DNA profile determination is based on unsite, and 646 mm (25 mm above normal) at the JU and MI known DNA sequences that are stable to the environsites. For the same 8 mo in 1998, total precipitation was 802 ment under field conditions and are inherited in a domimm at the HU site and 962 mm (290 mm above normal) at nant Mendelian fashion. These markers do not distinguish the JU and MI sites (data not available for September through between heterozygous and homozygous alleles, as do isoDecember, 1998). Normal precipitation for January through zyme markers, but a much greater number of markers August for the HU site was not available. can be generated. With this approach, we could identify The four quadrats on each of the pastures were sampled genotypes of sampled plants, assign samples to a clone, on five dates from May to September at the points indicated record the temporal occurrence of clones, follow tempoon the sampling grid shown in Fig. 1. The grid was in place only during sampling. We collected 2133 leaf samples, or 48% ral changes in genetic variance, and estimate genetic of the maximum of 4440 samples that could have been coldiversity in white clover populations. lected during the study (had there been a stolon with a trifoliate leaf at each sampling point for every quadrat in both MATERIALS AND METHODS years). The total numbers of samples collected in 1997 and The white clover populations used in this study were part 1998 were 1160 and 973, respectively. Trifoliate leaf samples of three managed permanent pastures in the ridge and valley were taken from up to 37 sampling points in a quadrat at each physiographic region of Pennsylvania (Table 1). The sites were date. Each sample for genomic DNA analysis consisted of similar in elevation but varied in soil types (Table 1). The one to four trifoliate leaves from the same stolon. If no stolon rotationally stocked swards were not treated with chemical was present within 2 cm of the sampling point, a sample could fertilizers. The pastures had been grazed for at least 5 yr since not be taken; if more than one stolon was present at the the previous seeding, and the white clover populations studied sampling point, trifoliate leaves were collected from a ranpresumably had developed from the viable seed pool (as dedomly selected stolon. Since we did not mark stolons, we did fined by Silvertown and Lovett Doust, 1993) of naturalized not know if a plant was sampled on more than one date. If a clover. The pastures in Juniata (JU) and Mifflin (MI) Counties stolon had no trifoliate leaves, the plant was not sampled. consisted of 10 to 60% white clover and 10 to 80% grasses, Leaf samples were stored on ice at the time of collection, which were composed of tall fescue (Festuca arundinacea Schreb.), quackgrass [Elytrigia repens (L.) Desv. ex Nevski], 1 Mention of a trademark, vendor, or proprietary product does and Kentucky bluegrass (Poa pratensis L.). The pasture in not constitute a guarantee or warranty of the product by the U.S. Huntingdon County (HU) consisted of 70% reed canarygrass Department of Agriculture, and does not imply its approval to the exclusion of other products that may also be suitable. (Phalaris arundinacea L.) and less than 25% white clover. The Table 1. Site characteristics at three Pennsylvania pastures where white clover populations were sampled. Annual Annual Seeding Year Year Pasture Location Lat. N Long. W Elevation Precip. mean temp. Soil Series Taxonomic Name and Species Established