Environmental response in gene expression and DNA methylation reveals factors influencing the adaptive potential of Arabidopsis lyrata.

Understanding what factors influence plastic and genetic variation is valuable for predicting how organisms respond to changes in the selective environment. Here, using gene expression and DNA methylation as molecular phenotypes, we study environmentally induced variation among Arabidopsis lyrata plants grown at lowland and alpine field sites. Our results show that gene expression is highly plastic, as many more genes are differentially expressed between the field sites than between populations. These environmentally responsive genes evolve under strong selective constraint - the strength of purifying selection on the coding sequence is high, while the rate of adaptive evolution is low. We find, however, that positive selection on cis-regulatory variants has likely contributed to the maintenance of genetically variable environmental responses, but such variants segregate only between distantly related populations. In contrast to gene expression, DNA methylation at genic regions is largely insensitive to the environment, and plastic methylation changes are not associated with differential gene expression. Besides genes, we detect environmental effects at transposable elements (TEs): TEs at the high-altitude field site have higher expression and methylation levels, suggestive of a broad-scale TE activation. Compared to the lowland population, plants native to the alpine environment harbor an excess of recent TE insertions, and we observe that specific TE families are enriched within environmentally responsive genes. Our findings provide insight into selective forces shaping plastic and genetic variation. We also highlight how plastic responses at TEs can rapidly create novel heritable variation in stressful conditions.

Inflorescence shoot elongation, but not flower primordia formation, is photoperiodically regulated in Arabidopsis lyrata.

BACKGROUND AND AIMS: Photoperiod contains information about the progress of seasons. Plants use the changing photoperiod as a cue for the correct timing of important life history events, including flowering. Here the effect of photoperiod on flowering in four Arabidopsis lyrata populations originating from different latitudes was studied, as well as expression levels of candidate genes for governing the between-population differences. METHODS: Flowering of plants from four A. lyrata populations was studied in three different photoperiods after vernalization. Flowering development was separated into three steps: flower primordia formation, inflorescence shoot elongation and opening of the first flower. Circadian expression rhythms of the A. lyrata homologues of GIGANTEA (GI), FLAVIN-BINDING, KELCH REPEAT, F-BOX 1 (FKF1), CONSTANS (CO) and FLOWERING LOCUS T (FT) were studied in three of the populations in the intermediate (14 h) photoperiod treatment. KEY RESULTS: Most plants in all populations formed visible flower primordia during vernalization. Further inflorescence development after vernalization was strongly inhibited by short days in the northern European population (latitude 61°N), only slightly in the central European population (49°N) and not at all in the North American populations (36°N and 42°N). In the 14 h daylength, where all plants from the three southernmost populations but only 60 % of the northernmost population flowered, the circadian expression rhythm of the A. lyrata FT was only detected in the southern populations, suggesting differentiation in the critical daylength for activation of the long-day pathway. However, circadian expression rhythms of A. lyrata GI, FKF1 and CO were similar between populations. CONCLUSIONS: The results indicate that in A. lyrata, transition to flowering can occur through pathways independent of long days, but elongation of inflorescences is photoperiodically regulated.

Role of seed germination in adaptation and reproductive isolation in Arabidopsis lyrata.

Seed germination is an important developmental and life history stage. Yet, the evolutionary impact of germination has mainly been studied in the context of dormancy, or for its role in reproductive isolation between species. Here, we aim to examine multiple consequences of genetic divergence on germination traits between two Arabidopsis lyrata subspecies: ssp. petraea (Eurasia) and ssp. lyrata (North America). Postdormancy germination time, a potentially adaptive trait, showed differentiation between the populations, and quantitative trait loci (QTL) mapping revealed that the trait variation is mainly controlled by two antagonistic loci. These QTL areas contain several candidate genes with known function in postdormancy germination in A. thaliana. The sequence variation of three genes was consistent with differential selection, and they also included fixed nonsynonymous substitutions with potential to account for the phenotypic differentiation. We further show that the divergence between the subspecies has led to a slight but significant reduction in hybrid germination proportions, indicating incipient reproductive isolation. Comparison of reciprocal F1 and F2 progenies suggests that Bateson-Dobzhansky-Muller incompatibilities likely act through uniparentally inherited factors. Examination of genomewide transmission ratio distortion further revealed that cytonuclear interactions cause substantial pregermination inviability in the hybrids. These results confirm that seed germination has adaptive potential beyond the dormancy stage and that hybrid seed inviability can be one of the first reproductive barriers to arise during divergence.

