Lineage-specific mapping of quantitative trait loci.

We present an approach for quantitative trait locus (QTL) mapping, termed as 'lineage-specific QTL mapping', for inferring allelic changes of QTL evolution along with branches in a phylogeny. We describe and analyze the simplest case: by adding a third taxon into the normal procedure of QTL mapping between pairs of taxa, such inferences can be made along lineages to a presumed common ancestor. Although comparisons of QTL maps among species can identify homology of QTLs by apparent co-location, lineage-specific mapping of QTL can classify homology into (1) orthology (shared origin of QTL) versus (2) paralogy (independent origin of QTL within resolution of map distance). In this light, we present a graphical method that identifies six modes of QTL evolution in a three taxon comparison. We then apply our model to map lineage-specific QTLs for inbreeding among three taxa of yellow monkey-flower: Mimulus guttatus and two inbreeders M. platycalyx and M. micranthus, but critically assuming outcrossing was the ancestral state. The two most common modes of homology across traits were orthologous (shared ancestry of mutation for QTL alleles). The outbreeder M. guttatus had the fewest lineage-specific QTL, in accordance with the presumed ancestry of outbreeding. Extensions of lineage-specific QTL mapping to other types of data and crosses, and to inference of ancestral QTL state, are discussed.

Postglacial history of a widespread conifer produces inverse clines in selective neutrality tests.

Deviations of the site frequency spectrum of mutations (SFS) from neutral expectations may be caused by natural selection or by demographic processes such as population subdivision or temporal changes in population size. As most widespread temperate and boreal tree species have expanded from glacial refugia in the past 13,000 years, colonization bottlenecks associated with this migration may have left variable demographic signatures among geographic populations corresponding to distance from the refugia. To determine whether the signature of postglacial re-colonization has skewed the SFS in the widely distributed conifer Sitka spruce (Picea sitchensis (Bong.) Carr.), we re-sequenced 153 nuclear genes in six populations from across the species range. We found that while the SFS for the pooled sample produced negative values for Tajima's D and Fay and Wu's H, these statistics exhibited strong clinal variation when populations were analysed separately (R(2) = 0.84, P = 0.007 for Tajima's D and R(2) = 0.65, P = 0.033 for Fay and Wu's H). When historical bottlenecks of varying age were simulated using approximate Bayesian computation, distance of populations from the southern range limit explained most of the variation in bottleneck timing among populations (R(2) = 0.89, P = 0.003). These data suggest that sequential population bottlenecks during postglacial re-colonization have resulted in diverse among-population signatures within the contemporary SFS in Sitka spruce, with rare variants more common in the south, and medium-frequency variants more common in the north. Our results also emphasize the need to consider sampling strategy and to explore population-specific null demographic models in surveys of nucleotide variation in widely distributed species.

Can clone size serve as a proxy for clone age? An exploration using microsatellite divergence in Populus tremuloides.

In long-lived clonal plants, the overall size of a clone is often used to estimate clone age. The size of a clone, however, might be largely determined by physical or biotic interactions, obscuring the relationship between clone size and age. Here, we use the accumulation of mutations at 14 microsatellite loci to estimate clone age in trembling aspen (Populus tremuloides) from southwestern Canada. We show that the observed patterns of genetic divergence are consistent with a model of increasing ramet population size, allowing us to use pairwise genetic divergence as an estimator of clone age. In the populations studied, clone size did not exhibit a significant relationship with microsatellite divergence, indicating that clone size is not a good proxy for clone age. In P. tremuloides, the per-locus per-year neutral somatic mutation rate across 14 microsatellite loci was estimated to lie between 6 x 10(-7) (lower bound) and 4 x 10(-5) (upper bound).

A novel mating system analysis for modes of self-oriented mating applied to diploid and polyploid arctic Easter daisies (Townsendia hookeri).

We have developed a new model for mating system analysis, which attempts to distinguish among alternative modes of self-oriented mating within populations. This model jointly estimates the rates of outcrossing, selfing, automixis and apomixis, through the use of information in the family structure given by dominant genetic marker data. The method is presented, its statistical properties evaluated, and is applied to three arctic Easter daisy populations, one consisting of diploids, the other two of tetraploids. The tetraploids are predominantly male sterile and reported to be apomictic while the diploids are male fertile. In each Easter daisy population, 10 maternal arrays of six progeny were assayed for amplified fragment length polymorphism markers. Estimates, confirmed with likelihood ratio tests of mating hypotheses, showed apomixis to be predominant in all populations (ca. 70%), but selfing or automixis was moderate (ca. 25%) in tetraploids. It was difficult to distinguish selfing from automixis, and simulations confirm that with even very large sample sizes, the estimates have a very strong negative statistical correlation, for example, they are not independent. No selfing or automixis was apparent in the diploid population, instead, moderate levels of outcrossing were detected (23%). Low but significant levels of outcrossing (2-4%) seemed to occur in the male-sterile tetraploid populations; this may be due to genotyping error of this level. Overall, this study shows apomixis can be partial, and provides evidence for higher levels of inbreeding in polyploids compared to diploids and for significant levels of apomixis in a diploid plant population.

