Demography and mating system shape the genome-wide impact of purifying selection in Arabis alpina

YesPlant mating systems have profound effects on levels and structuring of genetic variation and can affect the impact of natural selection. Although theory predicts that intermediate outcrossing rates may allow plants to prevent accumulation of deleterious alleles, few studies have empirically tested this prediction using genomic data. Here, we study the effect of mating system on purifying selection by conducting population-genomic analyses on whole-genome resequencing data from 38 European individuals of the arctic-alpine crucifer Arabis alpina. We find that outcrossing and mixed-mating populations maintain genetic diversity at similar levels, whereas highly self-fertilizing Scandinavian A. alpina show a strong reduction in genetic diversity, most likely as a result of a postglacial colonization bottleneck. We further find evidence for accumulation of genetic load in highly self-fertilizing populations, whereas the genome-wide impact of purifying selection does not differ greatly between mixed-mating and outcrossing populations. Our results demonstrate that intermediate levels of outcrossing may allow efficient selection against harmful alleles, whereas demographic effects can be important for relaxed purifying selection in highly selfing populations. Thus, mating system and demography shape the impact of purifying selection on genomic variation in A. alpina. These results are important for an improved understanding of the evolutionary consequences of mating system variation and the maintenance of mixed-mating strategies.This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1707492115/-/DCSupplemental

Data from: Evolution of the selfing syndrome: anther orientation and herkogamy together determine reproductive assurance in a self-compatible plant

Capacity for autonomous self-fertilization provides reproductive assurance, has evolved repeatedly in the plant kingdom, and typically involves several changes in flower morphology and development (the selfing syndrome). Yet, the relative importance of different traits and trait combinations for efficient selfing and reproductive success in pollinator-poor environments is poorly known. In a series of experiments, we tested the importance of anther-stigma distance and the less studied trait anther orientation for efficiency of selfing in the perennial herb Arabis alpina. Variation in flower morphology among eight self-compatible European populations was correlated with efficiency of self-pollination and with pollen limitation in a common-garden experiment. To examine whether anther-stigma distance and anther orientation are subject to directional and/or correlational selection, and whether this is because these traits affect pollination success, we planted a segregating F2 population at two native field sites. Selection strongly favored a combination of introrse anthers and reduced anther-stigma distance at a site where pollinator activity was low, and supplemental hand-pollination demonstrated that this was largely because of their effect on securing self-pollination. The results suggest that concurrent shifts in more than one trait can be crucial for the evolution of efficient self-pollination and reproductive assurance in pollinator-poor habitats

Data from: Evolution of the selfing syndrome: anther orientation and herkogamy together determine reproductive assurance in a self-compatible plant

Capacity for autonomous self-fertilization provides reproductive assurance, has evolved repeatedly in the plant kingdom, and typically involves several changes in flower morphology and development (the selfing syndrome). Yet, the relative importance of different traits and trait combinations for efficient selfing and reproductive success in pollinator-poor environments is poorly known. In a series of experiments, we tested the importance of anther-stigma distance and the less studied trait anther orientation for efficiency of selfing in the perennial herb Arabis alpina. Variation in flower morphology among eight self-compatible European populations was correlated with efficiency of self-pollination and with pollen limitation in a common-garden experiment. To examine whether anther-stigma distance and anther orientation are subject to directional and/or correlational selection, and whether this is because these traits affect pollination success, we planted a segregating F2 population at two native field sites. Selection strongly favored a combination of introrse anthers and reduced anther-stigma distance at a site where pollinator activity was low, and supplemental hand-pollination demonstrated that this was largely because of their effect on securing self-pollination. The results suggest that concurrent shifts in more than one trait can be crucial for the evolution of efficient self-pollination and reproductive assurance in pollinator-poor habitats

PEP1 of <em>Arabis alpina</em> Is Encoded by Two Overlapping Genes That Contribute to Natural Genetic Variation in Perennial Flowering

<div><p>Higher plants exhibit a variety of different life histories. Annual plants live for less than a year and after flowering produce seeds and senesce. By contrast perennials live for many years, dividing their life cycle into episodes of vegetative growth and flowering. Environmental cues control key check points in both life histories. Genes controlling responses to these cues exhibit natural genetic variation that has been studied most in short-lived annuals. We characterize natural genetic variation conferring differences in the perennial life cycle of <em>Arabis alpina</em>. Previously the accession Pajares was shown to flower after prolonged exposure to cold (vernalization) and only for a limited period before returning to vegetative growth. We describe five accessions of <em>A. alpina</em> that do not require vernalization to flower and flower continuously. Genetic complementation showed that these accessions carry mutant alleles at <em>PERPETUAL FLOWERING 1</em> (<em>PEP1</em>), which encodes a MADS box transcription factor orthologous to FLOWERING LOCUS C in the annual <em>Arabidopsis thaliana</em>. Each accession carries a different mutation at <em>PEP1</em>, suggesting that such variation has arisen independently many times. Characterization of these alleles demonstrated that in most accessions, including Pajares, the <em>PEP1</em> locus contains a tandem arrangement of a full length and a partial <em>PEP1</em> copy, which give rise to two full-length transcripts that are differentially expressed. This complexity contrasts with the single gene present in <em>A. thaliana</em> and might contribute to the more complex expression pattern of <em>PEP1</em> that is associated with the perennial life-cycle. Our work demonstrates that natural accessions of <em>A. alpina</em> exhibit distinct life histories conferred by differences in PEP1 activity, and that continuous flowering forms have arisen multiple times by inactivation of the floral repressor <em>PEP1</em>. Similar phenotypic variation is found in other herbaceous perennial species, and our results provide a paradigm for how characteristic perennial phenotypes might arise.</p> </div

