Pine cones that remain closed and retain seeds until fire causes the cones to open (cone serotiny) represent a key adaptive trait in a variety of pine species. In lodgepole pine, there is substantial geographical variation in serotiny across the Rocky Mountain region. This variation in serotiny has evolved as a result of geographically divergent selection, with consequences that extend to forest communities and ecosystems. An understanding of the genetic architecture of this trait is of interest owing to the wide-reaching ecological consequences of serotiny and also because of the repeated evolution of the trait across the genus. Here, we present and utilize an inexpensive and time-effective method for generating population genomic data. The method uses restriction enzymes and PCR amplification to generate a library of fragments that can be sequenced with a high level of multiplexing. We obtained data for more than 95,000 single nucleotide polymorphisms across 98 serotinous and nonserotinous lodgepole pines from three populations. We used a Bayesian generalized linear model (GLM) to test for an association between genotypic variation at these loci and serotiny. The probability of serotiny varied by genotype at 11 loci, and the association between genotype and serotiny at these loci was consistent in each of the three populations of pines. Genetic variation across these 11 loci explained 50% of the phenotypic variation in serotiny. Our results provide a first genome-wide association map of serotiny in pines and demonstrate an inexpensive and efficient method for generating population genomic data.

Abstract. The geographic mosaic theory of coevolution posits that the form of selection between interacting species varies across a landscape with coevolution important and active in some locations (i.e., coevolutionary hotspots) but not in others (i.e., coevolutionary coldspots). We tested the hypothesis that the presence of red squirrels (Tamiasciurus hudsonicus) affects the occurrence of coevolution between red crossbills (Loxia curvirostra complex) and Rocky Mountain lodgepole pine (Pinus contorta ssp. latifolia) and thereby provides a mechanism giving rise to a geographic mosaic of selection. Red squirrels are the predominant predispersal seed predator and selective agent on lodgepole pine cones. However, in four isolated mountain ranges east and west of the Rocky Mountains, red squirrels are absent and red crossbills are the main predispersal seed predator. These isolated populations of pine have apparently evolved without Tamiasciurus for about 10,000 to 12,000 years. Based on published morphological, genetic, and paleobotanical studies, we infer that cone traits in these isolated populations that show parallel differences from cones in the Rocky Mountains have changed in parallel. We used data on crossbill and conifer cone morphology and feeding preferences and efficiency to detect whether red crossbills and lodgepole pine exhibit reciprocal adaptations, which would imply coevolution. Cone traits that act to deter Tamiasciurus and result in high ratios of cone mass to seed mass were less developed in the isolated populations. Cone traits that act to deter crossbills include larger and thicker scales and perhaps increased overlap between successive scales and were enhanced in the isolated populations. In the larger, isolated mountain ranges crossbills have evolved deeper, shorter, and therefore more decurved bills to exploit these cones. This provides crossbills with higher feeding rates, and the change in bill shape has improved efficiency by reducing the concomitant increases in body mass and daily energy expenditures that would have resulted if only bill size had increased. These parallel adaptations and counter adaptations in red crossbills and lodgepole pine are interpreted as reciprocal adaptations and imply that these crossbills and pine are in coevolutionary arms races where red squirrels are absent (i.e., coevolutionary hotspots) but not where red squirrels are present (i.e., coevolutionary coldspots). The view that coevolution is a prominent evolutionary process has been contentious. Nonetheless, evidence from diverse areas of evolutionary biology indicate that coevolution is a fundamental process in evolution and that a better understanding of the coevolutionary process is critical for progress in evolutionary biology, conservation, and health-related issues (Thompson 1994(Thompson , 1999a. One recent advance in coevolutionary theory has risen from the recognition that the form of an interaction between species often varies across a landscape (e.g., Thompson and Pellmyr 1992). This geographic v...

Asymmetrical competition determines which of two seed predators drives the evolution of lodgepole pine (Pinus contorta ssp. latifolia) cones. Red squirrels (Tamiasciurus hudsonicus) are effective preemptive competitors in lodgepole pine forests so that red crossbills (Loxia curvirostra) are uncommon and selection from Tamiasciurus drives cone evolution. When Tamiasciurus are absent, crossbills increase in abundance and coevolve in an evolutionary arms race with pine. Similarly, Tamiasciurus alters the evolutionary trajectories of large-seeded pines, many of which rely on birds (Corvidae) for their seed dispersal. Populations therefore exhibit a selection mosaic with coevolutionary hot spots. In the coevolutionary hot spots, divergent selection on crossbills potentially leads to reproductive isolation and speciation. This results in a subsequent reduction in the geographic mosaic but diversifies the adaptive landscape on which crossbills have radiated. Thus, divergent selection is a double-edged sword. Divergent selection is critical in creating a selection mosaic but erodes the selection mosaic when it promotes reproductive isolation and speciation.