Estimating heritabilities and genetic correlations with marker-based methods: an experimental test in Mimulus guttatus.

The calculation of heritabilities and genetic correlations, which are necessary for predicting evolutionary responses, requires knowledge about the relatedness between individuals. This information is often not directly available, especially not for natural populations, but can be inferred by using molecular markers such as allozymes. Several methods based on inferred relatedness from marker data have been developed to estimate heritabilities and genetic correlations in natural populations. Most methods use maximum-likelihood procedures to assign pairs or groups of individuals to predefined discrete relatedness classes (e.g., half sibs and unrelated individuals). The Ritland method, on the other hand, uses method of moments estimators to estimate pairwise relatedness among individuals as continuous values. We tested both the Ritland method and a maximum-likelihood method by applying them to a greenhouse population consisting of seed families of the herb Mimulus guttatus and comparing the results to the ones from a frequently used standard method based on half-sib families. Estimates of genetic correlations were far from accurate, especially when we used the Ritland method. However, this study shows that even with a few variable allozyme loci, it is possible to get qualitatively good indications about the presence of heritable genetic variation from marker-based methods, even though both methods underestimated it.

Predicting evolution of floral traits associated with mating system in a natural plant population.

Evolution of floral traits requires that they are heritable, that they affect fitness, and that they are not constrained by genetic correlations. These prerequisites have only rarely been examined in natural populations. For Mimulus guttatus, we found by using the Riska-method that corolla width, anther length, ovary length and number of red dots on the corolla were heritable in a natural population. Seed production (maternal fitness) was directly positively affected by corolla width and anther size, and indirectly so by ovary length and number of red dots on the corolla. The siring success (paternal fitness), as estimated from allozyme data, was directly negatively affected by anther-stigma separation, and indirectly so by the corolla length-width ratio. Genetic correlations, estimated with the Lynch-method, were positive between floral size measures. We predict that larger flowers with larger reproductive organs, which generally favour outcrossing, will evolve in this natural population of M. guttatus.

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.

Sexual reproduction in the white pine weevil (Pissodes strobi [Peck] [Coleoptera: Curculionidae]): implications for population genetic diversity.

Controlled mating experiments in the white pine weevil (Pissodes strobi [Peck]) indicated that female weevils either stored sperm or fertilized eggs from one season to the next, and were able to colonize Sitka spruce (Picea sitchensis [Bong.] Carr.) trees without additional mating events. This was interpreted as being beneficial for the insect, in that population establishment in a new habitat could be initiated by dispersing previously mated females without participation of the male. This makes colonization and population/outbreak development more likely as it reduces the need for mate searching in the second season. Paternity identification, based on microsatellite molecular markers, established that the progeny produced in year 2 by females mated only in year 1, were often fathered by more than one male. Multiple paternity, coupled with a lack of parthenogenesis, which was also demonstrated herein, may help to account for the high degree of genetic diversity evidenced in this species.

Genetic diversity and differentiation of Kermode bear populations.

The Kermode bear is a white phase of the North American black bear that occurs in low to moderate frequency on British Columbia's mid-coast. To investigate the genetic uniqueness of populations containing the white phase, and to ascertain levels of gene flow among populations, we surveyed 10 highly polymorphic microsatellite loci, assayed from trapped bear hairs. A total of 216 unique bear genotypes, 18 of which were white, was sampled among 12 localities. Island populations, where Kermodes are most frequent, show approximately 4% less diversity than mainland populations, and the island richest in white bears (Gribbell) exhibited substantial genetic isolation, with a mean pairwise FST of 0.14 with other localities. Among all localities, FST for the molecular variant underlying the coat-colour difference (A893G) was 0.223, which falls into the 95th percentile of the distribution of FST values among microsatellite alleles, suggestive of greater differentiation for coat colour than expected under neutrality. Control-region sequences confirm that Kermode bears are part of a coastal or western lineage of black bears whose existence predates the Wisconsin glaciation, but microsatellite variation gave no evidence of past population expansion. We conclude that Kermodism was established and is maintained in populations by a combination of genetic isolation and somewhat reduced population sizes in insular habitat, with the possible contribution of selective pressure and/or nonrandom mating.

