The fission yeast clade, comprising Schizosaccharomyces pombe, S. octosporus, S. cryophilus and S. japonicus, occupies the basal branch of Ascomycete fungi and is an important model of eukaryote biology. A comparative annotation of these genomes identified a near extinction of transposons and the associated innovation of transposon-free centromeres. Expression analysis established that meiotic genes are subject to antisense transcription during vegetative growth, suggesting a mechanism for their tight regulation. In addition, trans-acting regulators control new genes within the context of expanded functional modules for meiosis and stress response. Differences in gene content and regulation also explain why, unlike the Saccharomycotina, fission yeasts cannot use ethanol as a primary carbon source. These analyses elucidate the genome structure and gene regulation of fission yeast and provide tools for investigation across the Schizosaccharomyces clade. The fission yeast genus Schizosaccharomyces forms a broad and ancient clade within the Ascomycete fungi (Fig. 1A) with a distinct life history from other yeasts (1). Fission yeast grow preferentially as haploids, divide by medial fission rather than asymmetric budding, and have evolved a single-celled lifestyle independently from the budding yeasts (Saccharomycotina). Fission yeasts share important biological processes with metazoans, including chromosome structure and metabolism (relatively large chromosomes, large repetitive centromeres, heterochromatic histone methylation, chromodomain heterochromatin proteins, siRNA-regulated heterochromatin and TRF-family telomere binding proteins), G2/M cell cycle control, cytokinesis, the mitochondrial translation code, the RNAi pathway, the signalosome pathway and spliceosome components with metazoans. These features are absent or highly diverged in budding yeast. In general, core orthologous genes in fission yeast more closely resemble those of metazoans than do those of other Ascomycetes (2). Fission yeasts have also evolved innovations in carbon metabolism, including aerobic fermentation of glucose to ethanol (3). This convergent evolution with the Rhind et al. Page 2 Science. Author manuscript; available in PMC 2011 November 20. N IH PA Athor M anscript N IH PA Athor M anscript N IH PA Athor M anscript budding yeast Saccharomyces cerevisiae offers insight into the evolution of complex phenotypes. S. pombe is widely used as a model for basic cell-biological processes and to study genes implicated in human disease. To better understand its evolution and natural history, we have compared the genomes and transcriptomes of S. pombe, S. japonicus, S. octosporus and S. cryophilus, which constitute all known fission yeasts. Genome sequence and phylogeny We sequenced and assembled the genomes of S. octosporus, S. cryophilus and S. japonicus using clone-based and clone-free whole-genome shotgun (WGS) approaches (Table S1). Each genome is ~11.5 Mb in size. S. octosporus and S. cryophilus are 38% GC; S. japonicus is 44%. By comparison, the S. pombe genome is 12.5 Mb in size and 36% GC. We assembled the S. octosporus and S. japonicus scaffolds into 3 full-length chromosomes of similar quality to the finished S. pombe genome (Figs. 1B, S1, S2 and Tables S2, S3) and identified telomeric sequence using WGS data (4). Telomere-repeats in S. japonicus (GTCTTA), S. octosporus (GGGTTACTT) and S. cryophilus (GGGTTACTT) matched a one and a half repeat-unit sequence at the putative telomerase-RNA locus, similar to the configuration in S. pombe (GGTTAC) (5). Using these motifs, we extended the S. japonicus and S. octosporus chromosomes into subtelomeric and telomeric sequence (4). We constructed a phylogeny of the Schizosaccharomycetes within Ascomycota (Fig. 1A, S3) from 440 single-copy core orthologs, placing the monophyletic Schizosaccharomyces species as a basal sister group to the clade including the filamentous fungi (Pezizomycotina) and budding yeast (Saccharomycotina). We found an average amino acid identity of 55% between all 1:1 orthologs between S. pombe and S. japonicus, similar to that between humans and the cephalochordate amphioxus (Table S4). For the most closely related species, S. cryophilus and S. octosporus, 1:1 orthologs share 85% identity on average, similar to humans and dogs. The genetic diversity within S. pombe is low. Comparing S. pombe 972 to WGS of S. pombe NCYC132 and S. pombe var kambucha, two phenotypically distinct strains, revealed less than 1% nucleotide difference between the three strains (Fig. S4, Table S5). Eradication of transposons and reorganization of centromere structure Transposons and other repetitive sequences are thought to be crucial for centromeric function through the maintenance of heterochromatin (6). These sequences evolve rapidly, but the evolutionary relationship between centromeres, transposons and heterochromatin is unclear, in part because fungal centromeres have not generally been