Author Summary The spliceosomal introns that interrupt eukaryotic genes show great number and sequence variation across species, from the rare, highly uniform yeast introns to the ubiquitous and highly variable vertebrate intron sequenc- es. The causes of these differences remain mysterious. We studied sequences of intron branch points and 39 termini in 50 eukaryotic species. All intron-rich species exhibit variable 39 sequences. However, intron-poor species range from variable sequences, to uniform branch point motifs, to uniform branch point motifs in uniform positions along the intronic sequence. This is a more complex pattern than the clear relationship between intron number and 59 intron sequence uniformity found previously. The corre- spondence of sequence uniformity and intron number extends to species of the green algal genus Ostreococcus, in which the single intron-rich genomic region shows far more variable intron sequences than in the otherwise intron-poor genome. We suggest that different concen- trations of spliceosomal complexes may explain these differences. In addition, we report the existence of 39 polyT tails in diverse eukaryotic protists, suggesting that this structure is ancestral. Together, these results underscore the complexity of ancestral eukaryotic splicing, the qualitatively different evolutionary forces acting on intron sequences in modern eukaryotes, and the impressive evolutionary malleability of eukaryotic genes. Results Branch Point Consensus Strength in Intron-Poor Species The branch point is an internal intronic sequence that initiates the splicing event through a hydrophilic attack by an adenosine 29 hydroxyl group at the 59 splice site [12,13]. As with donor sites, branch point consensus strength varies across species: 84% of Saccharomyces cerevisiae introns utilize the sequence TACTAAC and 94% have ACTAAC, compared to fewer than 20% of human introns with the exact same sixmer at the branch point site Convergent Evolution of Intron Structures (Figure 1), and with Caenorhabditis nematodes, where no branch points have been identified. We first studied intron branch points in available genomes from intron-poor species (Table 1; defined, as before, as those species with ,0.1 or fewer introns per gene on average [9]). Many of these species exhibited clear branch point consensus sequences. First, we found that the S. cerevisiae branch point consensus ACTAAC (throughout, the putative branch point A is underlined) is found in all fully-sequenced hemiascomycetous yeasts, with 66–100% of introns in each species containing this motif. Extended branch points of up to eight bases were found in some species (Table 1). We also found an extended single ACTAACC branch point motif in all 26 known introns of the red alga Cyanidioschyzon merolae. Other intron- poor species containing strong putative branch point motifs were the two excavate species: 32/34 (94.12%) probable introns (see Materials and Methods) in the flagellated protist Trichomonas vaginalis use WCTAAC, and all five known introns in Giardia lamblia (four published plus one unpublished instance) contain CTRACA. Finally, excluding two questionable predicted introns (see Discus- sion), all 13 introns in the microsporidium parasite Encephalitozoon cuniculi contain a TAAYTT hexamer (9/13 have CTAAYTT). Thus all lineages with strong branch points conformed to a general WCTRAYN consensus. However, in at least one intron-poor lineage we failed to find such clear branch point sequences. Visual inspection and computational analysis (see below) of the intron-poor apicom- plexan parasite Cryptosporidium parvum failed to reveal a potential branch point site. The case for the few introns of the Guillardia theta nucleomorph is less clear. 14/16 predicted G. theta introns contain a YAAY branch point-like sequence between 2 and 6 nucleotides from the acceptor AG (compared to only 2/16 that contain such a motif within the next 10 positions (7 to 17)). Intriguingly, a second YAAY signal exists further upstream – 8/16 introns have a YAAY 24–28 nt from the 39 terminus. The single known intron in each of the sequenced Trypanosomatid genomes does not show a clear branch point structure; however it is of note that despite having few cis-spliced introns, these species do have large numbers of 39 splicing boundaries due to the ubiquity of spliceosome-mediated spliced leader trans-splicing [14,15]. Figure 1. Intron sequence structures. Examples of a species with (A) strong consensus structures (S. cerevisiae), and (B) weakly conserved structures (human). doi:10.1371/journal.pgen.1000148.g001 PLoS Genetics | www.plosgenetics.org 2 August 2008 | Volume 4 | Issue 8 | e1000148