How do threatened plant species with low genetic diversity respond to environmental stress? Insights from comparative conservation epigenomics and phenotypic plasticity.

Many threatened plants have low genetic diversity, which may reduce their capacity for genetically based adaptation, increasing their extinction risk. Non-genetic variation (e.g. epigenomic modifications such as DNA methylation) and plasticity may facilitate the persistence of threatened plants, yet are rarely incorporated into conservation assessments. We present a case study investigating variation and plasticity in DNA methylation and phenotypic traits in four genetically depauperate species of Leavenworthia (Brassicaceae), including one widespread species and one asexual, threatened species. We grew individuals from several maternal lines and populations per species in contrasting watering treatments, measured phenotypic traits and analysed DNA methylation using whole-genome bisulphite sequencing. We addressed four questions: (1) How do patterns of DNA methylation differ within and among species? (2) Within species, how do phenotypic traits and patterns of DNA methylation vary in response to drought? (3) Does variation in DNA methylation correspond to phenotypic variation? (4) What are the implications for conservation? We found that taxa were epigenomically distinct and that each species exhibited variation in DNA methylation among populations that could be relevant for conservation. Within species, the DNA methylation response to environmental stress corresponded to its phenotypic response. Species differed in their DNA methylation and phenotypic responses to environmental stress, with the extent of plasticity possibly related to species geographic range size. We also found phenotypic and DNA methylation variation in the asexual, threatened species that may be relevant for conservation. Our results suggest that variation in DNA methylation may promote the persistence of genetically depauperate threatened plants, highlighting its potential as a novel conservation target to reduce extinction risk.

Beyond the usual climate? Factors determining flowering and fruiting phenology across a genus over 117 years.

PREMISE: Although changes in plant phenology are largely attributed to changes in climate, the roles of other factors such as genetic constraints, competition, and self-compatibility are underexplored. METHODS: We compiled >900 herbarium records spanning 117 years for all eight nominal species of the winter-annual genus Leavenworthia (Brassicaceae). We used linear regression to determine the rate of phenological change across years and phenological sensitivity to climate. Using a variance partitioning analysis, we assessed the relative influence of climatic and nonclimatic factors (self-compatibility, range overlap, latitude, and year) on Leavenworthia reproductive phenology. RESULTS: Flowering advanced by ~2.0 days and fruiting by ~1.3 days per decade. For every 1°C increase in spring temperature, flowering advanced ~2.3 days and fruiting ~3.3 days. For every 100 mm decrease in spring precipitation, each advanced ~6-7 days. The best models explained 35.4% of flowering variance and 33.9% of fruiting. Spring precipitation accounted for 51.3% of explained variance in flowering date and 44.6% in fruiting. Mean spring temperature accounted for 10.6% and 19.3%, respectively. Year accounted for 16.6% of flowering variance and 5.4% of fruiting, and latitude for 2.3% and 15.1%, respectively. Nonclimatic variables combined accounted for <11% of the variance across phenophases. CONCLUSIONS: Spring precipitation and other climate-related factors were dominant predictors of phenological variance. Our results emphasize the strong effect of precipitation on phenology, especially in the moisture-limited habitats preferred by Leavenworthia. Among the many factors that determine phenology, climate is the dominant influence, indicating that the effects of climate change on phenology are expected to increase.

Self-incompatibility and the genetic architecture of inbreeding depression.

Inbreeding depression plays a fundamental role in evolution. To help detect and characterize the loci that underlie inbreeding depression, we used bud pollination and salt treatments to circumvent self-incompatibility (SI) in plants from populations of Leavenworthia alabamica and produced families of progeny that were then genotyped at genetically mapped single-nucleotide polymorphism (SNP) loci. Using Bayesian inference, the segregation patterns for each SNP were used to explore support for different dominance and selection coefficients at linked viability loci in different genomic regions. There was support for several partially recessive viability loci in one of the populations, and one such locus mapped to the genomic region of the novel SI locus in L. alabamica. These results are consistent with earlier findings that showed purging of inbreeding depression for germination rate in L. alabamica. They are also consistent with expectations from evolutionary genetic theory that recessive, deleterious alleles linked to loci under balancing selection can be sheltered from selection.

Inbreeding depression is difficult to purge in self-incompatible populations of Leavenworthia alabamica.

The extent to which inbreeding depression can be purged is a major determinant of mating system evolution and is important to conservation and crop improvement. Studies of inbreeding depression purging have not been conducted in self-incompatible plants before. An experimental ('ancestral') treatment was first created from self-incompatible plants of Leavenworthia alabamica. Lines derived from this population were maintained by self-pollination for three generations in the attempt to create a 'purged' population with fewer recessive, deleterious mutations of large effect. Fitness components and the frequency of malformed phenotypes were monitored in progeny derived from selfing and outcrossing in the ancestral and purged treatments. Fitness component means and inbreeding depression were largely unchanged by three generations of forced self-pollination, and there was no reduction in the frequency of plants exhibiting malformed phenotypes. Our findings indicate that inbreeding depression in this species is largely a result of mutations of mild effect, consistent with the observation that self-incompatibility is maintained in most populations of L. alabamica, despite the presence of genetic variants with weaker self-incompatibility. Moreover, although population theory suggests that deleterious mutations of large effect should be sheltered from selection in the region of self-incompatibility locus, our results do not support this prediction.

