Highly structured nucleotide variation within and among Arabidopsis lyrata populations at the FAH1 and DFR gene regions

Nucleotide variation at the FAH1 and DFR gene regions was surveyed in four populations of Arabidopsis lyrata (two European A. l. petraea and two North American A. l. lyrata populations). In contrast to previous results, levels of variation were not consistently lower in A. l. lyrata than in A. l. petraea, and similar degrees of genetic differentiation were detected between and within subspecies. These observations and the significant genetic differentiation detected among populations suggest population substructure and no real subdivision between subspecies. For each gene studied, genotypic data were obtained, which allowed comparing nucleotide diversity within individuals (between sequences from the same individual) and within populations (between sequences from the same population). The generally lower level of variation within than among individuals detected in each population yielded a significant deviation from panmixia within populations. In three of the four populations studied, two highly divergent alleles were detected within populations at the highly variable DFR locus. This pattern and the significant excess of derived variants detected in most populations suggest that most variation segregating within populations results from rare migration events between relatively small and isolated populations exhibiting reduced panmixia.     

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

Species share homologous genes to a large extent, but it isnot yet known to what degree the same loci have been targetsfor natural selection in different species. Natural variationin 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 floweringtime between and within natural populations of Arabidopsis lyrata,a close outcrossing perennial relative of A. thaliana. We found2 FRIGIDA sequence variants producing potentially functionalproteins but with a length difference of 14 amino acids. Thesevariants conferred a 15-day difference in flowering time inan association experiment in 2 Scandinavian populations. Thedifference in flowering time between alleles was confirmed withtransformation to A. thaliana. Because the north European late-floweringpopulations harbor both late- and early sequence variants atintermediate frequencies and the late-flowering variant is mostfrequent in the southern early flowering European population,other genetic factors must be responsible for the floweringtime differences between the populations. The length polymorphismoccurs at high frequencies also in several North American populations.The occurrence of functional variants at intermediate frequenciesin several populations suggests that the variation may be maintainedby balancing selection. This is in contrast to A. thaliana,where independent loss-of-function mutations at the FRIGIDAgene are responsible for differences between populations andlocal adaptation.     

MAM3 catalyzes the formation of all aliphatic glucosinolate chain lengths in Arabidopsis

Chain elongated, methionine (Met)-derived glucosinolates are a major class of secondary metabolites in Arabidopsis (Arabidopsis thaliana). The key enzymatic step in determining the length of the chain is the condensation of acetyl-coenzyme A with a series of omega-methylthio-2-oxoalkanoic acids, catalyzed by methylthioalkylmalate (MAM) synthases. The existence of two MAM synthases has been previously reported in the Arabidopsis ecotype Columbia: MAM1 and MAM3 (formerly known as MAM-L). Here, we describe the biochemical properties of the MAM3 enzyme, which is able to catalyze all six condensation reactions of Met chain elongation that occur in Arabidopsis. Underlining its broad substrate specificity, MAM3 also accepts a range of non-Met-derived 2-oxoacids, e.g. converting pyruvate to citramalate and 2-oxoisovalerate to isopropylmalate, a step in leucine biosynthesis. To investigate its role in vivo, we identified plant lines with mutations in MAM3 that resulted in a complete lack or greatly reduced levels of long-chain glucosinolates. This phenotype could be complemented by reintroduction of a MAM3 expression construct. Analysis of MAM3 mutants demonstrated that MAM3 catalyzes the formation of all glucosinolate chain lengths in vivo as well as in vitro, making this enzyme the major generator of glucosinolate chain length diversity in the plant. The localization of MAM3 in the chloroplast suggests that this organelle is the site of Met chain elongation.     

