Opposing effects of plant traits on diversification.

Species diversity can vary dramatically across lineages due to differences in speciation and extinction rates. Here, we explore the effects of several plant traits on diversification, finding that most traits have opposing effects on diversification. For example, outcrossing may increase the efficacy of selection and adaptation but also decrease mate availability, two processes with contrasting effects on lineage persistence. Such opposing trait effects can manifest as differences in diversification rates that depend on ecological context, spatiotemporal scale, and associations with other traits. The complexity of pathways linking traits to diversification suggests that the mechanistic underpinnings behind their correlations may be difficult to interpret with any certainty, and context dependence means that the effects of specific traits on diversification are likely to differ across multiple lineages and timescales. This calls for taxonomically and context-controlled approaches to studies that correlate traits and diversification.

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.

Studying flowers in 3D using photogrammetry.

Flowers are intricate and integrated three-dimensional (3D) structures predominantly studied in 2D due to the difficulty in quantitatively characterising their morphology in 3D. Given the recent development of analytical methods for high-dimensional data, the reconstruction of flower models in three dimensions represents the limiting factor to studying flowers in 3D. We developed a floral photogrammetry protocol to reconstruct 3D models of flowers based on images taken with a digital single-lens reflex camera, a turntable and a portable lightbox. We demonstrate that photogrammetry allows a rapid and accurate reconstruction of 3D models of flowers from 2D images. It can reconstruct all visible parts of flowers and has the advantage of keeping colour information. We illustrated its use by studying the shape and colour of 18 Gesneriaceae species. Photogrammetry is an affordable alternative to micro-computed tomography (micro-CT) that requires minimal investment and equipment, allowing it to be used directly in the field. It has the potential to stimulate research on the evolution and ecology of flowers by providing a simple way to access 3D morphological data from a variety of flower types.

Repeated evolution of a reproductive polyphenism in plants is strongly associated with bilateral flower symmetry.

Polyphenisms are a special type of phenotypic plasticity in which the products of development are not continuous but instead are separate and distinct phenotypes produced in the same genetic background. One of the most widespread polyphenisms in the flowering plants is cleistogamy, in which the same individual plant produces both open, cross-pollinated flowers as well as highly reduced and closed, self-pollinated (cleistogamous) flowers.1-5 Cleistogamy is not a rare evolutionary phenomenon. It has evolved independently at least 41 times.1 But what favors the evolution of cleistogamy is still largely unknown.1 Darwin6 proposed a hypothesis that has never been properly tested. He observed that cleistogamy is more common in taxa with bilaterally symmetric (zygomorphic) flowers than in those with radially symmetric (actinomorphic) flowers. Moreover, Darwin suggested that cleistogamous flowers help to ensure pollination, which he postulated is less certain in zygomorphic taxa that rely on more specialized groups of pollinators. Here, we combined the largest datasets on floral symmetry and cleistogamy and used phylogenetic approaches to show that cleistogamy is indeed disproportionately associated with zygomorphic flowers and that zygomorphic species are more likely to evolve cleistogamy than actinomorphic species. We also show that zygomorphic species are less capable of autonomous open-flower self-pollination (lower autofertility), suggesting that selection of cleistogamy via reproductive assurance in zygomorphic taxa could help account for Darwin's observation. Our results provide support for the hypothesis that polyphenisms are favored when organisms encounter contrasting environments.

The Evolutionary Forces Shaping Cis- and Trans-Regulation of Gene Expression within a Population of Outcrossing Plants.

Understanding the persistence of genetic variation within populations has long been a goal of evolutionary biology. One promising route toward achieving this goal is using population genetic approaches to describe how selection acts on the loci associated with trait variation. Gene expression provides a model trait for addressing the challenge of the maintenance of variation because it can be measured genome-wide without information about how gene expression affects traits. Previous work has shown that loci affecting the expression of nearby genes (local or cis-eQTLs) are under negative selection, but we lack a clear understanding of the selective forces acting on variants that affect the expression of genes in trans. Here, we identify loci that affect gene expression in trans using genomic and transcriptomic data from one population of the obligately outcrossing plant, Capsella grandiflora. The allele frequencies of trans-eQTLs are consistent with stronger negative selection acting on trans-eQTLs than cis-eQTLs, and stronger negative selection acting on trans-eQTLs associated with the expression of multiple genes. However, despite this general pattern, we still observe the presence of a trans-eQTL at intermediate frequency that affects the expression of a large number of genes in the same coexpression module. Overall, our work highlights the different selective pressures shaping variation in cis- and trans-regulation.

