Rob W. Ness

GENOMIC CONSEQUENCES OF OUTCROSSING AND SELFING IN PLANTS

Evolutionary transitions from outcrossing to selfing are expected to cause a reduction in the effective population size and a corresponding increase in fixation rates of slightly deleterious mutations and decrease in fixation of advantageous mutations. Despite these predictions, evidence from genomic data does not suggest a significant reduction in the efficacy of selection associated with high levels of self‐fertilization. Here, we discuss opportunities for selfing populations to avoid an irreversible decline in fitness toward extinction and the implications for genome evolution. Most directly, large population sizes and the purging of deleterious recessive mutations can reduce genetic loads and slow the effects of genetic drift. Theory suggests that recombination rates may also evolve in response to the evolution of mating system, which can offset the harmful effects of inbreeding. Cytological data supporting the evolution of higher recombination rates in selfing species should be supplemented with genetic and molecular methods for estimating this parameter. Mutation rates may also evolve to be higher in selfing plants as a result of hitchhiking with advantageous mutations, although this is unlikely to lead to increased fitness. Finally, the abundance and activity of selfish genetic elements may also be reduced in selfing lineages, reducing the accumulation of transposable elements, B chromosomes, biased gene conversion, and the spread of cytoplasmic male sterility mutations. This reduction in genomic conflict can increase mean fitness, reduce deleterious mutation rates, and reduce genome size. We show, using comparative data, that highly selfing plants have genomes significantly smaller than those of outcrossing relatives, consistent with reduced activity and spread of repetitive elements in inbred plants. We discuss opportunities for tests of theory as plant genomic data accumulate and argue that a genomic perspective on reproductive transitions in a phylogenetic context should provide important insights into the diversity of reproductive systems in flowering plants.

De novo sequence assembly and characterization of the floral transcriptome in cross- and self-fertilizing plants

BackgroundThe shift from cross-fertilization to predominant self-fertilization is among the most common evolutionary transitions in the reproductive biology of flowering plants. Increased inbreeding has important consequences for floral morphology, population genetic structure and genome evolution. The transition to selfing is usually characterized by a marked reduction in flower size and the loss of traits involved in pollinator attraction and the avoidance of self-fertilization. Here, we use short-read sequencing to assemble, de novo, the floral transcriptomes of three genotypes of Eichhornia paniculata, including an outcrosser and two genotypes from independently derived selfers, and a single genotype of the sister species E. paradoxa. By sequencing mRNA from tissues sampled at various stages of flower development, our goal was to sequence and assemble the floral transcriptome and identify differential patterns of gene expression.ResultsOur 24 Mbp assembly resulted in ~27,000 contigs that averaged ~900 bp in length. All four genotypes had highly correlated gene expression, but the three E. paniculata genotypes were more correlated with one another than each was to E. paradoxa. Our analysis identified 269 genes associated with floral development, 22 of which were differentially expressed in selfing lineages relative to the outcrosser. Many of the differentially expressed genes affect floral traits commonly altered in selfing plants and these represent a set of potential candidate genes for investigating the evolution of the selfing syndrome.ConclusionsOur study is among the first to demonstrate the use of Illumina short read sequencing for de novo transcriptome assembly in non-model species, and the first to implement this technology for comparing floral transcriptomes in outcrossing and selfing plants.

Mating-System Variation, Demographic History and Patterns of Nucleotide Diversity in the Tristylous Plant Eichhornia paniculata

Inbreeding in highly selfing populations reduces effective size and, combined with demographic conditions associated with selfing, this can erode genetic diversity and increase population differentiation. Here we investigate the role that variation in mating patterns and demographic history play in shaping the distribution of nucleotide variation within and among populations of the annual neotropical colonizing plant Eichhornia paniculata, a species with wide variation in selfing rates. We sequenced 10 EST-derived nuclear loci in 225 individuals from 25 populations sampled from much of the geographic range and used coalescent simulations to investigate demographic history. Highly selfing populations exhibited moderate reductions in diversity but there was no significant difference in variation between outcrossing and mixed mating populations. Population size interacted strongly with mating system and explained more of the variation in diversity within populations. Bayesian structure analysis revealed strong regional clustering and selfing populations were highly differentiated on the basis of an analysis of Fst. There was no evidence for a significant loss of within-locus linkage disequilibrium within populations, but regional samples revealed greater breakdown in Brazil than in selfing populations from the Caribbean. Coalescent simulations indicate a moderate bottleneck associated with colonization of the Caribbean from Brazil ∼125,000 years before the present. Our results suggest that the recent multiple origins of selfing in E. paniculata from diverse outcrossing populations result in higher diversity than expected under long-term equilibrium.

