Can Yeast (<i>S. cerevisiae</i>) Metabolic Volatiles Provide Polymorphic Signaling?

<div><p>Chemical signaling between organisms is a ubiquitous and evolutionarily dynamic process that helps to ensure mate recognition, location of nutrients, avoidance of toxins, and social cooperation. Evolutionary changes in chemical communication systems progress through natural variation within the organism generating the signal as well as the responding individuals. A promising yet poorly understood system with which to probe the importance of this variation exists between <i>D. melanogaster</i> and <i>S. cerevisiae</i>. <i>D. melanogaster</i> relies on yeast for nutrients, while also serving as a vector for yeast cell dispersal. Both are outstanding genetic and genomic models, with <i>Drosophila</i> also serving as a preeminent model for sensory neurobiology. To help develop these two genetic models as an ecological model, we have tested if - and to what extent - <i>S. cerevisiae</i> is capable of producing polymorphic signaling through variation in metabolic volatiles. We have carried out a chemical phenotyping experiment for 14 diverse accessions within a common garden random block design. Leveraging genomic sequences for 11 of the accessions, we ensured a genetically broad sample and tested for phylogenetic signal arising from phenotypic dataset. Our results demonstrate that significant quantitative differences for volatile blends do exist among <i>S. cerevisiae</i> accessions. Of particular ecological relevance, the compounds driving the blend differences (acetoin, 2-phenyl ethanol and 3-methyl-1-butanol) are known ligands for <i>D. melanogaster</i>s chemosensory receptors, and are related to sensory behaviors. Though unable to correlate the genetic and volatile measurements, our data point clear ways forward for behavioral assays aimed at understanding the implications of this variation.</p></div

Genetic relationships between the 11 yeast accessions for which genomic sequence is available.

<p>Left: Inferred proportion of ancestry estimated for 2–5 genetic clusters. Right: A Neighbor Joining tree for the same yeast accessions. All branches have bootstrap values greater than 95% except for the two marked with red lines (upper branch = 55.8, lower branch = 74.9). Color-coding on tree tips indicate the grouping of the strains according to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070219#pone-0070219-t001" target="_blank">Table 1</a>.</p

Principle component analyses and loading summaries for yeast volatile data.

<p>Principle component analyses and loading summaries for yeast volatile data.</p

Relating olfactory receptors to significant compounds resulting from the PCA analysis.

<p>Relating olfactory receptors to significant compounds resulting from the PCA analysis.</p

Summary information for the 14 yeast accessions used in this study.

<p>Summary information for the 14 yeast accessions used in this study.</p

A Model for the Origin and Evolution of <i>Hun</i>

<p>Striped boxes indicate newly acquired UTR regions, white boxes indicate the <i>Bällchen</i> 5′ region included in duplication, grey boxes indicate newly acquired protein-coding regions, green boxes indicate regions deleted from the D. sechellia copy, and red bars represent premature stop codons.</p

Gene Tree for <i>D. simulans Hun</i> and <i>D. simulans, D. melanogaster,</i> and D. yakuba's <i>Bällchen</i>

<p>The tree includes measurements of divergence as measured by Ka/Ks (red ratios), nonsynonymous and synonymous fixations found along the <i>Hun</i> and <i>Bällchen</i> branches depicted by colored bars (red represents nonsynonymous changes and green represents synonymous changes, black ratio), and polymorphisms found in the D. simulans population data (black ratios below triangles, nonsynonymous/synonymous). The low divergence estimates suggest that all genes are constrained. The most notable feature of the tree is the significant excess of nonsynonymous substitutions along <i>Hun</i>'s branch. This excess was detected by McDonald-Kreitman tests, and is significantly different than that of the pooled <i>Bällchen</i> and <i>Hun</i> data, and marginally significantly different than that of the <i>Hun</i>-only data (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0020077#pgen-0020077-t002" target="_blank">Table 2</a>).</p

Expression Study Results for the <i>Bällchen and Hun</i> gene

<div><p>(A) <i>Bällchen</i> is expressed ubiquitously in both <i>D.melanogaster</i> and <i>D. simulans:</i> whole body, D. melanogaster (1), D. simulans (2); head, D. melanogaster (3), D. simulans (4); thorax, D. melanogaster (5), <i>D. simulans</i> (6); testes, D. melanogaster (7), D. simulans (8); and accessory gland with ejaculatory duct, D. melanogaster (9), D. simulans (10). Lane 11 is a genomic control.</p><p>(B) RT-PCR results for <i>Hun</i> from adult male and female <i>D. simulans, D. mauritiana,</i> and D. sechellia flies. <i>Hun</i>'s expression is limited to <i>D. simulans, D. mauritiana,</i> and D. sechellia males, lanes 1, 5, and 9, respectively. Lanes 3, 7, and 11 are (D) <i>D. simulans, D. mauritiana,</i> and D. sechellia females. Lanes 2, 4, 6, 8, 10, and 12 are corresponding RT-controls. (C–E) Tissue specific RT-PCR results for the <i>Hun</i> gene in D. simulans (C), D. mauritiana (D), and D. sechellia (E). For these RT-PCR experiments testes were dissected from the rest of body. <i>Hun</i>'s expression is testes-specific for each species: testes, lane 1; RT-control, lane 2; <i>Gapdh-2</i> positive control, lane 3; rest of body, lane 4; RT-control, lane 5; <i>Gapdh-2</i> positive control, lane 6.</p></div

Southern Hybridization Verifying the <i>Hun</i> Duplication in <i>D. sechellia, D. simulans,</i> and D. mauritiana

<p>Hybridizations, using genomic DNA from the D. melanogaster subgroup and the FISH cDNA probes, are contained in lanes 1–8: (1) <i>D. teissieri,</i> (2) <i>D. santomea,</i> (3) <i>D. yakuba,</i> (4) <i>D. simulans,</i> (5) <i>D. sechellia,</i> (6) <i>D. mauritiana,</i> (7) <i>D. erecta,</i> and (8) D. melanogaster. Species for which two signals were recovered <i>(D. mauritiana, D. sechellia,</i> and <i>D. simulans)</i> are noted with red numbers. Hybridization A (top) was carried out using the BamH I restriction enzyme; hybridization B (bottom) was carried out using the Xho I restriction enzyme. These results are in agreement with the FISH results (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0020077#pgen-0020077-g001" target="_blank">Figure 1</a>).</p