that time. Unexpectedly, we observed that this second subset of introns, shared only with older relatives, show stronger signal than those introns shared with the closest intron-poor ancestor in T. gondii (Table 4). This suggests that boundaries with greater strength in intron-poor species does not reflect retained ancestral signal. (There was an insufficient number of introns in the A. fumigatus distantly-related group for comparison). Another argument also argues that these intron subsets’ stronger boundaries reflects not retention of ancestral boundary strength, but something else: A. fumigatus and Y. lipolytica exhibit different 59 splice site consensus sequences, thus while A. fumigatus introns with homology to retained Y. lipolytica introns do show greater homogeneity in 59 splice site boundaries (matching the consensus GTAAGT), they do not more closely resemble Y. lipolytica boundaries (consensus GTGAGT). Indeed, we observe the opposite trend: only 15.2% (26/171) of the A. fumigatus introns shared with Y. lipolytica have a G in position +3, whereas in the whole set of A. fumigatus introns, 21.4% have a G in that position. Convergent Evolution of Intron Structures PolyT Distributions Finally, we studied the distribution of characteristic intronic polyT motifs along intron length. For each species, we calculated frequencies of intronic minimal ‘‘polyT motifs’’ (following previous studies, we define these as six consecutive nucleotides containing at least 3 T’s and no A’s [19,21,22]) as a function of distance from the acceptor site. Almost all species conformed to one of 3 broad patterns, which tend to be conserved within large phylogenetic groups (Figure 6). For the most common distribution (found in metazoans, plants, most apicomplexa and the heterokont Phytophtera species) polyT motifs concentrate near the intron terminus (Figure 6A). The 59 limit of the distribution is likely determined by branch point position in some species (,30 nt, similar to mean BP-AG distance 31.5 nt in mammals [23] and 27.6 nt in Arabidopsis thaliana [24]). In other species (Caenorhabditis elegans, Ciona intestinalis, Drosophila melanogaster, and the apicomplexan Theileria parva) polyTs are concentrated in the last ,10 nt, and are underrepresented 10– 15 nt from the terminus. This pattern could suggest a more 39 Figure 6. Characteristic patterns of polyT motif distributions across eukaryotes. Examples of: (A) Most common and widely distributed pattern. PolyT motifs concentrate at the 39 of the introns. Observed in plants, animals, apicomplexa and heterokonts. (B) Pattern characteristic of most fungi and in T. pseudonana, characterized by relatively uniform polyT distribution. (C) Pattern of some intron-poor species. (D) Pattern observed in amebozoa and in the fungus U. maydis. Species included in the study: Homo sapiens (Hsap); Caenorhabditis elegans (Cele); Drosophila melanogaster (Dmel); Strongylocentrotus purpuratus (Spur); Arabidopsis thaliana (Atha); Oryza sativa (Osat); Plasmodium falciparum (Pfal); Phytophthora ramorum (Pram); Schizosaccharomyces pombe (Spo); Aspergillus fumigatus (Afu); Giberella zeae (Gzeae); Cryptococcus neoformans (Cne); Candida glabrata (Cgla); Eremothecium gossypii (Egos); Debaryomyces hansenii (Dhans); Yarrowia lipolytica (Ylip); Cryptosporidium parvum (Cpar); Saccharomyces cerevisiae (Sce); Kluyveromyces lactis (Klact); Dictyostelium discoideum (Ddis); Entamoeba hystolitica (Ehys); Ustilago maydis (Umay). doi:10.1371/journal.pgen.1000148.g006 PLoS Genetics | www.plosgenetics.org 9 August 2008 | Volume 4 | Issue 8 | e1000148