Gene, phenotype and function: GLABROUS1 and resistance to herbivory in natural populations of Arabidopsis lyrata

The molecular genetic basis of adaptive variation is of fundamental importance for evolutionary dynamics, but is still poorly known. Only in very few cases has the relationship between genetic variation at the molecular level, phenotype and function been established in natural populations. We examined the functional significance and genetic basis of a polymorphism in production of leaf hairs, trichomes, in the perennial herb Arabidopsis lyrata. Earlier studies suggested that trichome production is subject to divergent selection. Here we show that the production of trichomes is correlated with reduced damage from insect herbivores in natural populations, and using statistical methods developed for medical genetics we document an association between loss of trichome production and mutations in the regulatory gene GLABROUS1. Sequence data suggest that independent mutations in this regulatory gene have provided the basis for parallel evolution of reduced resistance to insect herbivores in different populations of A. lyrata and in the closely related Arabidopsis thaliana. The results show that candidate genes identified in model organisms provide a valuable starting point for analysis of the genetic basis of phenotypic variation in natural populations.     

Genetic variation within and among populations of Arabidopsis thaliana.

We investigated levels of nucleotide polymorphism within and among populations of the highly self-fertilizing Brassicaceous species, Arabidopsis thaliana. Four-cutter RFLP data were collected at one mitochondrial and three nuclear loci from 115 isolines representing 11 worldwide population collections, as well as from seven commonly used ecotypes. The collections include multiple populations from North America and Eurasia, as well as two pairs of collections from locally proximate sites, and thus allow a hierarchical geographic analysis of polymorphism. We found no variation at the mitochondrial locus Nad5 and very low levels of intrapopulation nucleotide diversity at Adh, Dhs1, and Gpa1. Interpopulation nucleotide diversity was also consistently low among the loci, averaging 0.0014. gst, a measure of population differentiation, was estimated to be 0.643. Interestingly, we found no association between geographical distance between populations and genetic distance. Most haplotypes have a worldwide distribution, suggesting a recent expansion of the species or long-distance gene flow. The low level of polymorphism found in this study is consistent with theoretical models of neutral mutations and background selection in highly self-fertilizing species.     

Latitudinal trait variation and responses to drought in Arabidopsis lyrata

Species may respond in three ways to environmental change: adapt, migrate, or go extinct. Studies of latitudinal clines can provide information on whether species have adapted to abiotic stress such as temperature and drought in the past and what the traits underlying adaptation are. We investigated latitudinal trait variation and response to drought in North American populations of Arabidopsis lyrata. Plants from nine populations collected over 13° latitude were grown under well-watered and dry conditions. A total of 1,620 seedlings were raised and 12 phenological, physiological, morphological, and life history traits were measured. Two traits, asymptotic rosette size and the propensity to flower, were significantly associated with latitude: plants from northern locations grew to a larger size and were more likely to flower in the first season. Most traits displayed a plastic response to drought, but plasticity was never related linearly with latitude nor was it enhanced in populations from extreme latitudes with reduced water availability. Populations responded to drought by adopting mixed strategies of resistance, tolerance, and escape. The study shows that latitudinal adaptation in A. lyrata involves the classic life history traits, size at and timing of reproduction. Contrary to recent theoretical predictions, adaptation to margins is based on fixed trait differences and not on phenotypic plasticity, at least with respect to drought.     

Genetic control of natural variation in Arabidopsis glucosinolate accumulation

Glucosinolates are biologically active secondary metabolites of the Brassicaceae and related plant families that influence plant/insect interactions. Specific glucosinolates can act as feeding deterrents or stimulants, depending upon the insect species. Hence, natural selection might favor the presence of diverse glucosinolate profiles within a given species. We determined quantitative and qualitative variation in glucosinolates in the leaves and seeds of 39 Arabidopsis ecotypes. We identified 34 different glucosinolates, of which the majority are chain-elongated compounds derived from methionine. Polymorphism at only five loci was sufficient to generate 14 qualitatitvely different leaf glucosinolate profiles. Thus, there appears to be a modular genetic system regulating glucosinolate profiles in Arabidopsis. This system allows the rapid generation of new glucosinolate combinations in response to changing herbivory or other selective pressures. In addition to the qualitative variation in glucosinolate profiles, we found a nearly 20-fold difference in the quantity of total aliphatic glucosinolates and were able to identify a single locus that controls nearly three-quarters of this variation.     