Estimate of the Spontaneous Mutation Rate in Chlamydomonas reinhardtii

The nature of spontaneous mutations, including their rate, distribution across the genome, and fitness consequences, is of central importance to biology. However, the low rate of mutation has made it difficult to study spontaneous mutagenesis, and few studies have directly addressed these questions. Here, we present a direct estimate of the mutation rate and a description of the properties of new spontaneous mutations in the unicellular green alga Chlamydomonas reinhardtii. We conducted a mutation accumulation experiment for ∼350 generations followed by whole-genome resequencing of two replicate lines. Our analysis identified a total of 14 mutations, including 5 short indels and 9 single base mutations, and no evidence of larger structural mutations. From this, we estimate a total mutation rate of 3.23 × 10−10/site/generation (95% C.I. 1.82 × 10−10 to 5.23 × 10−10) and a single base mutation rate of 2.08 × 10−10/site/generation (95% C.I., 1.09 × 10−10 to 3.74 × 10−10). We observed no mutations from A/T → G/C, suggesting a strong mutational bias toward A/T, although paradoxically, the GC content of the C. reinhardtii genome is very high. Our estimate is only the second direct estimate of the mutation rate from plants and among the lowest spontaneous base-substitution rates known in eukaryotes.

Contributions of Protein-Coding and Regulatory Change to Adaptive Molecular Evolution in Murid Rodents

The contribution of regulatory versus protein change to adaptive evolution has long been controversial. In principle, the rate and strength of adaptation within functional genetic elements can be quantified on the basis of an excess of nucleotide substitutions between species compared to the neutral expectation or from effects of recent substitutions on nucleotide diversity at linked sites. Here, we infer the nature of selective forces acting in proteins, their UTRs and conserved noncoding elements (CNEs) using genome-wide patterns of diversity in wild house mice and divergence to related species. By applying an extension of the McDonald-Kreitman test, we infer that adaptive substitutions are widespread in protein-coding genes, UTRs and CNEs, and we estimate that there are at least four times as many adaptive substitutions in CNEs and UTRs as in proteins. We observe pronounced reductions in mean diversity around nonsynonymous sites (whether or not they have experienced a recent substitution). This can be explained by selection on multiple, linked CNEs and exons. We also observe substantial dips in mean diversity (after controlling for divergence) around protein-coding exons and CNEs, which can also be explained by the combined effects of many linked exons and CNEs. A model of background selection (BGS) can adequately explain the reduction in mean diversity observed around CNEs. However, BGS fails to explain the wide reductions in mean diversity surrounding exons (encompassing ∼100 Kb, on average), implying that there is a substantial role for adaptation within exons or closely linked sites. The wide dips in diversity around exons, which are hard to explain by BGS, suggest that the fitness effects of adaptive amino acid substitutions could be substantially larger than substitutions in CNEs. We conclude that although there appear to be many more adaptive noncoding changes, substitutions in proteins may dominate phenotypic evolution.

Estimation of the Spontaneous Mutation Rate per Nucleotide Site in a Drosophila melanogaster Full-Sib Family

We employed deep genome sequencing of two parents and 12 of their offspring to estimate the mutation rate per site per generation in a full-sib family of Drosophila melanogaster recently sampled from a natural population. Sites that were homozygous for the same allele in the parents and heterozygous in one or more offspring were categorized as candidate mutations and subjected to detailed analysis. In 1.23 × 109 callable sites from 12 individuals, we confirmed six single nucleotide mutations. We estimated the false negative rate in the experiment by generating synthetic mutations using the empirical distributions of numbers of nonreference bases at heterozygous sites in the offspring. The proportion of synthetic mutations at callable sites that we failed to detect was <1%, implying that the false negative rate was extremely low. Our estimate of the point mutation rate is 2.8 × 10−9 (95% confidence interval = 1.0 × 10−9 − 6.1 × 10−9) per site per generation, which is at the low end of the range of previous estimates, and suggests an effective population size for the species of ∼1.4 × 106. At one site, point mutations were present in two individuals, indicating that there had been a premeiotic mutation cluster, although surprisingly one individual had a G→A transition and the other a G→T transversion, possibly associated with error-prone mismatch repair. We also detected three short deletion mutations and no insertions, giving a deletion mutation rate of 1.2 × 10−9 (95% confidence interval = 0.7 × 10−9 − 11 × 10−9).

Estimation of the spontaneous mutation rate in Heliconius melpomene

We estimated the spontaneous mutation rate in Heliconius melpomene by genome sequencing of a pair of parents and 30 of their offspring, based on the ratio of number of de novo heterozygotes to the number of callable site-individuals. We detected nine new mutations, each one affecting a single site in a single offspring. This yields an estimated mutation rate of 2.9 × 10−9 (95% confidence interval, 1.3 × 10−9–5.5 × 10−9), which is similar to recent estimates in Drosophila melanogaster, the only other insect species in which the mutation rate has been directly estimated. We infer that recent effective population size of H. melpomene is about 2 million, a substantially lower value than its census size, suggesting a role for natural selection reducing diversity. We estimate that H. melpomene diverged from its Müllerian comimic H. erato about 6 Ma, a somewhat later date than estimates based on a local molecular clock.