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.

Transitions from distyly to homostyly are associated with floral evolution in the buckwheat genus (Fagopyrum).

PREMISE OF THE STUDY: Documenting trait transitions among species with dimorphic flowers can help to test whether similar patterns of selection are responsible for divergence in floral traits among different species. Heterostyly is thought to promote outcrossing. Theory suggests that the evolutionary transition from heterostylous to homostylous flowers should be accompanied by a reduction in floral size in which pollen size and style length are expected to covary. Patterns of such correlated floral trait evolution have not, however, been widely examined. METHODS: The evolutionary history of heterostyly and homostyly was reconstructed from a molecular phylogeny of 13 species of Fagopyrum and two outgroups, based on one nuclear gene (nrITS) and three chloroplast regions (matK, atpB-rbcL, and psbA-trnH spacer). Floral traits of nine Fagopyrum species including pollen number and size, as well as stigma depth (length of the capitate stigma), were measured and ancestral characters of the herkogamic condition were reconstructed. KEY RESULTS: Three transitions from distyly to homostyly were observed. In two transitions, flower size (corolla diameter, pedicel length), herkogamy (stigma-anther distance) and pollen production decreased, but stigma depth and pollen size did not. Changes of anther height and style length were inconsistent. The predicted positive relationship between style length and pollen size in the two transitions to homostyly was not observed. CONCLUSIONS: Floral evolution accompanying transitions to homostyly in Fagopyrum were found to be consistent with predictions of mating system evolution theory, and the correlation of traits in distylous vs. homostylous species revealed that pollen size generally correlates with stigma depth rather than style length.

The Relationship between Selection, Network Connectivity, and Regulatory Variation within a Population of Capsella grandiflora.

Interactions between genes can have important consequences for how selection shapes sequence variation at these genes. Specifically, genes that have pleiotropic effects by affecting the expression level of many other genes may be under stronger selective constraint. We used coexpression networks to measure connectivity between genes and investigated the relationship between gene connectivity and selection in a natural population of the plant Capsella grandiflora. We observed that network connectivity was negatively correlated with genetic divergence due to stronger negative selection on highly-connected genes even when controlling for variation in gene expression level. However, the presence of local regulatory variation for a gene's expression level was also associated with reduced negative selection and lower gene connectivity. While it is difficult to disentangle the causal relationships between these factors, our results show that both connectivity and local regulatory variation are important factors for explaining variation in selection between genes.

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.

Spontaneous Mutation Accumulation in Daphnia pulex in Selection-Free vs. Competitive Environments.

Understanding the rates, spectra, and fitness effects of spontaneous mutations is fundamental to answering key questions in evolution, molecular biology, disease genetics, and conservation biology. To estimate mutation rates and evaluate the effect of selection on new mutations, we propagated mutation accumulation (MA) lines of Daphnia pulex for more than 82 generations and maintained a non-MA population under conditions where selection could act. Both experiments were started with the same obligate asexual progenitor clone. By sequencing 30 genomes and implementing a series of validation steps that informed the bioinformatic analyses, we identified a total of 477 single nucleotide mutations (SNMs) in the MA lines, corresponding to a mutation rate of 2.30 × 10-9 (95% CI 1.90-2.70 × 10-9) per nucleotide per generation. The high overall rate of loss of heterozygosity (LOH) of 4.82 × 10-5 per site per generation was due to a large ameiotic recombination event spanning an entire arm of a chromosome (∼6 Mb) and several hemizygous deletion events spanning ∼2 kb each. In the non-MA population, we found significantly fewer mutations than expected based on the rate derived from the MA experiment, indicating purifying selection was likely acting to remove new deleterious mutations. We observed a surprisingly high level of genetic variability in the non-MA population, which we propose to be driven by balancing selection. Our findings suggest that both positive and negative selection on new mutations is powerful and effective in a strictly clonal population.

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.

Selection of sporophytic and gametophytic self-incompatibility in the absence of a superlocus.

