Depletion of SVs among highly conserved genes.

<p>We classified genes in deleted and duplicated regions (relative to the reference strain PS312) based on presence of protein domains as well as homology relationships with other nematodes including <i>C. elegans</i>. For the different homology classes, we find that predicted one-to-one orthologs with <i>C. elegans</i> are strongly depleted from SVs indicating the action of purifying selection. Conversely, conserved and orphan genes that are members of larger gene families are significantly enriched in SVs suggesting that similar processes that have generated different homology relationships at a cross-species level are operating on a microevolutionary level.</p

Identification of duplications and deletions in the <i>P. pacificus</i> strains.

<p>A) General phylogeny of <i>P. pacificus</i> strains and the sister species <i>P. exspectatus</i> (RS5522). Strains and species that are used in this study are colored in red. The tree was generated by building a neighbor joining tree on Hamming distances based on roughly 450,000 parsimony informative sites (singletons excluded). Excluding singletons leads to a vast underestimation of distances between strains and species but should increase the robustness of the tree topology. All internal nodes showed a perfect bootstrap support of 100/100. B) True and false positive rate for SV calls for various p-value and fold change cutoffs of the program cnv-seq. The evaluation is based on 100 manually classified SV calls per strain per SV type and final cutoff combinations were chosen subjectively. C) The graphs show the fraction of genomic sequence within non-overlapping 100kb windows, predicted to be duplicated (positive values) and deleted (negative values) relative to the reference strain PS312. Contigs were concatenated using the genetic map of <i>P. pacificus</i> to produce chromosome-scale plots [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0131136#pone.0131136.ref036" target="_blank">36</a>]. Gray boxes indicate conserved syntenic regions with the sister species <i>P. exspectatus</i>. While duplications exhibit a much more even distribution, we identified two almost megabase sized regions with high fraction of missing sequence on the X chromosome.</p

Gene families enriched in SVs.

<p>We defined gene families based on the presence of protein domains (PFAM) and tested whether genes of a given gene family are enriched in SVs. Panels A and D shows significantly enriched gene families for deletions and duplications relative to the reference strain PS312 as pooled data (unpolarized analysis). The other panels show the enriched gene families after restricting to perfectly collinear regions and interpreting the SVs as derived events (polarized analysis).</p

Depletion of SVs among highly conserved genes.

<p>We classified genes in deleted and duplicated regions (relative to the reference strain PS312) based on presence of protein domains as well as homology relationships with other nematodes including <i>C. elegans</i>. For the different homology classes, we find that predicted one-to-one orthologs with <i>C. elegans</i> are strongly depleted from SVs indicating the action of purifying selection. Conversely, conserved and orphan genes that are members of larger gene families are significantly enriched in SVs suggesting that similar processes that have generated different homology relationships at a cross-species level are operating on a microevolutionary level.</p

Effect of duplications and deletions on gene expression levels.

<p>Distribution of expression categories of duplicated and deleted genes for the reference strain and the strain of interest. Only genes within perfectly collinear regions between the reference genome and the sister species were used for this analysis. While deletions lead to a strong increase in genes without expression, i.e. more than 80% (RS5200) and 65% (RS5410) of genes in predicted deletions show indeed no evidence of expression. In contrast, we do not see the opposite trend for duplicated genes.</p

phylogenetic analysis of antisense gene pairs in nematodes

This archived folder contains sequences, alignments and trees that were used for the publication "First insights into the nature and evolution of antisense transcription in nematodes" (Figure 3,4,5) by Rödelsperger, Menden, Serobyan, Witte, Baskaran (BMC Evolutionary Biology, 2016). The protein sequences (.fa files) were obtained by aligning C. elegans reference sequences against 19 other nematode genomes with the help of the Software exonerate. Multiple sequence alignments (*_alignment.fa) for homologous proteins were generated by the MUSCLE software and Maximum-likelihood trees were estimated by the phangorn R-package (.nexml files)

Developmental timing and survival curves of <i>P</i>. <i>pacificus</i> after exposure to <i>C</i>. <i>albidus</i> and <i>C</i>. <i>curvatus</i>.

