Convergent Evolution of Intron Structures Figure 2. Phylogenetic relationship between the species included in this study. Consensus phylogenetic tree of the species included in this study. Red species names and discontinuous branches indicate intron-poor species. Note that representatives of all eukaryotic supergroups have been included in this study. Based on [47,48]. doi:10.1371/journal.pgen.1000148.g002 Branch Point Consensus Strength in Intron-Rich Species We next studied conservation of branch points in 33 more intron-rich species. We studied the occurrence of motifs conforming to the consensus branch point WCTRAY or CTRAYN with varying levels of two-fold degeneracy (i.e. allowing two possible nucleotides at each site). For instance, the ACTAAC hexamer common to all C. merolae introns has no degeneracy, whereas the CTRACA motif of G. lamblia has one degenerate site, and the general consensus WCTRAY contains three two-fold degenerate sites. As with the other sites, either one or two nucleotides at a time were allowed at the final ‘‘N’’ site (CTRAYA, CTRAYC, CTRAYR, CTRAYY…). For each species, and for each level of degeneracy (zero to three degenerate sites), we calculated the fraction of introns that contain the same motif within the last 200 nt of the intron (see Methods). Then, for each level of degeneracy, we identified the motif that was present in the largest fraction of introns for that species. These values are given in Table 2. As expected, the intron-poor species discussed above give high values at all levels of degeneracy. All intron-poor species with strong consensus discussed above have values of at least 72% for one-site degenerate branch point motifs. By contrast, every studied intron-rich species shows much lower scores, with lower than 22% of introns with the same putative branch point motif (for example, 8.73% (ACTAAT) in Drosophila melanogaster or 10.97% (ACTGAC) in Aspergillus fumigatus), and less than 36% allowing one degenerate site. This relation between intron numbers and branch point consensus strength is underscored in Figure 3. The species are clearly distributed in two main groups (with two exceptions, G. thetha NM and C. parvum (red asterisks)): intron-rich/ weak branch point consensus and intron-poor/strong branch consensus (intron-poor, fewer than 0.15 introns per gene; strong branch points, same BP-like hexamer in more than 50% of introns (red lines in Figure 3A). Overall, there is a negative correlation (r = 20.75) between intron numbers and branch point consensus conservation (by a linear regression analysis, Figure 3B). The Peculiar Case of Ostreococcus Only the species Ostreococcus lucimarinus appeared to represent an intermediate between strong and weak branch point species, with PLoS Genetics | www.plosgenetics.org 3 August 2008 | Volume 4 | Issue 8 | e1000148