Impacts of forest fragmentation on the mating system and genetic diversity of white spruce (Picea glauca) at the landscape level.

We studied the mating system of white spruce (Picea glauca) in a landscape fragmented by agriculture in northern Ontario, Canada. We sampled 23 stands that ranged in size from 1 to >500 trees isolated by 250-3000 m from the nearest other stand. Six polymorphic allozyme loci from four enzyme systems were used to genotype approximately 10 000 embryos from 104 families. We detected no allele frequency heterogeneity in the pollen pool among stands or families (Phi(FT)=-0.025). Overall, estimates of outcrossing were high (t(m)=94% and mean t(s)=91%) but significantly different from unity. Bi-parental inbreeding (t(m)-t(s)=3.2%) was low but significantly different from zero. Allozyme-based outcrossing estimates did not differ significantly among three stand-size classes (SSCs): small (<10 trees), medium (10-100 trees) and large (> or =100 trees). The number of effective pollen donors was high in all SSCs, but was significantly lower in small stands (N(ep)=62.5) than in medium-sized and large stands (both N(ep)=143). The primary selfing rate was significantly higher in medium stands than in large stands. We found no significant difference in genetic diversity measures in the filial (seed) population among SSCs. Overall, these results indicate that white spruce stands in this fragmented landscape are resistant to genetic diversity losses, primarily through high pollen-mediated gene-flow and early selection against inbred embryos. We discuss the importance of using seed data, in conjunction with genetic data, to evaluate the impacts of fragmentation on natural populations.

Fine-scale estimation of outcrossing in western redcedar with microsatellite assay of bulked DNA.

Western redcedar (Thuja plicata, Cupressaceae) is a self-fertile conifer with a mixed mating system and significant variation for outcrossing among populations. In this paper, we conducted a fine-scale study of mating system variation to identify correlates of outcrossing in natural populations. We examined variation for outcrossing within and among individual trees, and describe a new method to estimate outcrossing using bulked DNA samples. Bulking (assaying DNA tissues from several individuals simultaneously) increases the experimental power without increasing the experimental effort. We sampled 80 trees from four natural populations in southwestern British Columbia. From each tree, we sampled from up to six crown positions (three heights and inner vs outer branches). From each position, two samples of three seedlings each were bulked before DNA extractions. Using four microsatellite loci, we obtained outcrossing rates for each tree and for each of the six crown positions. We found individual tree selfing rates to increase with tree height in all four populations, but selfing rates did not differ among crown positions. The higher selfing rate of larger trees is probably due to their greater proportional contribution to local pollen clouds. Individual tree outcrossing rates ranged from 22 to 100% and the population outcrossing rates from 66 to 78%. Missed alleles due to bulking and the estimation method used both cause a downward bias in outcrossing rates, so that these estimates are probably lower than the actual outcrossing rates. Nevertheless, the trends we observed are not affected by systematic biases of estimation.

Somatic mutations at microsatellite loci in western Redcedar (Thuja plicata: Cupressaceae).

A per-generation somatic mutation rate for microsatellites was estimated in western redcedar (Thuja plicata, Donn ex D. Don.: Cupressaceae). A total of 80 trees representative of the average size and age of reproductive trees were sampled in four natural populations in southwestern British Columbia. Samples of bulked haploid megagametophytes were collected from two or three positions on each tree, assuming that the collections were far enough apart that the same mutant sector was not sampled twice. All samples were genotyped at eight microsatellite loci. A single mutation corresponding to a stepwise increase in one dinucleotide repeat was detected. The estimated mutation rate for microsatellites was 6.3 x 10(-4) mutations per locus per generation (or 3.1 x 10(-4) per allele per generation), with a 95% confidence interval of 3.0 x 10(-5) to 4.0 x 10(-3) mutations per locus. Somatic mutations can contribute to a greater mutational load in trees, as compared to shorter lived plants, and genotypic mosaics within an individual have important implications for plant defense strategies and plant evolution.