<p>A) The boxplots show the time needed for <i>P</i>. <i>pacificus</i> nematodes to enter different developmental stage when growing on <i>E</i>. <i>coli</i> OP50 or <i>Cryptococcus</i> strains. The inlay shows the individual data points representing ten plates per food source. B) Survival of <i>P</i>. <i>pacificus</i> exposed to <i>C</i>. <i>albidus</i> (C3) and <i>C</i>. <i>curvatus</i> (C5) for 10 days. Standard lab food <i>E</i>. <i>coli</i> OP50 was used as a control.</p

Life history traits of <i>P</i>. <i>pacificus</i> cultured on the yeasts <i>C</i>. <i>albidus</i> and C. <i>curvatus</i>.

<p>The boxplots show the median, first and third quartile values from different life history traits experiments. A) Brood size of <i>P</i>. <i>pacificus</i> grown on <i>C</i>. <i>albidus</i> (C3), <i>C</i>. <i>curvatus</i> (C5) and <i>E</i>. <i>coli</i> OP50. Brood size was calculated as average number of progeny of 10 J4 larvae. B) Pharyngeal pumping rate results of <i>P</i>. <i>pacificus</i> exposed to <i>Cryptococcus</i> species. Assay was performed using <i>E</i>. <i>coli</i> OP50 as control. C) Defecation time (10 mins) of <i>P</i>. <i>pacificus</i> exposed to <i>C</i>. <i>albidus</i> (C3) and <i>C</i>. <i>curvatus</i> (C5) using <i>E</i>. <i>coli</i> OP50 as control. D) Eurystomatous (Eu) ratio of <i>P</i>. <i>pacificus</i> adults grown on <i>C</i>. <i>albidus</i> (C3) and <i>C</i>. <i>curvatus</i> (C5). Eu ratio was calculated from five biological replicates. Statistical significance was calculated using student t-test.</p

Gene expressions profiling of <i>P</i>. <i>pacificus</i> after a <i>Cryptoccocus</i> or <i>E</i>. <i>coli</i> diet.

<p>A) PCA of transcriptomes obtained from <i>P</i>. <i>pacificus</i> adult worms growing on <i>C</i>. <i>albidus</i> (C3), <i>C</i>. <i>curvatus</i> (C5) and <i>E</i>. <i>coli</i> OP50. Despite substantial variation in the control samples grown on <i>E</i>. <i>coli</i> OP50, different samples cluster together according to their food source indicating robust expression changes under different environments. B) Genes that are down-regulated upon <i>Cryptococcus</i> diet show a bias towards higher expression at early developmental stages. The graph shows mean expression and standard error of down-regulated genes in 10 developmental transcriptomes representing dauer, J2, J3, J4, and adult worms. C) Genes that are up regulated upon <i>Cryptococcus</i> tend to reflect transcriptomes from later developmental stages. D) Enrichment of significantly differentially expressed genes upon exposure to <i>C</i>. <i>albidus (C3)</i> in previous gene expression profiling studies. E) Enrichment of significantly differentially expressed genes upon exposure to <i>C</i>. <i>curvatus (C5)</i> in previous gene expression profiling studies. F) KEGG pathway enrichment of down-regulated genes under exposure to <i>C</i>. <i>albidus (C3)</i>. The x-axis shows the enrichment score indicating how much more genes of a given pathway are found as differentially expressed when compared to random gene sets. The y-axis shows the negative logarithm of the p-value indicating the significance of the overrepresentation. G) KEGG pathway enrichment of up-regulated genes under exposure to <i>C</i>. <i>albidus</i> (C3). H) Enrichment of KEGG pathways among down-regulated genes when fed with <i>C</i>. <i>curvatus</i> (C5). I) Enrichment of KEGG pathways among up-regulated genes after exposure to <i>C</i>. <i>curvatus</i> (C5).</p

Differential Interference Contrast microscopy (DIC) to study nature of yeast clumps.

<p>A) Yeast cells clumps of <i>C</i>. <i>albidus (C3)</i> in intestine of <i>P</i>. <i>pacificus</i>. B), C) Absence of yeast clumps in defecated worms after 3 hours off food, suggesting that yeasts cells can be fully digested or excreted by <i>P</i>. <i>pacificus</i>.</p