Selection for population-specific adaptation shaped patterns of variation in the photoperiod pathway genes in Arabidopsis lyrata during post-glacial colonization.

Spatially varying selection can lead to population-specific adaptation, which is often recognized at the phenotypic level; however, the genetic evidence is weaker in many groups of organisms. In plants, environmental shifts that occur due to colonization of a novel environment may require adaptive changes in the timing of growth and flowering, which are often governed by location-specific environmental cues such as day length. We studied locally varying selection in 19 flowering time loci in nine populations of the perennial herb Arabidopsis lyrata, which has a wide but patchy distribution in temperate and boreal regions of the northern hemisphere. The populations differ in their recent population demographic and colonization histories and current environmental conditions, especially in the growing season length. We searched for population-specific molecular signatures of directional selection by comparing a set of candidate flowering time loci with a genomic reference set within each population using multiple approaches and contrasted the patterns of different populations. The candidate loci possessed approximately 20% of the diversity of the reference loci. On average the flowering time loci had more rare alleles (a smaller Tajima's D) and an excess of highly differentiated sites relative to the reference, suggesting positive selection. The strongest signal of selection was detected in photoperiodic pathway loci in the colonizing populations of Northwestern Europe, whereas no evidence of positive selection was detected in the Central European populations. These findings emphasized the population-specific nature of selection and suggested that photoperiodic adaptation was important during postglacial colonization of the species.

Genetic changes in flowering and morphology in response to adaptation to a high-latitude environment in Arabidopsis lyrata.

BACKGROUND AND AIMS: The adaptive plastic reactions of plant populations to changing climatic factors, such as winter temperatures and photoperiod, have changed during range shifts after the last glaciation. Timing of flowering is an adaptive trait regulated by environmental cues. Its genetics has been intensively studied in annual plants, but in perennials it is currently not well characterized. This study examined the genetic basis of differentiation in flowering time, morphology, and their plastic responses to vernalization in two locally adapted populations of the perennial Arabidopsis lyrata: (1) to determine whether the two populations differ in their vernalization responses for flowering phenology and morphology; and (2) to determine the genomic areas governing differentiation and vernalization responses. METHODS: Two A. lyrata populations, from central Europe and Scandinavia, were grown in growth-chamber conditions with and without cold treatment. A QTL analysis was performed to find genomic regions that interact with vernalization. KEY RESULTS: The population from central Europe flowered more rapidly and invested more in inflorescence growth than the population from alpine Scandinavia, especially after vernalization. The alpine population had consistently a low number of inflorescences and few flowers, suggesting strong constraints due to a short growing season, but instead had longer leaves and higher leaf rosettes. QTL mapping in the F2 population revealed genomic regions governing differentiation in flowering time and morphology and, in some cases, the allelic effects from the two populations on a trait were influenced by vernalization (QTL × vernalization interactions). CONCLUSIONS: The results indicate that many potentially adaptive genetic changes have occurred during colonization; the two populations have diverged in their plastic responses to vernalization in traits closely connected to fitness through changes in many genomic areas.

Role of vernalization and of duplicated FLOWERING LOCUS C in the perennial Arabidopsis lyrata.

FLOWERING LOCUS C (FLC) is one of the main genes influencing the vernalization requirement and natural flowering time variation in the annual Arabidopsis thaliana. Here we studied the effects of vernalization on flowering and its genetic basis in the perennial Arabidopsis lyrata. Two tandemly duplicated FLC genes (FLC1 and FLC2) were compared with respect to expression and DNA sequence. The effect of vernalization on flowering and on the expression of FLC1 was studied in three European populations. The genetic basis of the FLC1 expression difference between two of the populations was further studied by expression quantitative trait locus (eQTL) mapping and sequence analysis. FLC1 was shown to have a likely role in the vernalization requirement for flowering in A. lyrata. Vernalization decreased its expression and the northern study populations showed higher FLC1 expression than the southern one. eQTL mapping between two of the populations revealed one eQTL affecting FLC1 expression in the genomic region containing the FLC genes. Most FLC1 sequence differences between the study populations were found in the promoter region and in the first intron. Variation in the FLC1 sequence may cause differences in FLC1 expression between late- and early-flowering A. lyrata populations.

