Generation-biased gene expression across the brown algae

A. Cladogram of the brown algae showing the ten species for which generation-biased gene expression was compared. Adapted from (Cock et al., 2014). B. Generation-biased gene expression in relation to life cycle dimorphism. Pie diagrams indicating the proportions of sporophyte-specific (dark red), sporophyte-biased (light red), gametophyte-specific (dark blue), gametophyte-biased (light blue) and unbiased (grey) genes in species with different haploid-diploid life cycles ranging from sporophyte-dominant through isomorphic to gametophyte-dominant (indicated by the red and blue wedges). sp., species. C. Overlaps (Jaccard index) between sets of gametophyte-biased/specific and sporophyte-biased/specific genes across the ten species.

Biological function gene ontology term enrichment in the generation-biased/specific gene sets of the ten species

Conserved general functional categories are indicated by coloured boxes. padj, p-value adjusted for multiple testing based on the Benjamini-Hochberg false discovery rate.; GA, gametophyte; SP, sporophyte.

Gene expression patterns during the Ectocarpus species 7 life cycle

A. Principal component analysis of the Ectocarpus species 7 gene expression across ten life cycle stages. The numbers in circles refer to the differential gene expression analyses illustrated in C and D. B. Percentage changes in gene expression (regression breakpoints) during Ectocarpus species 7 sporophyte development (from left to right, transitions between free-swimming male gamete, sporophyte initial cell (24 hours after gamete release), elongating sporophyte initial cell (48 hours after gamete release), sporophyte 2-5 cell stage, non-fertile adult sporophyte and fertile sporophyte). C. Proportions of differentially expressed genes in the transitions between 1) free-swimming male gamete and sporophyte initial cell stage, and 2) adult sporophyte and gametophyte stages, determined using DESeq2 (Table S1). D. Density distribution of the |log2(FoldChange)| for upregulated and downregulated genes in the differential gene expression analysis described in C. Dotted lines indicate mean values.

Characterisation of gene co-expression modules in Ectocarpus species 7

A. Functional categories manually assigned to modules based on Gene Ontology term enrichment analysis of each module. B. Enrichments in sub-cellular localisations based on HECTAR predictions: mitochondria (small ellipses), plastid (tube shape with thylakoids) and signal peptide/anchor peptide for secreted proteins. Note that HECTAR does not predict nuclear localisation. C. Proportion of differentially expressed genes (DESeq2 adjusted p-value < 0.05 and |log2FoldChange| > 1) in each gene module in the transitions between 1) free-swimming male gametes and sporophyte initial cell stage, and 2) adult sporophyte and gametophyte (Table S1). Significant enrichment (indicated by a darker shade of green or orange) means that the proportion of differentially expressed genes in the module was significantly greater (ClusterProfiler adjusted p-value < 0.05) than the proportion for the entire genome. D. Average module gene expression profile computed on genes with a WGCNA module MM > 0.86 for, from left to right: free-swimming male gamete, sporophyte initial cell (24 hours after gamete release), elongating sporophyte initial cell (48 hours after gamete release), sporophyte 2-5 cell stage, non-fertile adult sporophyte, fertile sporophyte, non-fertile female and male gametophytes, fertile female and male gametophytes developmental stages. E. Number of genes in the module positively or negatively correlated with the module eigengene. F. Module eigengene dendrogram showing the relationship between module eigengenes.

Expression of translation-related genes during Ectocarpus species 7 development

A. Mean expression profile computed for genes with a WGCNA module MM > 0.86 for (from left to right): free-swimming male gamete, sporophyte initial cell (24 hours after gamete release), elongating sporophyte initial cell (48 hours after gamete release), sporophyte 2-5 cell stage, non-fertile adult sporophyte, fertile sporophyte, non-fertile female and male gametophyte, fertile female and male gametophyte developmental stages in modules “purple”, “cyan”, “greenyellow”, “magenta”. B and C. Heatmaps showing log2(NormalisedCounts+1) values across the same developmental timepoints as in A for manually reannotated ribosomal protein genes corresponding to cytosolic, chloroplast and mitochondrial ribosomal subunits (B) and translation-related genes (C). Left annotation track: differential expression analysis results for the transitions between 1) free-swimming male gamete and sporophyte initial cell stage, and 2) adult sporophyte and adult gametophyte; right annotation track: WGCNA module colours. D. Schematic representation of translation-related functions enriched in the “greenyellow”, “purple”, “magenta”, “cyan” modules. When it was possible to manually assign a general function to a module, the annotation is indicated in brackets after the module name. E. Gene ontology terms significantly enriched in the sets of genes clustered within the “purple”, “greenyellow”, “cyan”, and “magenta” modules in Ectocarpus species 7. Enrichment is indicated as log2 of the ratio of the proportion of genes assigned to the GO term in the module divided by the proportion for the whole genome. CC, cellular component; BP, biological process; MF, molecular function.

Conservation of life-cycle-related gene co-expression patterns between Ectocarpus species 7 and D. dichotoma

A. WGCNA density and connectivity median rank statistics indicating the degree of conservation of gene co-expression patterns when of Ectocarpus species 7 genes in Ectocarpus species 7 co-expression modules were substituted with their D. dichotoma orthologues. Manually-assigned general biological functions are indicated in boxes. SDR, sex-determining region. Module size indicates the number of one-to-one orthologues in each module. The modules “darkred” and “darkturquoise” were not included in this analysis because they had only one and zero orthologues, respectively (Table S6). TF, transcription factor; SDR, sex-determining region. B. Correlation heatmap comparing three Ectocarpus species 7 modules between Ectocarpus species 7 and D. dichotoma: “midnightblue” (very poor conservation), “purple”, and “royalblue” (good conservation). The lower half of the heatmap was calculated based on the expression pattern of the D. dichotoma orthologue of each Ectocarpus species 7 gene in each module. C. Counts of shared one-to-one orthologues between gene co-expression modules defined for Ectocarpus species 7 (x-axis) and D. dichotoma (y-axis). The colour code represents the log10(p-value) for Fisher’s exact test (red bar), which was applied to determine whether pairs of modules shared a greater number of one-to-one orthologues than expected from a random distribution. Numbers after the module names indicate the number of one-to-one orthologues in each module. The “darkturquoise” module was not included in this analysis because it contained zero orthologues (Table S6) D. Heatmap showing the expression levels (log2(NormalisedCounts+1)) of 20 selected one-to-one orthologous flagellum-related genes in Ectocarpus species 7 and D. dichotoma. Ectocarpus species 7 timepoints: free-swimming male gamete, sporophyte initial cell (24 hours after gamete release), elongating sporophyte initial cell (48 hours after gamete release), sporophyte 2-5 cell stage, non-fertile adult sporophyte, fertile sporophyte, non-fertile female and male gametophytes, fertile female and male gametophytes. D. dichotoma timepoints: sperm, egg cell, zygote, embryo, adult non-fertile sporophytes, and female and male gametophytes. E. Computed z-score based on log2(NormalisedCounts+1) values for three TALE homeodomain transcription factors over six developmental stages shared between Ectocarpus species 7 and D. dichotoma, namely sperm, zygote, early sporophyte, non-fertile sporophyte, female and male gametophyte. The Pearson correlation coefficient between the two expression datasets (cor) and the associated p-value are indicated.