included in genome assemblies. The S. japonicus genome harbors 10 families of gypsy-type retrotransposons (4) (Figure S5 and Table S6). Sequence divergence of their reverse transcriptases suggests that these transposon families predate the last common ancestor of the Ascomycetes. However, a dramatic loss of transposons occurred after the divergence of S. japonicus; S. pombe harbors two related retrotransposon, Tf1 and Tf2; S. cryophilus has a single related retrotransposon, Tcry1; S. octosporus contains no transposons, but contains sequences related to reverse transcriptase and integrase that may represent extinct transposons (Fig. S5, Table S6). The disappearance of transposons in the post-S. japonicus fission yeast species correlates with the appearance of the cbp1 gene family, suggesting a transition in the control of centromere function. In S. pombe, Cbp1 proteins bind centromeric repeats and are required for transposon silencing and genome stability (7, 8). Although described as orthologs of CENP-B, a human centromere-binding protein, Cbp1 proteins apparently evolved independently within the Schizosaccharomyces lineage from a domesticated Pogo-like DNA Rhind et al. Page 3 Science. Author manuscript; available in PMC 2011 November 20. N IH PA Athor M anscript N IH PA Athor M anscript N IH PA Athor M anscript transposase (9). The appearance of the cbp1 gene family also correlates with the switch from RNAi-mediated transposon silencing in S. japonicus (see below) to a Cbp1-based mechanism in S. pombe, suggesting that this shift to Cbp1-based transposon control allowed the eradication of most transposons from the fission yeast genomes, possibly by promoting recombinational deletion between LTRs (8). Furthermore, the cbp1 family is evolving rapidly (Fig. S6), suggesting that Cbp1-based transposon silencing is a Schizosaccharomyces-specific innovation that arose after the divergence of S. japonicus. The loss of transposons was accompanied by a significant reorganization of chromosome architecture that conserves centromere function, suggesting evolution of novel centromere structures that compensate for the loss of transposons. In S. japonicus, transposons cluster next to telomeres and centromeres, as in metazoans (Fig. 1B,C). In the other Schizosaccharomycetes, the subtelomeres and pericentromeres are also repetitive, but lack transposons (Fig. 1C). However, like S. japonicus, the centromeric and subtelomeric repeats are confined to pericentromeric and subtelomeric regions, respectively, with one exception — a centromeric repeat involved in transcriptional silencing at the S. pombe mating-type locus (10). We confirmed that the centromeres are heterochromatic by histone H3 lysine-9 methylation mapping (Fig. S7), and by showing that the S. japonicus centromeres are functional by meiotic mapping (Table S2). Although centromeric repeats evolve rapidly, differing even between related strains (11), individual repeat sequences tend to be similar within strains (Fig. 1C). No similarity was observed between the centromeric repeats of S. pombe, S. octosporus or S. cryophilus. However, both S. pombe and S. octosporus centromeres contain repeated elements, highly similar between chromosomes, that are arrayed in a larger inverted repeat structure around a unique core sequence (Fig. 1C), suggesting that they are homogenized by non-reciprocal recombination. This contrasts with a lack of symmetry in S. japonicus, and implies that transposition occurs more rapidly than homogenization by recombination. Thus, the suppression of transposition likely led both to the degeneration of transposon sequences and to the evolution of symmetric centromeric repeats. Despite the divergence of centromere sequence and of gene order on the chromosome arms, karyotype and pericentromeric gene order are conserved between S. pombe and S. octosporus (Fig. S8). Thus, although gene conversion maintains the similarity of centromeric repeats between the different centromeres, crossover recombination between centromeres is suppressed. We observed neither centromeric translocations nor neocentromere events within these lineages, despite the fact that centromeres can occur at novel locations in manipulated S. pombe strains. The retention of repetitive elements in the centromeres of (12) S. pombe, S. octosporus and S. cryophilus, even as they have lost their transposons, implies that centromeric repeats have an important function. Since siRNAs are involved in both transposon silencing and centromere function (13), we investigated these roles in the Schizosaccharomyces lineage. In S. pombe, the centromeric repeats produce dicer-dependent siRNAs required for maintenance of centromeric structure, function and transcriptional silencing via Argonaute-dependent heterochromatin formation (14). However, transposons are silenced in S. pombe by RNAi-independent mechanisms and do not produce abundant siRNAs (Figs. 1B, S9) (7). To investigate whether centromeredirec