Natural variation in Arabidopsis lyrata vernalization requirement conferred by a FRIGIDA indel polymorphism.

Species share homologous genes to a large extent, but it is not yet known to what degree the same loci have been targets for natural selection in different species. Natural variation in flowering time is determined to a large degree by 2 genes, FLOWERING LOCUS C and FRIGIDA, in Arabidopsis thaliana. Here, we examine whether FRIGIDA has a role in differences in flowering time between and within natural populations of Arabidopsis lyrata, a close outcrossing perennial relative of A. thaliana. We found 2 FRIGIDA sequence variants producing potentially functional proteins but with a length difference of 14 amino acids. These variants conferred a 15-day difference in flowering time in an association experiment in 2 Scandinavian populations. The difference in flowering time between alleles was confirmed with transformation to A. thaliana. Because the north European late-flowering populations harbor both late- and early sequence variants at intermediate frequencies and the late-flowering variant is most frequent in the southern early flowering European population, other genetic factors must be responsible for the flowering time differences between the populations. The length polymorphism occurs at high frequencies also in several North American populations. The occurrence of functional variants at intermediate frequencies in several populations suggests that the variation may be maintained by balancing selection. This is in contrast to A. thaliana, where independent loss-of-function mutations at the FRIGIDA gene are responsible for differences between populations and local adaptation.

Comparative gene mapping in Arabidopsis lyrata chromosomes 1 and 2 and the corresponding A. thaliana chromosome 1: recombination rates, rearrangements and centromere location.

To add detail to the genetic map of Arabidopsis lyrata, and compare it with that of A. thaliana, we have developed many additional markers in the A. lyrata linkage groups, LG1 and LG2, corresponding to A. thaliana chromosome 1. We used a newly developed method for marker development for single nucleotide polymorphisms present in gene sequences, plus length differences, to map genes in an A. lyrata family, including variants in several genes close to the A. thaliana centromere 1, providing the first data on the location of an A. lyrata centromere; we discuss the implications for the evolution of chromosome 1 of A. thaliana. With our larger marker density, large rearrangements between the two Arabidopsis species are excluded, except for a large inversion on LG2. This was previously known in Capsella; its presence in A. lyrata suggests that, like most other rearrangements, it probably arose in the A. thaliana lineage. Knowing that marker orders are similar, we can now compare homologous, non-rearranged map distances to test the prediction of more frequent crossing-over in the more inbreeding species. Our results support the previous conclusion of similar distances in the two species for A. lyrata LG1 markers. For LG2 markers, the distances were consistently, but non-significantly, larger in A. lyrata. Given the two species' large DNA content difference, the similarity of map lengths, particularly for LG1, suggests that crossing-over is more frequent across comparable physical distances in the inbreeder, A. thaliana, as predicted.

Studying genetics of adaptive variation in model organisms: flowering time variation in Arabidopsis lyrata.

Arabidopsis thaliana has emerged as a model organism for plant developmental genetics, but it is also now being widely used for population genetic studies. Outcrossing relatives of A. thaliana are likely to provide suitable additional or alternative species for studies of evolutionary and population genetics. We have examined patterns of adaptive flowering time variation in the outcrossing, perennial A. lyrata. In addition, we examine the distribution of variation at marker genes in populations form North America and Europe. The probability of flowering in this species differs between southern and northern populations. Northern populations are much less likely to flower in short than in long days. A significant daylength by region interaction shows that the northern and southern populations respond differently to the daylength. The timing of flowering also differs between populations, and is made shorter by long days, and in some populations, by vernalization. North American and European populations show consistent genetic differentiation over microsatellite and isozyme loci and alcohol dehydrogenase sequences. Thus, the patterns of variation are quite different from those in A. thaliana, where flowering time differences show little relationship to latitude of origin and the genealogical trees of accessions vary depending on the genomic region studied. The genetic architecture of adaptation can be compared in these species with different life histories.

Comparing the linkage maps of the close relatives Arabidopsis lyrata and A. thaliana.

