Genome-wide association study (GWAS) has revolutionized the search for the genetic basis of complex traits. To date, GWAS have generally relied on relatively sparse sampling of nucleotide diversity, which is likely to bias results by preferentially sampling high-frequency SNPs not in complete linkage disequilibrium (LD) with causative SNPs. To avoid these limitations we conducted GWAS with >6 million SNPs identified by sequencing the genomes of 226 accessions of the model legume Medicago truncatula. We used these data to identify candidate genes and the genetic architecture underlying phenotypic variation in plant height, trichome density, flowering time, and nodulation. The characteristics of candidate SNPs differed among traits, with candidates for flowering time and trichome density in distinct clusters of high linkage disequilibrium (LD) and the minor allele frequencies (MAF) of candidates underlying variation in flowering time and height significantly greater than MAF of candidates underlying variation in other traits. Candidate SNPs tagged several characterized genes including nodulation related genes SERK2, MtnodGRP3, MtMMPL1, NFP, CaML3, MtnodGRP3A and flowering time gene MtFD as well as uncharacterized genes that become candidates for further molecular characterization. By comparing sequence-based candidates to candidates identified by in silico 250K SNP arrays, we provide an empirical example of how reliance on even high-density reduced representation genomic makers can bias GWAS results. Depending on the trait, only 30–70% of the top 20 in silico array candidates were within 1 kb of sequence-based candidates. Moreover, the sequence-based candidates tagged by array candidates were heavily biased towards common variants; these comparisons underscore the need for caution when interpreting results from GWAS conducted with sparsely covered genomes.

Local adaptation and adaptive clines are pervasive in natural plant populations, yet the effects of these types of adaptation on genomic diversity are not well understood. With a data set of 202 accessions of Medicago truncatula genotyped at almost 2 million single nucleotide polymorphisms, we used mixed linear models to identify candidate loci responsible for adaptation to three climatic gradientsannual mean temperature (AMT), precipitation in the wettest month (PWM), and isothermality (ITH)-representing the major axes of climate variation across the species' range. Loci with the strongest association to these climate gradients tagged genome regions with high sequence similarity to genes with functional roles in thermal tolerance, drought tolerance, or resistance to herbivores of pathogens. Genotypes at these candidate loci also predicted the performance of an independent sample of plant accessions grown in climatecontrolled conditions. Compared to a genome-wide sample of randomly drawn reference SNPs, candidates for two climate gradients, AMT and PWM, were significantly enriched for genic regions, and genome segments flanking genic AMT and PWM candidates harbored less nucleotide diversity, elevated differentiation between haplotypes carrying alternate alleles, and an overrepresentation of the most common haplotypes. These patterns of diversity are consistent with a history of soft selective sweeps acting on loci underlying adaptation to climate, but not with a history of long-term balancing selection. L OCAL and clinal adaptation is widespread in natural populations (Clausen et al. 1941;Leimu and Fischer 2008), which, by definition, results from selection that varies across a species' range. Most methods to search for the targets of adaptation are designed to identify gene regions that have experienced "hard" selective sweeps, in which selection acts on new mutations that confer a selective advantage across the entire range of a sample (Maynard Smith and Haigh 1974;Nielsen 2005;Pritchard and Di Rienzo 2010;Kelly et al. 2013). These methods are not designed to identify targets of adaptation to selective environments that vary across the range of sampled populations. The targets of locally variable selection either may be maintained as stable polymorphisms or experience partial, or "soft," sweeps either because local adaptation involves fixation of different alleles in different portions of a species' range or because selection acts on standing variation (Hermisson and Pennings 2005;Pavlidis et al. 2012;Messer and Petrov 2013).Identifying the molecular targets of clinal adaptation offers an opportunity not only to identify functionally important genes, but also to further our understanding of adaptation itself. If the selective environment that drives clinal adaptation is stable, and alleles responsible for adaptation are at stable equilibria, then the loci responsible for adaptation may bear population genetic signatures of balancing selection: elevated differentiation between haplotypes linked to the ...

Ecologists and biogeographers are currently expending great effort forecasting shifts in species geographical ranges that may result from climate change. However, these efforts are problematic because they have mostly relied on presence‐only data that ignore within‐species genetic diversity. Technological advances in high‐throughput sequencing have now made it cost‐effective to survey the genetic structure of populations sampled throughout the range of a species. These data can be used to delineate two or more genetic clusters within the species range, and to identify admixtures of individuals within genetic clusters that reflect different patterns of ancestry. Species distribution models (SDMs) applied to the presence and absence of genetic clusters should provide more realistic forecasts of geographical range shifts that take account of genetic variability. High‐throughput sequencing and spatially explicit models may be used to further refine these projections.

It is often assumed that the geographic distributions of species match their climatic tolerances, but this assumption is not frequently tested. Moreover, few studies examine the relative importance of abiotic and biotic factors for limiting species ranges. We combined multiple approaches to assess the extent to which fitness of a widespread native annual legume, Chamaecrista fasciculata, decreases at and beyond its northern and western range edges, and how this is influenced by the presence of neighbors. First, we examined plant fitness and the effect of neighbors in natural populations at different geographic range locations for three years. Fitness decreased toward the northern range edge, but not the western edge. Neighbor removal had a consistently positive effect on seedpod production across all years and sites. Second, we established experimental populations at sites within the range, and at and beyond the northern and western range edges. We tracked individual fitness and recorded seedling recruitment in the following year (a complete generation) to estimate population growth rate. Individual fitness and population growth declined to near zero beyond both range edges, indicating that C. fasciculata with its present genetic composition will not establish in these regions, given conditions currently. We also carried out a neighbor removal treatment. Consistent with the natural populations, neighbors reduced seedpod production of reproductive adults. However, neighbors also increased early-season survival, and this positive effect early in life history resulted in a net positive effect of neighbors on lifetime fitness at most range locations. Our data show that the population growth rate of C. fasciculata includes values above replacement, and populations are well adapted to conditions up to the edge of the range, whereas the severely compromised fitness at sites beyond the edge precludes immediate establishment of populations and thereby impedes adaptation to these conditions.

