The S. pombe genome was divided in to five different feature classes: protein coding sequence, intron sequence, untranslated sequence (5' and 3' UTRs) and intergenic sequence (all nucleotides between UTRs of protein coding genes). The frequency of RNA-Seq reads was calculated over sequential 20 bp windows across these features; for coding sequence, the frequency of antisense reads was also calculated. Frequency was normalized to the maximum frequency within each feature class to compensate for the different class sizes.

A) Expression clusters for each species. Gene expression profiles for each species were clustered (4). The size of each heat map is proportional to the number of genes in the cluster and the number of genes in each is indicated. Similar cluster sizes and patterns reflect similar expression patterns between the species. The heat-shock transcription profile is similar to log-phase growth because the transcriptional response on the 15-minute timescale used here is limited to a relatively small number of genes.
B) A selection of enriched GO terms for each cluster. The color intensity is proportional to the negative logarithm of the hyper-geometric p-value enrichment on a continuous scale of 0–10. Complete GO term enrichments are shown in Table S26.

A) A maximum-likelihood phylogeny of 12 fungal species from 440 core orthologs (each occurring once in each of the genomes) from fly to yeast. A maximum-parsimony analysis produces the same topology. Both approaches have 100% bootstrap support for all nodes.
B) The chromosome structure of S. pombe, S. octosporus and S. japonicus. The middle bar in each figure represents the chromosome and its centromere: red for Chromosome 1, blue for Chromosome 2 and yellow for Chromosome 3. Above and below each chromosome are depicted the chromosomes in the other two species to which the genes on the chromosome of interest map, using the same color scheme. Above the S. pombe and S. japonicus chromosomes are depicted the distributions of transposons and mapping of siRNAs. S. cryophilus is not included because its genome has not been assembled into complete chromosomes.
C) The centromeric repeat structures of S. pombe, S. octosporus and S. japonicus.

A) Examples of antisense transcription of meiotic genes. Above and below the chromosome coordinates are the coding sequence annotations on the top and bottom strand, respectively. Above and below these are the strand-specific RNA-Seq read densities on a 0–300 scale; signal above 300 is truncated to make the low amplitude signal visible.
B) Enrichment of GO annotations within the set of protein-coding genes with more antisense than sense transcription. All terms with a p value of less than .01 are included, except for high-level terms (i.e. biological process and molecular function).

A) The enrichment of gene functional modules regulated by the Rtg3-biding motif in 23 Ascomycota. This motif is enriched upstream of amino acid metabolism genes in all Ascomycota. However, in fission yeast, it is specifically enriched upstream of stress-response genes. S. cerevisiae (Scer), S. paradoxus (Spar), S. mikatae (Smik), S. bayanus (Sbay), C. glabrata (Cgla), S. castellii (Scas), K. waltii (Kwal), A. gossypii (Agos), K. lactis (Klac), S. kluyveri (Sklu), D. hansenii (Dhan), C. guilliermondii (Cgui), C. lusitaniae (Clus), C. albicans (Calb), C. tropicalis (Ctro), C. parapsilosis (Cpar), C. elongosporus (Celo), Y. lipolytica (Ylip), N. crassa (Ncra), A. nidulans (Anid), S. japonicus (Sjap), S. octosporus (Soct), S. pombe (Spom)
B) Enrichment of Rtg3- and Aft1-binding sites in the promoters of stress response genes. Each row represents a gene. The strength of the strongest regulatory site upstream of the gene is indicated in the blue heat map. The expression of the gene in glucose depletion (gd) and early-stationery phase (es) relative to log phase is indicated in the blue-yellow heat map. Genes specific to the fission yeast clade are indicated in orange.
C) Enrichment of Fkh2/Mei4- and MBF-binding sites in front of antisense-transcribed genes. As in B, but each row represents a gene with greater antisense than sense transcription. Gene associated with meiosis (44) are indicated in magenta.