We have constructed a genetic map of Arabidopsis lyrata, a self-incompatible relative of the plant model species A. thaliana. A. lyrata is a diploid (n = 8) species that diverged from A. thaliana (n = 5) approximately 5 MYA. Mapping was conducted in a full-sib progeny of two unrelated F(1) hybrids between two European populations of A. lyrata ssp. petraea. We used the least-squares method of the Joinmap program for map construction. The gross chromosomal differences between the two species were most parsimoniously explained with three fusions, two reciprocal translocations, and one inversion. The total map length was 515 cM, and the distances were 12% larger than those between corresponding markers in the linkage map of A. thaliana. The 72 markers, consisting of microsatellites and gene-based markers, were spaced on average every 8 cM. Transmission ratio distortion was extensive, and most distortions were specific to each reciprocal cross, suggesting cytoplasmic interactions. We estimate locations and most probable genotype frequencies of transmission ratio distorting loci (TRDL) with a Bayesian method and discuss the possible reasons for the observed distortions.

Quantifying latitudinal clines to light responses in natural populations of Arabidopsis thaliana (Brassicaceae).

Evidence of adaptation in Arabidopsis thaliana (Brassicaceae) phenotypic traits has rarely been shown. We demonstrate latitudinal clines in two A. thaliana traits: hypocotyl responses to red and far-red light. Natural populations of A. thaliana were sampled along a latitudinal gradient from southern to northern Norway. Seeds from maternal families within each population were subjected to 1 wk of constant red, far-red, blue, white, and dark treatments. Hypocotyl lengths were measured for each maternal family within each population. Significant variability within and among populations in hypocotyl responses for the various treatments was found. There was a significant latitudinal cline in hypocotyl responses for red and far-red treatments, with northern populations being more de-etiolated than southern populations. These results suggest that northern populations are more responsive to red and far-red light than southern populations. Thus, differentiation of seedling traits in natural populations of A. thaliana seems in part to be mediated by the phytochrome pathway. There was no correlation between hypocotyl responses and flowering time for any treatment. This suggests that flowering time variability and variability in hypocotyl responses may not be governed by genes shared between the pathways, such as those involved in photoreception or the circadian clock.

GENETIC BASIS OF INBREEDING DEPRESSION IN ARABIS PETRAEA.

Inbreeding depression may be caused by (partially) recessive or overdominant gene action. The relative evolutionary importance of these two modes has been debated; the former mode is emphasized in the "dominance hypothesis," the latter in the "overdominance hypothesis." We analyzed the genetic basis of inbreeding depression in the self-incompatible herb Arabis petraea (L.) Lam.: In the selfed progeny of twelve parental plants, we studied the proportion of chlorophyll-deficient seedlings, the genotypic distributions of marker genes, and associations of marker genotypes with viability and quantitative traits. Early components of fitness were examined by scoring seed size, germination time, and early growth rate and by observing the proportion of chlorophyll-deficient seedlings. Later components of fitness, flowering, and root and aboveground biomass were also measured. Marker genotypes of young seedlings were scored for 11 enzyme loci and three microsatellite markers. We found a high proportion (about 70%) of families with chlorophyll-deficient seedlings, indicating a high mutational load. We found six significant deviations from 1:2:1 ratio at marker loci of 60 tests in seedlings, with three of these significant at the experimentwide level. Deviations from the expected ratio were assumed to be due to linked viability loci. A graphical and a Bayesian method were used to distinguish between the overdominance and dominance hypotheses. Most of the deviant segregation ratios suggested overdominance instead of recessivity of the deleterious allele. Neither the early (seed size, germination time, or early growth trait) nor the late quantitative traits (flowering, and root and aboveground biomass) showed significant linkage to markers at the experimentwide level. Presence of significant associations between markers and early viability, but lack thereof for quantitative traits expressed late, suggests either that there may be relatively low inbreeding depression in later life stages or that individual quantitative trait loci may have smaller effects than loci contributing to early viability.

Picea omorika is a self-fertile but outcrossing conifer.

Outcrossing rates were estimated in a natural Yugoslavian and in a cultivated Finnish population of Serbian spruce [Picea omorika (Pancic) Purk.]. The outcrossing rates in the cultivated stand in two years were 0.98 ±0.03 and 1.02 ±0.04, and in the natural stand 0.84 ±0.05. The relative self-fertility was estimated in seven trees in the cultivated population. The results indicate high self-fertility, which agrees with the earlier information. The high self-fertility combined with high outcrossing rate shows that Serbian spruce, in contrast to most other conifers, employs other means than early acting inbreeding depression to avoid selfing.