Evolutionary pathways to self‐fertilization in a tristylous plant species

Evolutionary transitions from outcrossing to selfing occur commonly in heterostylous genera. The morphological polymorphisms that characterize heterostyly provide opportunities for different pathways for selfing to evolve. Here, we investigate the origins and pathways by which selfing has evolved in tristylous Eichhornia paniculata by providing new evidence based on morphology, DNA sequences and genetic analysis. The primary pathway from outcrossing to selfing involves the stochastic loss of the short-styled morph (S-morph) from trimorphic populations, followed by the spread of selfing variants of the mid-styled morph (M-morph). However, the discovery of selfing variants of the long-styled morph (L-morph) in Central America indicates a secondary pathway and distinct origin for selfing. Comparisons of multi-locus nucleotide sequences from 27 populations sampled from throughout the geographical range suggest multiple transitions to selfing. Genetic analysis of selfing variants of the L- and M-morphs demonstrates recessive control of the loss of herkogamy, although the number of factors appears to differ between the forms. Early stages in the establishment of selfing involve developmental instability in the formation of flowers capable of autonomous self-pollination. The relatively simple genetic control of herkogamy reduction and frequent colonizing episodes may often create demographic conditions favouring transitions to selfing in E. paniculata.

Reconciling Gene and Genome Duplication Events: Using Multiple Nuclear Gene Families to Infer the Phylogeny of the Aquatic Plant Family Pontederiaceae

Most plant phylogenetic inference has used DNA sequence data from the plastid genome. This genome represents a single genealogical sample with no recombination among genes, potentially limiting the resolution of evolutionary relationships in some contexts. In contrast, nuclear DNA is inherently more difficult to employ for phylogeny reconstruction because major mutational events in the genome, including polyploidization, gene duplication, and gene extinction can result in homologous gene copies that are difficult to identify as orthologs or paralogs. Gene tree parsimony (GTP) can be used to infer the rooted species tree by fitting gene genealogies to species trees while simultaneously minimizing the estimated number of duplications needed to reconcile conflicts among them. Here, we use GTP for five nuclear gene families and a previously published plastid data set to reconstruct the phylogenetic backbone of the aquatic plant family Pontederiaceae. Plastid-based phylogenetic studies strongly supported extensive paraphyly of Eichhornia (one of the four major genera) but also depicted considerable ambiguity concerning the true root placement for the family. Our results indicate that species trees inferred from the nuclear genes (alone and in combination with the plastid data) are highly congruent with gene trees inferred from plastid data alone. Consideration of optimal and suboptimal gene tree reconciliations place the root of the family at (or near) a branch leading to the rare and locally restricted E. meyeri. We also explore methods to incorporate uncertainty in individual gene trees during reconciliation by considering their individual bootstrap profiles and relate inferred excesses of gene duplication events on individual branches to whole-genome duplication events inferred for the same branches. Our study improves understanding of the phylogenetic history of Pontederiaceae and also demonstrates the utility of GTP for phylogenetic analysis.

Comparative population genomics in Collinsia sister species reveals evidence for reduced effective population size, relaxed selection, and evolution of biased gene conversion with an ongoing mating system shift.

Selfing species experience reduced effective recombination rates and effective population size, which can lead to reductions in polymorphism and the efficacy of natural selection. Here, we use illumina transcriptome sequencing and population resequencing to test for changes in polymorphism, base composition, and selection in the selfing angiosperm Collinsia rattanii (Plantaginaceae) compared with its more outcrossing sister species Collinsia linearis. Coalescent analysis indicates intermediate species divergence (500,000–1 million years) with no ongoing gene flow, but also evidence that the C. rattanii clade remains polymorphic for floral morphology and mating system, suggesting either an ongoing shift to selfing or a potential reversal from selfing to outcrossing. We identify a significant reduction in polymorphism in C. rattanii, particularly within populations. Analysis of polymorphisms suggests an elevated ratio of unique nonsynonymous to synonymous polymorphism in C. rattanii, consistent with relaxed selection in selfing lineages. We additionally find higher linkage disequilibrium and differentiation, lower GC content at variable sites, and reduced expression of genes important in pollen production and pollinator attraction in C. rattanii compared with C. linearis. Together, our results highlight the potential for rapid shifts in the efficacy of selection, gene expression and base composition associated with ongoing evolution of selfing.