BackgroundMassively parallel sequencing of cDNA is now an efficient route for generating enormous sequence collections that represent expressed genes. This approach provides a valuable starting point for characterizing functional genetic variation in non-model organisms, especially where whole genome sequencing efforts are currently cost and time prohibitive. The large and complex genomes of pines (Pinus spp.) have hindered the development of genomic resources, despite the ecological and economical importance of the group. While most genomic studies have focused on a single species (P. taeda), genomic level resources for other pines are insufficiently developed to facilitate ecological genomic research. Lodgepole pine (P. contorta) is an ecologically important foundation species of montane forest ecosystems and exhibits substantial adaptive variation across its range in western North America. Here we describe a sequencing study of expressed genes from P. contorta, including their assembly and annotation, and their potential for molecular marker development to support population and association genetic studies.ResultsWe obtained 586,732 sequencing reads from a 454 GS XLR70 Titanium pyrosequencer (mean length: 306 base pairs). A combination of reference-based and de novo assemblies yielded 63,657 contigs, with 239,793 reads remaining as singletons. Based on sequence similarity with known proteins, these sequences represent approximately 17,000 unique genes, many of which are well covered by contig sequences. This sequence collection also included a surprisingly large number of retrotransposon sequences, suggesting that they are highly transcriptionally active in the tissues we sampled. We located and characterized thousands of simple sequence repeats and single nucleotide polymorphisms as potential molecular markers in our assembled and annotated sequences. High quality PCR primers were designed for a substantial number of the SSR loci, and a large number of these were amplified successfully in initial screening.ConclusionsThis sequence collection represents a major genomic resource for P. contorta, and the large number of genetic markers characterized should contribute to future research in this and other pines. Our results illustrate the utility of next generation sequencing as a basis for marker development and population genomics in non-model species.

I quantitatively test the hypothesis that four taxa or "types" (species or subspecies) of Red Crossbills (Loxia curvirostra) in the Pacific Northwest have diversified morphologically in bill characters in response to alternative adaptive peaks presented by their food: seeds in conifer cones. Hypothetically, each adaptive peak corresponds to one conifer species whose seeds are (1) produced regularly from year to year, (2) held in cones through late winter when seed is most limiting, and (3) protected from depletion by potential noncrossbill competitors. Four such conifers, termed "key conifers," are present (Tsuga heterophylla, Pseudotsuga menziesii, Pinus ponderosa, and Pinus contorta var. latifolia). I use data on foraging efficiency for 31 captive crossbills of four types to determine the optimal bill size and palate structure for foraging on the key conifers. As predicted, if each type is adapted for foraging on a key conifer, the observed morphology of a given type is often the predicted optimal size morphology of foraging on its respective key conifer. Two of the types have mean bill sizes (bill depth) equalling their predicted optimal size. For one of the remaining types, the observed differs from the optimum by 0.4 mm; I was unable to predict an optimal size for the remaining type. Optimal bill size varies with season. Bill sizes corresponded more closely to the optima for winter (lean period) than for summer. Observed mean width of the palate groove, in which crossbills hold conifer seeds while the seeds are being husked, was consistently close to the estimated optimal groove width. Optimal groove width was correlated (r2 = 1.00, n = 4) with seed size (cube root of mass), suggesting optimal groove width is determined by seed size. Overall, each crossbill type has either the optimal bill size or optimal husking groove width, or both, for foraging on their key conifers. Fitness set analyses indicate that there are substantial trade—offs in foraging efficiency. The best phenotype for foraging on one conifer is often only one—half as efficient on other conifers. All four fitness sets are concave, implying selection against intermediate phenotypes. I conclude, first, that reliability of seeds on key conifers during periods of food scarcity is a critical feature in the ecology and evolution of crossbills. Second, optimization of morphological traits occurs even in populations in highly variable environments. Third, disruptive selection against intermediate phenotypes is likely. This should maintain, if not reinforce, the distinctiveness of types. Fourth, the diversity of cone structure and seed size among key conifers is ultimately responsible for the diversification of crossbills.