Patterns of genetic diversity in outcrossing and selfing populations of Arabidopsis lyrata

Arabidopsis lyrata is normally considered an obligately outcrossing species with a strong self-incompatibility system, but a shift in mating system towards inbreeding has been found in some North American populations (subspecies A. lyrata ssp. lyrata). This study provides a survey of the Great Lakes region of Canada to determine the extent of this mating system variation and how outcrossing rates are related to current population density, geographical distribution, and genetic diversity. Based on variation at microsatellite markers (progeny arrays to estimate multilocus outcrossing rates and population samples to estimate diversity measures) and controlled greenhouse pollinations, populations can be divided into two groups: (i) group A, consisting of individuals capable of setting selfed seed (including autogamous fruit set in the absence of pollinators), showing depressed outcrossing rates (T(m) = 0.2-0.6), heterozygosity (H(O) = 0.02-0.06) and genetic diversity (H(E) = 0.08-0.10); and (ii) group B, consisting of individuals that are predominantly self-incompatible (T(m) > 0.8), require pollinators for seeds set, and showing higher levels of heterozygosity (H(O) = 0.13-0.31) and diversity (H(E) = 0.19-0.410). Current population density is not related to the shift in mating system but does vary with latitude. Restricted gene flow among populations was evident among all but two populations (F(ST) = 0.11-0.8). Group A populations were more differentiated from one another (F(ST) = 0.78) than they were from group B populations (F(ST) = 0.59), with 41% of the variation partitioned within populations, 47% between populations, and 12% between groups. No significant relationship was found between genetic and geographical distance. Results are discussed in the context of possible postglacial expansion scenarios in relation to loss of self-incompatibility.     

Local adaptation in European populations of Arabidopsis lyrata (Brassicaceae)

We studied local adaptation to contrasting environments using an organism that is emerging as a model for evolutionary plant biology-the outcrossing, perennial herb Arabidopsis lyrata subsp. petraea (Brassicaceae). With reciprocal transplant experiments, we found variation in cumulative fitness, indicating adaptive differentiation among populations. Nonlocal populations did not have significantly higher fitness than the local population. Experimental sites were located in Norway (alpine), Sweden (coastal), and Germany (continental). At all sites after one year, the local population had higher cumulative fitness, as quantified by survival combined with rosette area, than at least one of the nonlocal populations. At the Norwegian site, measurements were done for two additional years, and fitness differences persisted. The fitness components that contributed most to differences in cumulative fitness varied among sites. Relatively small rosette area combined with a large number of inflorescences produced by German plants may reflect differentiation in life history. The results of the current study demonstrate adaptive population differentiation in A. lyrata along a climatic gradient in Europe. The studied populations harbor considerable variation in several characters contributing to adaptive population differentiation. The wealth of genetic information available makes A. lyrata a highly attractive system also for examining the functional and genetic basis of local adaptation in plants.     

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.     

Intraspecfic variation in cold-temperature metabolic phenotypes of Arabidopsis lyrata ssp. petraea

Atmospheric temperature is a key factor in determining the distribution of a plant species. Alongside this, plant populations growing at the margin of their range may exhibit traits that indicate genetic differentiation and adaptation to their local abiotic environment. We investigated whether geographically separated marginal populations of Arabidopsis lyrata ssp. petraea have distinct metabolic phenotypes associated with exposure to cold temperatures. Seeds of A. petraea were obtained from populations along a latitudinal gradient, namely Wales, Sweden and Iceland and grown in a controlled cabinet environment. Mannose, glucose, fructose, sucrose and raffinose concentrations were different between cold treatments and populations, especially in the Welsh population, but polyhydric alcohol concentrations were not. The free amino acid compositions were population specific, with fold differences in most amino acids, especially in the Icelandic populations, with gross changes in amino acids, particularly those associated with glutamine metabolism. Metabolic fingerprints and profiles were obtained. Principal component analysis (PCA) of metabolite fingerprints revealed metabolic characteristic phenotypes for each population and temperature. It is suggested that amino acids and carbohydrates were responsible for discriminating populations within the PCA. Metabolite fingerprinting and profiling has proved to be sufficiently sensitive to identify metabolic differences between plant populations at different atmospheric temperatures. These findings show that there is significant natural variation in cold metabolism among populations of A. l. petraea which may signify plant adaptation to local climates. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Trichome production and spatiotemporal variation in herbivory in the perennial herb Arabidopsis lyrata