Chloroplast phylogeography and evolution of highly polymorphic microsatellites in lodgepole pine ( Pinus contorta).

We employed a novel set of six highly polymophic chloroplastic simple sequence repeat (cpSSR) loci to investigate the phylogeography of lodgepole pine ( Pinus contorta Dougl. Ex. Loud.), and to examine aspects of the evolutionary process operating on these repetitive DNA sequences. Chloroplast haplotypes of 500 trees, sampled throughout the range of lodgepole pine, were determined. We found a marked association of genetic distance with physical distance within the scale of 0 to 1,000 km, but no association beyond that range. Likewise, geographic clustering was observed only among recent clades in a dendrogram. These phylogeographic patterns are consistant with a rapid rangewide expansion ("big-bang") followed by recent, local population differentiation ("galaxy formation"). In support of this expansion, coalescent simulations of the genealogical process gave a long-term effective population size in the low thousands, and a time to common ancestry of about 1,500 generations (12,000 years), consistent with a post-Pleistocene population expansion as documented by previous pollen-sediment analyses. Two lines of evidence (mapping mutational events onto a phylogeny, and evaluation of observed versus expected gene diversity) suggest that five of the cpSSR loci evolve primarily by a stepwise model of evolution of single repeat changes (but with a small proportion of changes involving two or more repeats), and the coalescent simulations point to a mutation rate of about 10(-3).

Inheritance and population structure of the white-phased "Kermode" black bear.

We report that a single nucleotide replacement in the melanocortin 1 receptor gene [1] (mc1r) is responsible for the white coat color of the "Kermode" bear [2], a color phase of the black bear (Ursus americanus Pallus) found in the rainforests along the north coast of British Columbia. In a sample of 220 bears, of which 22 were white, there was complete association of a recessive Tyr-to-Cys replacement at codon 298 with the white phase. This variant has not been yet been reported in other mammals, and it also is the lightest-colored variant yet found at mc1r. Also, we found that heterozygotes, which act as a hidden reservoir for the allele among black bears, were infrequent outside of the three islands where Kermodes are common and that, within these three islands, heterozygotes were less frequent than expected under random mating. Immigration of black bears into Kermode populations can depress the occurrence of the white phase, and management practices should be designed to avoid facilitating higher immigration rates.

Diversity and genetic structure in populations of Pseudotsuga menziesii (Pinaceae) at chloroplast microsatellite loci.

Genetic variation was compared between uniparentally-inherited (chloroplast simple sequence repeats, cpSSRs) vs. biparentally-inherited (isozyme and random amplified polymorphic DNA, RAPD) genetic markers in Douglas-fir (Pseudotsuga mensiezii) from British Columbia. Three-hundred twenty-three individuals from 11 populations were assayed. In Douglas-fir, the cpSSR primer sites were well-conserved relative to Pinus thunbergii (11 of 17 loci clearly amplified), but only 3 loci were appreciably polymorphic. At these cpSSR loci, we found an unexpectedly low level of polymorphism within populations, and no genetic differentiation among populations. By contrast, the nuclear markers showed variation typical of conifers, with significant among-population differentiation. This difference is likely the outcome of both historical factors and high pollen dispersal.

Heritability of phenolics in Quercus laevis inferred using molecular markers.

Studies of quantitative inheritance of phenotypes do not generally encompass the range of environmental conditions to which a population may be exposed in a natural setting and are rarely conducted on long-lived species due to the time required for traditional crossing experiments. We used a marker-based method to estimate relatedness with microsatellite markers in a natural population of a long-lived oak, then used this inferred relatedness to examine quantitative genetic variation in the concentration of foliar phenolics. Estimating heritability using this method requires both significant relatedness and variance in relatedness over distance. However, this population did not show significant variance of relatedness, so only the presence of heritability, and its ranking among traits and environments, could be estimated. Seven foliar phenolics showed a significant relationship between phenotypic similarity and relatedness. The significance of this relationship varied among individual phenolic compounds, as well as by season. Genetic factors appeared to have a more measurable influence on the production of secondary compounds early in the season. After leaf expansion, covariance of relatedness and phenotypic variance appear to become less significant. Therefore heritability may vary seasonally for these traits.

Marker-inferred relatedness as a tool for detecting heritability in nature.

