Supplementary Materials01. dotted lines for each species in a. In the case of cDNA assemblies [1-3] for each (Fig. 1), and validated their accuracy with the high quality genome assembly and annotations (Fig. S1, Table S2). The three outcrossing species consistently yielded many more contigs (Fig. 1a) with a greater total assembly size (Fig 1b) than and We carefully excluded assembly artifacts as a source of these differences. First, the transcriptome assembly size difference held over a wide range of sequence read numbers, indicating it was not an artifact of undersampling. Second, the larger outcrossing species assemblies were also not due to the residual heterozygosity known to exist in the strains used , as transcriptome assembly sizes and heterozygosity estimates in Cidofovir pontent inhibitor the three gonochorists  were not correlated. Additionally, when we stripped the transcriptomes of potentially allelic contigs with high similarity to another, the relative assembly sizes were similar to those obtained with the entire sets (5.55 Mbp, 6.01 Mbp, 9.40 Mbp, 9.01 Mbp, 7.84 Mbp). Given the evolutionary relationships of these species [Fig. 1; 5], we conclude that the reliably detectable L4/adult transcriptomes of the selfing species have shrunk approximately one-third relative to their obligately outcrossing relatives. Having established a link between self-fertility and transcriptome shrinkage, we next examined whether sexual specialization might have changed as well. We compared the male and female partitions of the datasets to determine sex bias in transcript levels (Fig. 2). To allow direct comparisons between hermaphrodites and females, we used and mutants, which lack XX spermatogenesis but have normal female fertility and development [6, 7]. We examined sex bias by two methods. One used the cDNA contigs as a reference onto which the Cidofovir pontent inhibitor reads were mapped (Fig. 2a). The other used the publicly available genome sequences and associated gene predictions (Fig. 2b). The estimates of bias using these two approaches were highly concordant (Fig. S2, Table S3). Open in a separate window Figure 2 Sex-biased transcripts in outcrossing and selfing assembly analysis (top) and for gene predictions (bottom). d. Expression values of genes with highly male-biased expression in Males (top) and Females (bottom) of and pseudo-females of expressed as log2 FPKM. The heavy line in the boxes indicates the median. The top and bottom of the boxes indicate the upper and lower quartile respectively. Significant differences between distributions as assessed by the Kolmogorov-Smirnov test are indicated above (p 0.001; Cidofovir pontent inhibitor ** 0.01; *** 0.001). In both analyses, the broadest patterns of sex bias were similar in all five species, regardless of mating system (Fig. 2). Contigs (Fig. 2a) or gene models (Fig. 2b) (collectively transcribed units) with significantly male-biased expression were more abundant and had a wider range of expression values than those with female-biased expression. A similar pattern was found previously for  and other animals with heterogametic males [9, 10]. However, and differed from the obligate outcrossers as a group in two ways. First, the distributions of male-to-female expression ratios for highly male-biased transcribed units were less male-biased (Fig. 2c). For pseudo-females (Fig. 2d). In contrast, expression ratios for highly male-biased transcribed units had a broader distribution (Fig. 2c), largely explained by lower and more variable expression levels in males (Fig. 2d). Thus, while the expression of the most male-biased genes evolved to be less male-specific in both selfing species, this occurred in distinct ways. A second aspect of transcriptome-wide sex bias that distinguished the selfing species was that a lower fraction of their detected transcribed units were highly female-biased (Fig. 2a,b, see red numbers in lower right corner of each panel). This could be because genes with highly female-biased expression were eliminated disproportionately from the and genomes, or because they are being created in male-female lineages at a higher rate. Consistent with the idea of ongoing gene formation, and genesa. Comparison of the patterns of conservation of genes whose expression is detected (white), sex-biased Cryab (grey), highly sex-biased (over 10 fold, black), highly male-biased (blue) or highly female-biased (pink). The fraction of genes with a homolog in at least one other gonochoric species but none in one or both of the selfing species are represented, as well as those missing in another outcrossing species, bars.