The unusual S locus of Leavenworthia is composed of two sets of paralogous loci.

The Leavenworthia self-incompatibility locus (S locus) consists of paralogs (Lal2, SCRL) of the canonical Brassicaceae S locus genes (SRK, SCR), and is situated in a genomic position that differs from the ancestral one in the Brassicaceae. Unexpectedly, in a small number of Leavenworthia alabamica plants examined, sequences closely resembling exon 1 of SRK have been found, but the function of these has remained unclear. BAC cloning and expression analyses were employed to characterize these SRK-like sequences. An SRK-positive Bacterial Artificial Chromosome clone was found to contain complete SRK and SCR sequences located close by one another in the derived genomic position of the Leavenworthia S locus, and in place of the more typical Lal2 and SCRL sequences. These sequences are expressed in stigmas and anthers, respectively, and crossing data show that the SRK/SCR haplotype is functional in self-incompatibility. Population surveys indicate that < 5% of Leavenworthia S loci possess such alleles. An ancestral translocation or recombination event involving SRK/SCR and Lal2/SCRL likely occurred, together with neofunctionalization of Lal2/SCRL, and both haplotype groups now function as Leavenworthia S locus alleles. These findings suggest that S locus alleles can have distinctly different evolutionary origins.

Genetic variation for pseudo-self-compatibility in self-incompatible populations of Leavenworthia alabamica (Brassicaceae).

Self-incompatibility (SI) promotes outcrossing, but transitions to self-compatibility (SC) are frequent. Population genetic theory describing the breakdown of SI to SC suggests that, under most conditions, populations should be composed of either SI or SC individuals. Under a narrow range of conditions, theory suggests that SI may persist alongside reduced expression of SI (pseudo-SI, PSI) in mixed-mating populations. We studied genetic variation for PSI segregating in four SI populations of Leavenworthia alabamica by measurement of the heritability of pollen tube number after self-pollination. We tested for the role of the S-locus in this variation by sequencing seven S-alleles from plants with high pseudo-SC (PSC) and testing for the co-segregation of these alleles with PSC. We found a continuous distribution of PSC in all populations and 90% of plants exhibited PSC. The heritability ranged from 0.39 to 0.57. All seven S-alleles from plants with high PSC exhibited trans-specific polymorphism, and no stop codons were observed within the c. 600-bp region sequenced. One of these S-alleles was directly associated with the inheritance of PSC. We conclude that heritable variation in PSC is largely a result of genetic variation in the signaling cascade downstream of the S-locus reaction, together with the presence of one leaky S-allele.

Recent selection for self-compatibility in a population of Leavenworthia alabamica.

The evolution of self-compatibility (SC) is the first step in the evolutionary transition in plants from outcrossing enforced by self-incompatibility (SI) to self-fertilization. In the Brassicaceae, SI is controlled by alleles of two tightly linked genes at the S-locus. Despite permitting inbreeding, mutations at the S-locus leading to SC may be selected if they provide reproductive assurance and/or gain a transmission advantage in a population when SC plants self- and outcross. Positive selection can leave a genomic signature in the regions physically linked to the focus of selection when selection has occurred recently. From an SC population of Leavenworthia alabamica with a known nonfunctional mutation at the S-locus, we collected sequence data from a ∼690 Kb region surrounding the S-locus, as well as from regions not linked to the S-locus. To test for recent positive selection acting at the S-locus, we examined polymorphism and the site-frequency spectra. Using forward simulations, we demonstrate that recent selection of the strength expected for SC at a locus formerly under balancing selection can generate patterns similar to those seen in our empirical data.

Genetic factors associated with mating system cause a partial reproductive barrier between two parapatric species of leavenworthia (brassicaceae).

Reproductive barriers play a major role in the origin and maintenance of biodiversity by restricting gene flow between species. Although both pre- and postzygotic barriers often isolate species, prezygotic barriers are thought to contribute more to reproductive isolation. We investigated possible reproductive barriers between Leavenworthia alabamica and L. crassa, parapatric species with high morphological and ecological similarity and the ability to hybridize. Using greenhouse and field experiments, we tested for habitat isolation and genetic incompatibilities. From controlled crosses, we identified unilateral incompatibility (a partial prezygotic barrier associated with the self-incompatibility system), but no evidence of other genetic incompatibilities. We found a small reduction in pollen viability of F(1) hybrids and early germination of F(1), F(2), and BC hybrids relative to L. alabamica and L. crassa in a common garden experiment, but the effect on fitness was not tested. Field studies of hybrid pollen viability and germination are needed to determine if they contribute to reproductive isolation. In a reciprocal transplant, we found no evidence of habitat isolation or reduced hybrid survival (from seedling to adult stage) or reproduction. These data suggest unilateral incompatibility partially reproductively isolates L. alabamica and L. crassa, but no other reproductive barriers could be detected.