This paper presents a perspective of how inferred relatedness, based on genetic marker data such as microsatellites or amplified fragment length polymorphisms (AFLPs), can be used to demonstrate quantitative genetic variation in natural populations. Variation at two levels is considered: among pairs of individuals within populations, and among pairs of subpopulations within a population. In the former, inferred pairwise relatedness, combined with trait measures, allow estimates of heritability 'in the wild'. In the latter, estimates of QST are obtained, in the absence of known heritabilities, via estimates of pairwise FST. Estimators of relatedness based on the 'Kronecker operator' are given. Both methods require actual variation of relationship, a rarely studied aspect of population structure, and not necessarily present. Some conditions for appropriate population structures in the wild are identified, in part through a review of recent studies.

Estimation of pairwise relatedness with molecular markers.

Applications of quantitative genetics and conservation genetics often require measures of pairwise relationships between individuals, which, in the absence of known pedigree structure, can be estimated only by use of molecular markers. Here we introduce methods for the joint estimation of the two-gene and four-gene coefficients of relationship from data on codominant molecular markers in randomly mating populations. In a comparison with other published estimators of pairwise relatedness, we find these new "regression" estimators to be computationally simpler and to yield similar or lower sampling variances, particularly when many loci are used or when loci are hypervariable. Two examples are given in which the new estimators are applied to natural populations, one that reveals isolation-by-distance in an annual plant and the other that suggests a genetic basis for a coat color polymorphism in bears.

Microsatellite variation and evolution in the Mimulus guttatus species complex with contrasting mating systems.

Mutational variability at microsatellite loci is shaped by both population history and the mating system. In turn, alternate mating systems in flowering plants can resolve aspects of microsatellite loci evolution. Five species of yellow monkeyflowers (Mimulus sect. Simiolis) differing for historical rates of inbreeding were surveyed for variation at six microsatellite loci. High levels of diversity at these loci were found in both outcrossing and selfing taxa. In line with allozyme studies, inbreeders showed more partitioning of diversity among populations, and diversity in selfing taxa was lower than expected from reductions in effective population size due to selfing alone, suggesting the presence of either population bottlenecks or background selection in selfers. Evaluation of the stepwise mutation model (a model of DNA replication slippage) suggests that these loci evolve in a stepwise fashion. Inferred coalescent times of microsatellite alleles indicate that past bottlenecks of population size or colonization events were important in reducing diversity in the inbreeding taxon.

Quantitative trait loci differentiating the outbreeding Mimulus guttatus from the inbreeding M. platycalyx.

Theoretical predictions about the evolution of selfing depend on the genetic architecture of loci controlling selfing (monogenic vs. polygenic determination, large vs. small effect of alleles, dominance vs. recessiveness), and studies of such architecture are lacking. We inferred the genetic basis of mating system differences between the outbreeding Mimulus guttatus and the inbreeding M. platycalyx by quantitative trait locus (QTL) mapping using random amplified polymorphic DNA and isozyme markers. One to three QTL were detected for each of five mating system characters, and each QTL explained 7.6-28.6% of the phenotypic variance. Taken together, QTL accounted for up to 38% of the variation in mating system characters, and a large proportion of variation was unaccounted for. Inferred QTL often affected more than one trait, contributing to the genetic correlation between those traits. These results are consistent with the hypothesis that quantitative variation in plant mating system characters is primarily controlled by loci with small effect.

Marker-based inferences about epistasis for genes influencing inbreeding depression.

We describe a multilocus, marker-based regression method for inferring interactions between genes controlling inbreeding depression in self-fertile organisms. It is based upon selfing a parent heterozygous for several unlinked codominant markers, then analyzing the fitness of progeny marker genotypes. If loci causing inbreeding depression are linked to marker loci, then viability selection is manifested by distorted segregation of markers, and fecundity selection by dependence of the fecundity character upon the marker genotype. To characterize this selection, fitness is regressed on the proportion of loci homozygous for markers linked to deleterious alleles, and epistasis is detected by nonlinearity of the regression. Alternatively, fitness can be regressed on the proportion of heterozygous loci. Other modes of selection can be incorporated with a bivariate regression involving both homozygote and heterozygote marker genotypes. The advantage of this marker-based approach is that "purging" is minimized and specific chromosomal segments are identified; its disadvantage lies in low statistical power when linkage is not strong and/or the linkage phase between marker and selected loci is uncertain. Using this method in the wildflower Mimulus guttatus, we found predominant multiplicative gene interaction determining fecundity and some negative synergistic (nonmultiplicative) interaction for viability.