O. lucimarinus All introns Intron subset EST confirmed doi:10.1371/journal.pgen.1000148.t003 All Chrom Chrom 2 Non-Chrom 2 All Chrom Chrom 2 Non-Chrom 2 2148 1748 396 560 241 319 1.6 1.8 1.8 3.0 1.9 4.8 Table 3. Intron signal features for different intron subsets of O. lucimarinus. Convergent Evolution of Intron Structures Introns studied 59ss strength (bits) % of introns containing most common branch point motif for given number of degenerate sites 0 41.15% (actgac) 5.53% (ctgacg) 49.12% (actgac) 50.71% (actgac) 4.56% (ctgacg) 87.15% (actgac) 1 42.97% (actrac) 8.79% (ctgack) 50.65% (actrac) 53.21% (actrac) 8.30% (ctgack) 89.97% (actrac) 2 44.93% (wctrac) 15.08% (wctrac) 51.56% (wctrac) 57.32% (wctrac) 12.45% (ctrack) 91.54% (wctrac) 3 47.16% (wctray) 20.35% (wctray) 53.09% (wctray) 60.36% (wctray) 17.01% (wctray) 93.10% (wctray) introns with larger BP-AG distances. Y. lipolytica did not show differences in strength of 39 sequence consensus for different BP- AG distances. Mapping of BP-AG distances across hemiascomyces shows a complex phylogenetic pattern (Figure 5). The five species with strong BP-AG constraint are intermingled on the tree with the three less constrained lineages, suggesting convergent evolution of BP-AG constraint. Importantly, the ‘‘preferred’’ BP-AG distance varies across species – for Y. lipolytica, the most common BP-AG distance is 2 nt, compared to 3 in C. guilliermondii, 4–5 for C. lusitaniae, 6–8 for E. gossypii and 7–8 for D. hanseii. It seems unlikely that a species with a strong preference for a certain BP-AG distance would convert to a different distance, since this would require indels of a very specific length occurring in dozens of already constrained introns. It seems more likely that this condition reflects ancestrally relatively unconstrained BP-AG distance, and convergent evolution of constrained BP-AG distances in different lineages. Introns in Intron-Rich Species Orthologous to Retained Introns in Intron-Poor Species Convergent evolution of retained intron sequences in intron- poor species is likely due to a combination of two factors: preferential retention of introns with consensus-like (i.e. strong) sequences and change of retained intron sequences to consensus boundaries. However, the relative impact of these two factors is unknown. We attempted to address this issue by identifying introns in intron-rich species that were present at the exact homologous position to introns in any available intron-poor species, and thus are likely to be ancestral to both species. If strong consensus sequences in intron-poor species are due to differential retention of introns with conserved ancestral sequences, it is possible that orthologous introns in intron-rich species could retain some of this signal. For each intron-rich species (the apicomplexan Toxoplasma gondii, H. sapiens, S. pombe, A. fumigatus, and A. thaliana), we compared 59 splice site strength and branch point conservation between introns putatively orthologous to introns retained in at least one intron-poor species and the total set of introns in these species. Significant differences for both intron structures were found for A. fumigatus and T. gondii introns (Table 4). Despite this analysis being perhaps the most direct way to test the hypothesis of preferential intron retention available, it is deeply undermined by the great phylogenetic distances between intron- poor species and even their closest relatives (T. gondii-C. parvum and A. fumigatus-hemiascomycetes diverged both many hundred million years ago) and associated large amounts of sequence change. Therefore, the finding of a positive signal for any of these Figure 4. Highly constraint BP-AG distances in Y. lipolytica and T. vaginalis. (A) 39 intron boundary in Yaworria lipolytica. (B) 39 intron boundary in Trichomonas vaginalis. Note that the difference between both consensus is the existance of 1 (Y. lipolytica) or 2 (T. vaginalis) AC dinucleotides between the BP motif and the terminal AG. Consensus sequence for G. lamblia is similar to T. vaginalis. (C) 39 intron boundary for confirmed introns from non-chromosome 2 genes in O. lucimarinus. (D) 39 intron boundary in C. elegans. doi:10.1371/journal.pgen.1000148.g004 PLoS Genetics | www.plosgenetics.org 7 August 2008 | Volume 4 | Issue 8 | e1000148