Allocation theory suggests that the optimal level of resistance against herbivores should vary with the risk of herbivory if allocation to resistance is costly. The perennial herb Arabidopsis lyrata has a genetically based polymorphism for trichome production and occurs in a glabrous and a trichome-producing form. Leaf trichomes (hairs) can protect plants against insect herbivores, and may increase tolerance to drought and UV-radiation. To examine the functional significance of trichome production, we documented the frequency of glabrous plants and damage by insect herbivores in 30 A. lyrata populations in Sweden and Norway. The proportion of glabrous plants ranged from 0.10 to 0.71 (median=0.44) in polymorphic populations; 7 of 12 populations in Norway and 14 of 18 populations in Sweden were monomorphic glabrous, i.e. with fewer than 5% trichome-producing plants. The mean proportion of the leaf area removed by herbivores varied substantially among populations and years. With few exceptions, glabrous plants were more damaged than trichome-producing plants in polymorphic populations. The intensity of herbivory quantified as the mean damage to glabrous plants tended to be higher in polymorphic populations than in populations monomorphic for the glabrous morph and was higher in Sweden than in Norway. In Norway, both the magnitude of herbivore damage and the frequency of trichome-producing plants tended to decrease with increasing altitude. The results indicate that leaf trichomes contribute to resistance against herbivorous insects in A. lyrata , and suggest that herbivore-mediated selection contributes to the maintenance of the polymorphism in trichome production.     

Geographical variation in the response to nitrogen deposition in Arabidopsis lyrata petraea

The adaptive responses to atmospheric nitrogen deposition for different European accessions of Arabidopsis lyrata petraea were analysed using populations along a strong atmospheric N-deposition gradient. Plants were exposed to three N-deposition rates, reflecting the rates at the different locations, in a full factorial design. Differences between accessions in the response to N were found for important phenological and physiological response variables. For example, plants from low-deposition areas had higher nitrogen-use efficiencies (NUE) and C : N ratios than plants from areas high in N deposition when grown at low N-deposition rates. The NUE decreased in all accessions at higher experimental deposition rates. However, plants from high-deposition areas showed a limited capacity to increase their NUE at lower experimental deposition rates. Plants from low-deposition areas had faster growth rates, higher leaf turnover rates and shorter times to flowering, and showed a greater increase in growth rate in response to N deposition than those from high-deposition areas. Indications for adaptation to N deposition were found, and results suggest that adaptation of plants from areas high in N deposition to increased N deposition has resulted in the loss of plasticity.     

Haplotype structure of the stigmatic self-incompatibility gene in natural populations of Arabidopsis lyrata

We describe analyses of almost full-length sequences (including both the kinase domain and the S-domain) of the putative SRK incompatibility gene of the self-incompatible plant Arabidopsis lyrata. In A. lyrata, the SRK S-domain controls the pistil recognition specificity, as in self-incompatible Brassica species. In alleles from plants derived from natural A. lyrata populations, nonsynonymous and synonymous site diversity values are very high in both domains; even in exons 3 to 7 of the kinase domain, which probably have no recognition functions, 39% of the amino acids are polymorphic. Within populations, diversity between alleles is high, as expected for an incompatibility locus, which should be under frequency-dependent selection within populations, whereas within the different putative allelic classes polymorphism is very low, as predicted from theoretical models when recombination is rare. Nonsynonymous site variability declines in the kinase domain with increasing distance from the S-domain border, although synonymous diversity remains high, and the introns are unalignable. A decline in nonsynonymous diversity is expected due to selective constraints in the kinase domain, in combination with recombination (allowing diversity to decrease at sites distant from those under balancing selection). However, it is unclear whether recombination occurs in the SRK locus, and interpretation of the observed diversity pattern is complicated by apparent gene conversion with a paralogous gene (or genes). Patterns of linkage disequilibrium in our SRK sequences do not support the conclusion that recombination occurs, which was suggested from previous analyses based on Brassica SLG sequences.     