Silanization of silica nanoparticles and their processing as nanostructured micro-raspberry powders - a route to control the mechanical properties of isoprene rubber composites

Depending on interparticle structure and chemical behavior, fillers greatly influence the mechanical properties in rubber compounds. In this work, the influence of the filler–matrix versus filler–filler interaction on the mechanical properties of a silica nanoparticle–rubber composite was investigated. For this purpose, nanostructured microraspberry particles with different surface properties were designed and prepared using colloidal silica and two different kinds of silane agents, one coating agent (triethoxyoctylsilane, OCTEO) and one coupling agent (bis(triethoxysilylpropyl)tetrasulfide, Si69™). In addition, the degree of silane coverage of the nanoparticles was adjusted in a precisely controlled way. This sophisticated particle system allowed for straightforward integration into the composite formation process while ensuring that a redispersion of the nanoparticles in the rubber matrix occurred during compounding. With this microraspberry particle system, it was possible to influence the mechanical properties by the degree of silane surface coverage on the nanoparticles while the filler content could be kept constant. The two silane systems were carefully compared and the impact of the particle/particle and the particle/rubber interactions with respect to the mechanical properties of the composite was studied. Ultimately, an overall picture of the influence of the type and the amount of silane on mechanical properties in silica–rubber composites could be obtained

Comparison of <i>PEP1</i> coding sequences of five early-flowering <i>A. alpina</i> accessions and the obligate-vernalization requiring Paj accession.

<p>Multiple cDNAs were analyzed for the accessions Dor, Tot, Wca, Cza and Mug and the numbers of cDNA sequences recovered is shown in the “Clones” column. The full-length <i>PEP1</i> cDNA sequence of the vernalization-requiring accession Paj is used as a reference (row highlighted in grey). Nucleotide polymorphisms compared to Paj <i>PEP1</i> cDNA sequence obtained for each accession are presented. Nucleotide (nucl.) position and aminoacid (a.a.) changes compared to Paj mentioned in rows above the grey row. For most accessions, different <i>PEP1</i> spicing forms were also recovered and are presented in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003130#pgen.1003130.s005" target="_blank">Table S2</a>.</p

<i>PEP1a</i> and <i>PEP1b</i> genes in the accession Dor are independently transcribed and have different transcriptional start sites.

<p>(A,B) Number of clones containing G to A polymorphism on exon 1 (A) or not (B) after 5′ RACE using apices from Dor plants growing for 3 weeks in long days. Schematic representation of exon1 and 5′ UTR regions (top), exon1a and exon 1b (black boxes), sequence present in 5′ UTR upstream of both exon 1a and 1b (white box), sequence specific to 5′ UTR upstream of exon 1a (red box), sequence specific to 5′ UTR upstream exon 1b on the 248 bp insertion (grey box). Horizontal lines represent individual clones. Numbers on the top represent bp upstream of ATGs. (B,C) Percentage of clones with the A or G polymorphism after 5′ RACE in apices and leaves before (3 week long days) and after vernalization (5 weeks in long days after 12 weeks vernalization). (D)–(F) <i>PEP1</i> mRNA levels on 3 week old Dor plants, vernalized for 12 weeks and subsequently grown for several week in long days. (D) <i>PEP1</i> (a+b) expression, primers used similar as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003130#pgen.1003130-Wang1" target="_blank">[5]</a> to detect both transcripts (E) <i>PEP1a</i> expression, primers used to detect only <i>PEP1a</i> transcripts. (F) <i>PEP1b</i> expression, primers used to detect only <i>PEP1b</i> transcripts.</p

Flowering behavior of <i>A. alpina</i> accessions.

<p>(A) Accession Paj grown vegetatively for four years in long day glasshouse. Paj has an obligate requirement for vernalization to flower. (B)–(I) Non-vernalization requiring <i>A. alpina</i> accessions at flowering under long days. Accession Dor (B), Tot (C), Wca (D), Cza (E) and Mug (F). (G) Flowering times of non-vernalization requiring <i>A. alpina</i> accessions under long days (16 hours light) compared to <i>pep1-1</i> mutant and the accession Paj. Flowering time is measured as days to flower (DTF). <i>pep1-1</i> mutant (H) and the accession Dor (I) flower perpetually after 6 months in long days. (J) Duration of flowering in non-vernalization requiring <i>A. alpina</i> accessions.</p

List of primers used for expression studies.

<p>List of primers used for expression studies.</p