branch point position, although branch points in these species are difficult to determine [25]. The rhizarian Plasmodiophora brassicae seems consistent with this broad pattern; however, the small number of available introns renders confident conclusions difficult. Second, in most fungi, polyTs are roughly equally common across the intron (with the exception of the position of the branch point site, which typically falls ,13–25 nt from the terminus [19]) (Figure 6B). This pattern resembles that found in some intron-poor species, including the T-rich introns of C. parvum as well as the more moderate T-rich introns of hemiascomycete yeasts (Figure 6C). S. cerevisiae shows a partial exception to the pattern, with a pronounced peak ,10 nt from the terminus. The third pattern is found in the two fully-sequenced amoebozoans (Dictyostelium discoideum and Entamoeba histolytica) and in the intron-poor fungus Ustilago maydis (Figure 6D). This pattern shows a single peak in polyT occurrence, centered between 15 and 40 nt from the acceptor site. Although the numerous exceptions make firm conclusions difficult, the broad phylogenetic distribution of the first pattern (in animals, plants, apicomplexans and heterokonts, and perhaps rhizarians), suggests that the ancestral intronic structure had polyT motifs concentrated between the BP and the terminal AG, and that broader polyT distributions evolved early in fungal evolution. Discussion Evolutionary Convergence to Highly-Conserved Intron- Exon Structures in Distant Eukaryotes We report convergent evolution of strong branch point consensus sequences and constrained branch points positions in eukaryotic lineages ranging from fungi to plants to protists. These observations join our previous findings of convergent evolution to strong 59 splice site boundaries [9], as well as the pattern of recurrent nearly-complete intron loss, as examples of convergent intron-exon structure evolution across eukaryotes [26]. Interest- ingly, these five patterns appear to be closely related. Those lineages that are highly derived in intron number, having lost most of their introns, are the same ones that exhibit constraint of their few remaining introns’ sequences. However, different intron sequence characteristics show differ- ent degrees of co-evolution with intron number. Whereas strong splice sites show a one-to-one correspondence with low intron number across species [9], only a subset of intron-poor lineages have strong branch point sequences, of which only a subset have highly constrained branch point positions. Thus while intron paucity may be necessary for the emergence of branch point sequence and position constraint, it is not sufficient. Difference in levels of intron structure constraint likely is associated with (even perhaps driven by; see below) changes in the spliceosomal machinery that have led to increased requirements for adherence to consensus sequences [9,27]. Indeed, the intron- poor species whose splicing apparatus has been most extensively studied, S. cerevisiae, shows considerable alterations in the mechanisms and protein components of its spliceosome [2,19]. Future work should explore spliceosomal changes in other intron- poor lineages, in particular the possibility in evolutionary convergence in spliceosomal machinery across lineages. Notably, we failed to find intermediate stages. 59 splice site strength shows a clear gap between intron-poor lineages (at least 5 bits of information content), and intron-rich (1–4 bits) [9]. Almost all species have either clear and strong branch point consensus (66–100% introns with same branch point hexamer) or much weaker conservation (,30%). Branch point position also seems bimodal, as clearly seen among the hemiascomycetous yeasts, Convergent Evolution of Intron Structures where either .80% of branch points fall within a few base pairs, or fewer than 40%. For 59 splice sites, this lack of intermediates is consistent with some qualitative difference in the selective regimes acting within intron- poor and intron-rich species, leading to a lack of intermediate strengths. For branch point sequences and positions the case is more subtle. Do weak branch points in some intron-poor lineages reflect an ongoing process, or are these lineages somehow refractory 39 intron convergence? Repeated evolution of constraint in hemi- ascomycetes may suggest an ongoing process, however in this case we might expect to observe intermediate stages. Possibly, once put in motion, intron structure constraint proceeds rapidly, which could explain the lack of observed intermediates. Opposed Intron Structure Evolution in Two Classes of Reduced Eukaryotes Widespread sequencing has underscored the complexity of eukaryotic genome structure. While some genomes seem generally complex (with large numbers of genes containing numerous long introns and ubiquitous transposable elements) or simple (with short intergenic regions flanking a modest complement of nearly intronless genes), many genomes defy such straight-forward characterization. Intron-exon structures provide a clear example: the three genomes with the shortest known intron structures each have relatively high intron densities (Paramecium tetraurelia, the nucleomorph of B. natans, and Dicyemids, so-called mesozoans), whereas introns in very intron- poor species are not particularly short (Table 2). Interestingly, these two classes of reduced lineages appear to show opposed patterns of intron evolution. Whereas intron-poor lineages tend towards highly-constrained intron sequence ele- ments, short-intron lineages seem to show very weak sequence constraint. Available Dicyemid introns give the weakest known score for 59 intron boundaries (0.5 bits) and 59 splice sites of P. tetraurelia and B. natans are largely restricted to GT(A). These three lineages also show no signature of branch points (Table 2). This does not simply reflect an inability of short introns to accommodate branch points or reduced splicing constraints associated with short introns per se: E. cuniculi introns (35.8 nt on average) and many T. vaginalis introns (,25 nt) are short, yet both show conserved 59 splice site and branch point sequences (note that this also suggests that species with both types of genome reduction exhibit strong consensus, reflecting their intron paucity). This pattern under- scores the importance of intron number, and not simply genome reduction, in driving the emergence of strong consensus sequences. Hypotheses for Intron Convergence and a Natural Experiment in Ostreococcus The finding of a general inverse correspondence between intron number and splicing signals’ strength is unexpected and remains unexplained. Previously, we suggested that in intron-poor species, selection against aberrant splicing of cryptic splice sites would drive changes in the spliceosome towards stricter splicing requirements, which would in turn drive sequence change in (or loss of) non-conforming introns [9]. In intron-rich species, this evolutionary pathway would not be available since increased spliceosomal strictness would imply deleterious inefficient splicing of a much larger number of non-consensus introns [9]. The genome of the ultra-small green alga Ostreococcus lucimarinus provides a rare natural experiment to test this hypothesis. While genes in the majority of the genome exhibit very low intron densities, the genes spanning roughly half of chromosome 2 show a much higher density, well within that of ‘‘intron-rich’’ species [18]. That the two sets diverge so clearly in level of intron sequence PLoS Genetics | www.plosgenetics.org 10 August 2008 | Volume 4 | Issue 8 | e1000148