Allozyme variation within and between populations inLolium (Poaceae)

Isozyme analysis was used to determine genetic variation within and between populations of sevenLolium species. All populations from the inbreeding species (L. temulentum, L. remotum, L. loliaceum, andL. persicum) were completely fixed for all enzymes scored. They also contained, for four of the five enzyme systems studied, exactly the same allelic variant. The three cross-breeding species showed large within-population variation and much less between-population variation. The great similarity of the allozymic variants found in all species, made the division of the genusLolium into species on basis of allozymic data difficult. It was not possible to separate the different inbreeding species from each other. Within the cross-breeding groupL. multiflorum andL. rigidum could be distinguished fromL. perenne. L. multiflorum, andL. rigidum could, with more difficulty, also be separated from each other. Allelic variation could have more relation with the provenance of the populations than with taxonomic classification.Genetic variation inLolium spp. II. For first part see Pl. Syst. Evol. 188: 87–99.     

A heritable glucosinolate polymorphism within natural populations of Barbarea vulgaris

In natural populations of Barbarea vulgaris we found two distinctly different glucosinolate profiles. The most common glucosinolate profile is dominated (94%) by the hydroxylated form, (S)-2-hydroxy-2-phenylethyl-glucosinolate (glucobarbarin, BAR-type), whereas in the other type 2-phenylethyl-glucosinolate (gluconasturtiin, NAS-type) was most prominent (82%). NAS-type plants have a 108-fold increase of gluconasturtiin concentration in rosette leaves compared to BAR-type plants. The glucosinolate composition of both chemotypes is consistent throughout all plant organs and after induction with jasmonic acid. Although the glucosinolate profile of the roots has a more diverse composition than other plant organs, it still matches the chemotype. In 12 natural populations that we sampled in Germany, Belgium, France and Switzerland solely BAR-type plants were found. However, eight out of the 15 Dutch populations that were sampled contained 2-22% NAS-type plants. Controlled crosses showed that the chemotype was heritable and determined by a single gene with two alleles. The allele coding for the BAR-type was dominant and the allele for the NAS-type was recessive. The different glucosinolate profiles will yield different hydrolysis products upon damage, and therefore we expect them to differentially affect the multitrophic interactions associated with B. vulgaris in their natural environment.     

Environmental and genetic effects on flowering differences between northern and southern populations of Arabidopsis lyrata (Brassicaceae)

Arabidopsis lyrata (Brassicaceae) is a close outcrossing relative of A. thaliana. We examine flowering time variation of northern and southern A. lyrata populations in controlled environmental conditions, in a common garden experiment with A. thaliana, and in the field. Southern populations of A. lyrata flowered earlier than northern ones in all environmental conditions. Individuals from southern populations were more likely to flower in short days (14 h light) than northern ones, and all populations had a higher probability of flowering and flowered more rapidly in long days (20 h). The interaction of population and day length significantly affected flowering probability, and flowering time in one of two comparisons. The common garden experiment demonstrated differences between populations in the response to seed cold treatment, but growth chamber experiments showed no vernalization effect after 4 wk of rosette cold treatment. In a field population in Norway, a high proportion of the plants flowered in each year of the study. The plants progressed to flowering more rapidly in the field and common garden than in the growth chamber. The genetic basis of these flowering time differences here can be further studied using A. thaliana genetic tools.