Self-incompatibility (SI) is a complex trait that enforces outcrossing in plant populations. SI generally involves tight linkage of genes coding for the proteins that underlie self-pollen detection and pollen identity specification. Here, we develop two-locus genetic models to address the question of whether sporophytic SI (SSI) and gametophytic SI (GSI) can invade populations of self-compatible plants when there is no linkage or weak linkage of the underlying pollen detection and identity genes (i.e., no S-locus supergene). The models assume that SI evolves as a result of exaptation of genes formerly involved in functions other than SI. Model analysis reveals that SSI and GSI can invade populations even when the underlying genes are loosely linked, provided that inbreeding depression and selfing rate are sufficiently high. Reducing recombination between these genes makes conditions for invasion more lenient. These results can help account for multiple, independent evolution of SI systems as seems to have occurred in the angiosperms.

An atlas of over 90,000 conserved noncoding sequences provides insight into crucifer regulatory regions.

Despite the central importance of noncoding DNA to gene regulation and evolution, understanding of the extent of selection on plant noncoding DNA remains limited compared to that of other organisms. Here we report sequencing of genomes from three Brassicaceae species (Leavenworthia alabamica, Sisymbrium irio and Aethionema arabicum) and their joint analysis with six previously sequenced crucifer genomes. Conservation across orthologous bases suggests that at least 17% of the Arabidopsis thaliana genome is under selection, with nearly one-quarter of the sequence under selection lying outside of coding regions. Much of this sequence can be localized to approximately 90,000 conserved noncoding sequences (CNSs) that show evidence of transcriptional and post-transcriptional regulation. Population genomics analyses of two crucifer species, A. thaliana and Capsella grandiflora, confirm that most of the identified CNSs are evolving under medium to strong purifying selection. Overall, these CNSs highlight both similarities and several key differences between the regulatory DNA of plants and other species.

Secondary evolution of a self-incompatibility locus in the Brassicaceae genus Leavenworthia.

Self-incompatibility (SI) is the flowering plant reproductive system in which self pollen tube growth is inhibited, thereby preventing self-fertilization. SI has evolved independently in several different flowering plant lineages. In all Brassicaceae species in which the molecular basis of SI has been investigated in detail, the product of the S-locus receptor kinase (SRK) gene functions as receptor in the initial step of the self pollen-rejection pathway, while that of the S-locus cysteine-rich (SCR) gene functions as ligand. Here we examine the hypothesis that the S locus in the Brassicaceae genus Leavenworthia is paralogous with the S locus previously characterized in other members of the family. We also test the hypothesis that self-compatibility in this group is based on disruption of the pollen ligand-producing gene. Sequence analysis of the S-locus genes in Leavenworthia, phylogeny of S alleles, gene expression patterns, and comparative genomics analyses provide support for both hypotheses. Of special interest are two genes located in a non-S locus genomic region of Arabidopsis lyrata that exhibit domain structures, sequences, and phylogenetic histories similar to those of the S-locus genes in Leavenworthia, and that also share synteny with these genes. These A. lyrata genes resemble those comprising the A. lyrata S locus, but they do not function in self-recognition. Moreover, they appear to belong to a lineage that diverged from the ancestral Brassicaceae S-locus genes before allelic diversification at the S locus. We hypothesize that there has been neo-functionalization of these S-locus-like genes in the Leavenworthia lineage, resulting in evolution of a separate ligand-receptor system of SI. Our results also provide support for theoretical models that predict that the least constrained pathway to the evolution of self-compatibility is one involving loss of pollen gene function.

Phylogeny of Leavenworthia S-alleles suggests unidirectional mating system evolution and enhanced positive selection following an ancient population bottleneck.

The adoption of self-fertilization from an ancestral outcrossing state is one of the most common evolutionary transitions in the flowering plants. In the mustard family, outcrossing is typically enforced by sporophytic self-incompatibility (SI), but there are also many self-compatible species. The genus Leavenworthia contains taxa that either possess or lack SI. Here, we present data showing that SI is associated with strict outcrossing and that there is widespread trans-specific sequence polymorphism at the locus involved in the recognition of self-pollen (the S-locus). This ancestral polymorphism is consistent with the presence of an outcrossing mating system in the common ancestor of Leavenworthia species, and suggests that there have been several independent losses of SI in the group. When compared with other mustard species, the bulk of Leavenworthia S-allele sequences are highly diverged from those found in other Brassicaceae and show relatively low levels of nucleotide diversity, a pattern that suggests the common ancestor of the genus likely underwent a strong population bottleneck. The hypothesis of postbottleneck S-locus rediversification is supported by tests showing stronger positive selection acting on S-alleles from Leavenworthia than those found in other Brassicaceae.

