constraint is clearly not predicted by general alterations of splicing strictness due to changes in a (assumed) single spliceosome. One possibility is that the two intron sets are serviced by different spliceosomes (as is the case of U2 and U12 in different eukaryotic lineages). However, a computational search turned up only single copies of spliceosomal RNA components in congenitor O. tauri [28]. Conceivably snRNA changes to complement the divergent Ostreococcus 59 splice sites and branch points (GTGCGTG and GACTGACG in O. lucimarinus) could have thwarted their identification in the previous study, and this possibility is worth exploring. Alternatively (though more difficult to test), a single core RNA splicing machinery could associate with different sets of protein components in distinct spliceosomes with different splicing activities. More likely, however, O. lucimarinus contains a single spliceo- some, strongly suggesting that the constrained introns through most of the genome, as well as those of intron-poor species that they so closely resemble, do not reflect inherent changes in the spliceosomal machinery. A simpler possibility is that differences in local (in O. lucimarinus) or cellular (in other species) concentrations of spliceosomal complexes is the driving factor. It seems possible or even likely that spliceosomal complexes in intron-poor species are downregulated. Such downregulation could reflect either selection to reduce incorrect splicing of truly exonic sequence (i.e. fewer spliceosomes, less chance of false splice boundaries being spliced), or could be favored by reducing energetic costs associated with transcription, processing, and translation of spliceosomal compo- nents. If so, the lowered concentration of spliceosomal components would require stronger binding affinity of individual splice sites to corresponding snRNAs for efficient splicing, which would in turn drive the evolution of stronger boundaries (or intron loss). Differential local concentrations across genomic regions in Ostreococcus could be maintained if spliceosomes were preferentially recruited to the intron-rich genomic region. This scenario is similar to our previous hypothesis in invoking a tradeoff between the costs of efficient splicing of weak boundaries (maintenance of high spliceosome concentration) and the costs of mis-splicing, which we argue would likely be different in intron-rich and -poor species. This hypothesis makes the testable prediction that spliceosomal components should show reduced expression in intron-poor species relative to intron-rich species. Another hypothesis concerning the concentration of spliceo- somal complexes, suggested to us by Tony Russell, sees a very different role for selection. A predicted consequence of increasing the length of snRNA-intron element base-pairing interactions is a reduction in the overall rate of splicing, simply due to a tighter association between intron and spliceosome. Such a decrease in splicing rate will be tolerated in intron-poor species if spliceosomal components are in excess relative to the number of introns. However, in intron-rich species spliceosomal components may not be in excess, in which case stronger base-pairing between intron and snRNAs could be disfavored. Notably, these two hypotheses make qualitatively different predictions. Whereas the latter hypothesis predicts that strong boundaries would be disfavored in intron-rich species, the former predicts that they would as or more fit than weaker boundaries. Comparative analysis of closely related species to test these predictions is underway. Sequence Convergence versus Preferential Loss Two factors could drive evolutionary convergence to strong intron boundaries: sequence changes in existing intron sequences to consensus sequences, and preferential loss of non-consensus introns. The relative contributions of these factors may depend on the precise evolutionary pathway from (ancestral) genomes with Convergent Evolution of Intron Structures many introns with weak boundaries and relatively lax splicing requirements to fewer introns with strong boundaries and stricter requirements. First, widespread (mostly random) intron loss could lead to selective conditions favoring the evolution of a spliceosome with stricter sequence requirements for splicing (as argued above and in reference [9]). Introns with non-consensus sequences would then impose a burden, which could be resolved by sequence change or intron loss. If intron loss rates in these lineages are at least comparable to substitution mutation rates (for instance, 90% intron loss over 500 million years is consistent with a constant loss rate of 561029 per year, comparable to some estimated mutation rates [29,30]), preferential loss could play an (or even the) important role in convergence. In this case, intron loss would be a self-catalyzing process, with widespread intron loss leading to increased splicing requirements driving yet faster intron loss. Alternatively, increased splicing requirements could come first, driving intron loss (consistent with [31]). However evolution of stricter splicing requirements in intron-rich organisms, where there are large numbers of non-consensus introns, would lead to widespread deleterious mis-splicing. Thus it is hard to imagine such strict requirements arising prior to widespread intron loss. Finally, even under lax splicing requirements, loss of non- consensus introns could be more highly selected due to the effects of the less efficient splicing of these introns (e.g. [32]), with stricter splicing requirements gradually enabled by loss of non-consensus introns. The viability of this scenario depends on significantly higher selective costs of suboptimal boundaries in intron-rich species respect to the optimal introns. Attempts to estimate these selective costs by comparative analysis are underway. In any case, it seems likely that intron loss, increased splicing constraints, and intron sequence change will all be reinforcing of one another, such that (perhaps past some critical threshold), the three will proceed in tandem. Differences in the relative contributions of the three phenomena will depend on mutation rates and selective coefficients for different kinds of changes (basepair substitutions, intron loss, spliceosomal changes), which may vary considerably across times and lineages. Convergent Evolution between U12-Type Introns and Nearly Intronless Species’ U2-Type Introns The convergent U2 intron structures observed here and elsewhere in some intron poor species – strong 59 and branch point consensus sequences, constrained BP-AG length – are strikingly reminiscent of the U12-type intron structures found across a wide variety of lineages [33,34]. One possibility is that the U12-type intron structures represent a derived state (possibly evolved in the ancestor of eukaryotes), and that these convergent cases have similar causes – that U12-type introns’ low genomic number has subjected them to the same pressures as those experienced by U2-type introns in intron-poor lineages. Notably, if the conservation across lineages of specific conserved U12 intron sequence elements, as opposed to the differences in consensus structures observed in U2-type introns across diverse intron poor lineages reflects the emergence of these structures early on in eukaryotic history, this interpretation would imply that U12-type introns have been rare since very early in eukaryotic history. Alternatively, the similar structures of U12 introns in general and U2 introns in intron-poor species could have different explana- tions. One possibility is that the U12 system represents an intermediate between the type II introns that initially proliferated in early eukaryotic ancestors, with their highly similar sequences and structures, and typical highly degenerate U2 introns. In this case the persistence of strong consensus sequences in U12-type but PLoS Genetics | www.plosgenetics.org 11 August 2008 | Volume 4 | Issue 8 | e1000148