Improving drought tolerance of crop plants is a major goal of plant breeders. In this study, we characterized biomass and drought-related traits of 220 Medicago truncatula HapMap accessions. Characterized traits included shoot biomass, maximum leaf size, specific leaf weight, stomatal density, trichome density and shoot carbon-13 isotope discrimination (δ(13) C) of well-watered M. truncatula plants, and leaf performance in vitro under dehydration stress. Genome-wide association analyses were carried out using the general linear model (GLM), the standard mixed linear model (MLM) and compressed MLM (CMLM) in TASSEL, which revealed significant overestimation of P-values by CMLM. For each trait, candidate genes and chromosome regions containing SNP markers were found that are in significant association with the trait. For plant biomass, a 0.5 Mbp region on chromosome 2 harbouring a plasma membrane intrinsic protein, PIP2, was discovered that could potentially be targeted to increase dry matter yield. A protein disulfide isomerase-like protein was found to be tightly associated with both shoot biomass and leaf size. A glutamate-cysteine ligase and an aldehyde dehydrogenase family protein with Arabidopsis homologs strongly expressed in the guard cells were two of the top genes identified by stomata density genome-wide association studies analysis.

The availability and quality of mutualists beyond a species' range edge may limit range expansion. With the legume Chamaecrista fasciculata, we asked to what extent the availability and quality of rhizobia beyond the range edge limits host range expansion. We tested the effect of rhizobia availability on plant growth by transplanting seed from three locations into five sites spanning C. fasciculata's range (interior, at the northern and western range edges, and beyond the range edges), and inoculating half the seeds with rhizobia. We recorded growth of all surviving plants, and, for the uninoculated plants, whether they had formed nodules or not. We isolated rhizobia from nodules collected on the uninoculated plants, and cross-inoculated seed from four populations (both range edge and interior populations) in the greenhouse to determine whether the quality of rhizobia differed between regions. We found that seeds transplanted beyond the range edge were less likely to be nodulated when they were not experimentally inoculated, and there was benefit to inoculation at all sites. In the greenhouse, the three inocula that formed nodules on plants, from the range interior, northern edge and beyond the northern edge, did not detectably differ in their effect on plant growth. These results suggest that low densities of suitable rhizobia beyond the range edge may limit range expansion of legume species.

Summary1. Heritability (h 2 ) represents the potential for short-term response of a quantitative trait to selection. Unfortunately, estimating h 2 through traditional crossing experiments is not practical for many species, and even for those in which mating can be manipulated, it may not be possible to assay them in ecologically relevant environments. 2.We evaluated an approach, GCTA, that uses relatedness estimated from genomic data to estimate the proportion of phenotypic variance due to genotyped SNPs, which can be used to infer h 2 . Using phenotypic and genotypic data from eight replicates of experimentally grown plants of the annual legume Medicago truncatula, we examined how h 2 estimates from GCTA (h 2 GCTA ) related to traditional estimates of heritability (clonal repeatability for these inbred lines). Further, we examined how h 2 GCTA estimates were affected by SNP number, minor allele frequency, the number of individuals assayed and the exclusion of causative SNPs. 3. We found that the average h 2 GCTA estimates for each trait made with the full data set (>5 million SNPs, 200 individuals) were strongly correlated (r = 0Á99) with estimates of clonal repeatability. However, this result masks considerable variation among replicate estimates of h 2 GCTA , even in relatively uniform greenhouse conditions. h 2 GCTA estimates with 250 000 and 25 000 SNPs were very similar to those obtained with >5 million SNPs, but with 2500 SNPs, h 2 GCTA were lower and had higher variance than those with ≥25 k SNPs. h 2 GCTA estimates were slightly lower when only common SNPs were used. Excluding putatively causative SNPs had little effect on the estimates of h 2 GCTA , suggesting that genotyping putatively causative SNPs is not necessary to obtain accurate estimates of h 2 . The number of accessions sampled had the greatest effect on h 2 GCTA estimates, and variance greatly increased as fewer accessions were included. With only 50 accessions sampled, the range of h 2 GCTA ranged from 0 to 1 for all traits. 4. These results indicate that the GCTA method may be useful for estimating h 2 using data sets of a size that are available from reduced-representation genotyping but that hundreds of individuals may need to be sampled to obtain robust estimates of h 2 .

Populations are often found on different habitats at different geographic locations. This habitat shift may be due to biased dispersal, physiological tolerances or biotic interactions. To explore how fitness of the native plant Chamaecrista fasciculata depends on habitat within, at and beyond its range edge, we planted seeds from five populations in two soil substrates at these geographic locations. We found that with reduced competition, lifetime fitness was always greater or equivalent in one habitat type, loam soils, though early-season survival was greater on sand soils. At the range edge, natural populations are typically found on sand soil habitats, which are also less competitive environments. Early-season survival and fitness differed among source populations, and when transplanted beyond the range edge, range edge populations had greater fitness than interior populations. Our results indicate that even when the optimal soil substrate for a species does not change with geographic range location, the realized niche of a species may be restricted to sub-optimal habitats at the range edge because of the combined effects of differences in abiotic and biotic effects (e.g. competitors) between substrates.