The Red Queen and the seed bank: pathogen resistance of ex situ and in situ conserved barley.

Plant geneticists have proposed that the dynamic conservation of crop plants in farm environments (in situ conservation) is complementary to static conservation in seed banks (ex situ conservation) because it may help to ensure adaptation to changing conditions. Here, we test whether collections of a traditional variety of Moroccan barley (Hordeum vulgare ssp. vulgare) conserved ex situ showed differences in qualitative and quantitative resistance to the endemic fungal pathogen, Blumeria graminis f.sp. hordei, compared to collections that were continuously cultivated in situ. In detached-leaf assays for qualitative resistance, there were some significant differences between in situ and ex situ conserved collections from the same localities. Some ex situ conserved collections showed lower resistance levels, while others showed higher resistance levels than their in situ conserved counterparts. In field trials for quantitative resistance, similar results were observed, with the highest resistance observed in situ. Overall, this study identifies some cases where the Red Queen appears to drive the evolution of increased resistance in situ. However, in situ conservation does not always result in improved adaptation to pathogen virulence, suggesting a more complex evolutionary scenario, consistent with several published examples of plant-pathogen co-evolution in wild systems.

Migration rates, frequency-dependent selection and the self-incompatibility locus in Leavenworthia (brassicaceae).

Loci subject to negative frequency-dependent selection are expected to exhibit higher effective migration rates compared to reference loci. Although the number of gene copies transferred between populations by migration is the same for all genes, those subject to negative frequency-dependent selection are more likely to be retained in the immigrant population because rare alleles are selectively favored. So far, evidence for this prediction has been indirect, based on summary statistics rather than on migration rate estimates. Here, we introduce an approximate Bayesian procedure to jointly estimate migration rates at two predefined sets of loci between two populations. We applied the procedure to compare migration rate estimates at the self-incompatibility locus (S-locus) with that at 10 reference loci in two plant species, Leavenworthia alabamica and L. crassa (Brassicaceae). The maximum likelihood estimate for the proportion of migrants (m) was four times higher at the S-locus than at reference loci, but the difference was not statistically significant. Lack of significance might be due to insufficient data, but perhaps also to the recent divergence of the two species (311 ka), because we also show using simulations that the effective migration rate at the S-locus is expected to increase with increasing divergence time. These findings aid in understanding the evolutionary dynamics of negative frequency-dependent selection and they suggest that divergence time should be accounted for when employing migration rates to help detect negative frequency-dependent selection.

Demographic signatures accompanying the evolution of selfing in Leavenworthia alabamica.

The evolution of selfing from outcrossing is a common transition, yet little is known about the mutations and selective factors that promote this shift. In the mustard family, single-locus self-incompatibility (SI) enforces outcrossing. In this study, we test whether mutations causing self-compatibility (SC) are linked to the self-incompatibility locus (S-locus) in Leavenworthia alabamica, a species where two selfing races (a2 and a4) co-occur with outcrossing populations. We also infer the ecological circumstances associated with origins of selfing using molecular sequence data. Genealogical reconstruction of the Lal2 locus, the putative ortholog of the SRK locus, showed that both selfing races are fixed for one of two different S-linked Lal2 sequences, whereas outcrossing populations harbor many S-alleles. Hybrid crosses demonstrated that S-linked mutations cause SC in each selfing race. These results strongly suggest two origins of selfing in this species, a result supported by population admixture analysis of 16 microsatellite loci and by a population tree built from eight nuclear loci. One selfing race (a4) shows signs of a severe population bottleneck, suggesting that reproductive assurance might have caused the evolution of selfing in this case. In contrast, the population size of race a2 cannot be distinguished from that of outcrossing populations after correcting for differences in selfing rates. Coalescent-based analyses suggest a relatively old origin of selfing in the a4 race (∼150 ka ago), whereas selfing evolved recently in the a2 race (∼12-48 ka ago). These results imply that S-locus mutations have triggered two recent shifts to selfing in L. alabamica, but that these transitions are not always associated with a severe population bottleneck, suggesting that factors other than reproductive assurance may play a role in its evolution.