Table 1. Putative branch point consensus in the 16 studied intron-poor species. Hemiacsomycete yeasts Candida albicans Candida glabrata Candida tropicalis Candida guilliermondii Candida lusitaniae Debaryomyces hansenii Eremothecium gossypii Kluyveromyces lactis Saccharomyces cerevisiae Yarrowia lipolytica Cyanidioschyzon merolae Guillardia theta NM Red algae Excavates Microsporidians Apicomplexans Trichomonas vaginalis Giardia lamblia Encephalitozoon cuniculi Cryptosporidium parvum CONSENSUS TACTAAC TACTAACA ACTAAC ACTAAC ACTAAC TACTAAC ACTAAC TACTAAC TACTAAC ACTAAC ACTAACC Not clear WCTAACA CTRACA CTAAYTT Not clear WCTRAYN The active adenosine is underlined. doi:10.1371/journal.pgen.1000148.t001 41.15% of predicted introns containing the same branch point-like ACTGAC sequence (Table 2). However, given the high evolution- ary distance of O. lucimarinus from other species with fully-sequenced genomes and the relatively small number of available transcript sequences, annotation in O. lucimarinus and distant congenitor O. tauri is difficult and some intron predictions may thus be unreliable. To identify a confident set of introns, we performed BLASTN searches of the predicted intron-containing coding sequences against the 17,592 available O. lucimarinus EST sequences. Further computa- tional and manual filtering for ambiguities and potential problems associated with reverse transcriptase [16,17] yielded a total of 560 confirmed intron sequences. The confirmed introns show a stronger signal, with 50.7% containing the branch point sequence ACTGAC (and 37.9% showing an extended branch point GACTGACG). The pattern becomes even more striking when intron sequences are divided along the lines of the previously reported genomic heterogeneity of O. lucimarinus, with roughly half of chromosome 2 differing from the rest of the genome in a variety of ways including much higher intron density [18]. Confirmed chromosome 2 introns show very weak branch point signal, with only 4.6% sharing the same sixmer (CTGACG), while 87.2% of introns outside of chromosome 2 contain ACTGAC (Table 3), and 66.5% contain an extended GACTGACG motif. Confirmed introns outside of chromosome 2 also show very strong 59 splice sites consistent with intron-poor structures (4.8 bits), whereas confirmed chromosome 2 introns show much weaker boundaries (1.9 bits) (Table 3). Notably, 59 splice boundaries outside chromosome 2 exhibit the atypical consensus GTGCGTG, whereas chromosome 2 introns prefer a more typical GTRNGT. Convergent Evolution of Intron Structures In contrast to O. lucimarinus, predicted introns in O. tauri show far lower conservation of branch points (Table 2). The lack of EST sequences for this species makes confirmation difficult, however a few putative intron sequences identified by TBLASTN searches of intron-containing non-chromosome 2 O. lucimarinus genes against the O. tauri genome exhibited sequences similar to O. lucimarinus introns, suggesting that O. tauri may exhibit a similar pattern. Given this seeming discrepancy between all predicted and confirmed O. tauri introns, we excluded O. tauri from the analysis. Acceptor Site Conservation Next, we studied acceptor sequence conservation within genomes. The 39 sequences of spliceosomal introns generally show more similarity across species, with most species showing a terminal YAG, sometimes preceded by a poly-pyrimidine tract (Figure 1), although most fungal species and some others lack the poly-pyrimidine tract [19]. A survey of 50 widely diverged eukaryotic species showed 5 clear exceptions to this pattern. Three species, the protists T. vaginalis and G. lamblia, and Yarrowia lipolytica, a hemiascomycetes fungus (relatives of the baker’s yeast S. cerevisiae), show strikingly similar patterns (Figure 4). The three species lack the 39 polypyrimidine tract, and show clear anchoring of the branch point site at a specific number of basepairs away from the 39 terminal AG (branchpoint- AG (BP-AG)) distance; 2 nt (AC) in Y. lipolytica, 4 nt (ACAC) in T. vaginalis and G. lamblia; Figure 4). Non-chromosome 2 O. lucimarinus introns show a preference for CGCAG, though notably weaker than that found in the in the other three species (Figure 4). Interestingly, O. lucimarinus introns’ conserved 39 boundaries are associated with conserved BP-AG distance, as branch points for confirmed non-chromosome 2 O. lucimarinus introns show a broad peak ranging from around 20– 35 bp (data not shown). It is interesting then that both O. lucimarinus and the constrained BP-AG distance species prefer a C at position –5 and a R at position –4. We were unable to find an explanation involving snRNA sequences for this preference. Finally, C. elegans introns also show stronger 39 consensus, matching TTTCAG with nucleotides -6 and -5 significantly more conserved (Figure 4), as had been previously shown [20]. 39 Evolution in Hemiascomycetous Yeasts To better understand this pattern, we next studied available relatives of Y. lipolytica. We studied all six hemiascomycetes species with full genome annotations, as well as three additional Candida species for which some intron sequences were available. The species show pronounced differences, with three (including S. cerevisiae) showing large variations in BP-AG length and five species showing clear BP-AG constraint (differences in BP-AG length across hemiascomycetes was previously reported in [10]). In Debaryomyces hansenii, 65% of introns show a BP-AG distance between 6 and 8, and 88% of introns have a BP-AG distance between 5 and 10 nt. 75% of introns in Eremothecium gossypii with well defined branch points have BP-AG between 6 and 9 nt, with 66% between 6 and 11. The small number of available introns in C. lusitaniae and C. guilliermondii suggested preferred BP-AG distances of 4–5 and 3 nt, respectively. This BP-AG constraint could partially reflect differences in intron lengths, as mean/median lengths are lower for some of these species across the clade (Figure 5). However, the species with the clearest pattern of constraint, Y. lipolytica, has rather long introns relative to the other species. Intriguingly, in E. gossypii introns, sequences between the BP and intron terminus varied considerably based on the BP-AG distance. The 30 introns with a BP-AG distance of 6 nt (the shortest distance with more than a few introns) showed a strong sequence consensus at the 39 end of the sequence, with 80.0% having a G at PLoS Genetics | www.plosgenetics.org 4 August 2008 | Volume 4 | Issue 8 | e1000148