Figure 7. Phylogenetic trees for all splice site types. Trees were constructed in a manner similar to the trees for the individual splice site subtypes, but the terminal dinucleotides of the intron were removed from the PWM (see text for details). GT–AG 50ss motif. The C.elegans GC–AG motif is not as conserved as the other GC–AG motifs, so it clusters closer to the GT–AG motif from the same species. The two C.elegans U2-type motifs and the A.thaliana U2-type GT–AG motif are close to each other due to the T-rich com- position of plant and nematode introns. The 50ss motifs for U12-type introns are distant from the U2-type introns, their closest relative being the GT–AG 50ss motif from A.thaliana, probably because these motifs are rich in pyrimidines from positions +7 to +10. The C.ele- gans U12-type-like GT–AG motif is located between the U2- type and the bona ﬁde U12-type GT–AG motifs, but it is clo- ser to the U12-type motifs in the other species, consistent with the idea that these introns could represent a small group of vestigial U12-type introns that were converted to the U2-type. Interestingly, in this tree the four AT–AC motifs are located on the same branch of the tree, because these motifs are slightly more conserved than those for U12-type GT–AG introns. The tree for 30ss shows that, in general, the 30ss tend to cluster by species rather than by subtype. Most remarkably, the three subtypes of C.elegans 30ss motifs, U2-type GT– AG, GC–AG and U12-type-like GT–AG, occupy a distinct branch on the tree, indicating that: (i) these motifs are strongly related to each other, in turn suggesting that the U12-type-like introns are indeed U2-dependent; (ii) these motifs are separated from the rest, in agreement with the idea of divergent evolution of C.elegans 30ss. C.elegans exhibits two distinct features, a short and highly conserved PPT in 30ss (72,73) and trans-splicing (36). The selection of 30ss for efﬁciency in cis- as well as trans-splicing might explain these features and the divergent evolution. Three of the motifs in A.thaliana, U2-type GT–AG and GC–AG and the U12-type GT–AG, also cluster together. In summary, the phylogenetic trees for the different splice- site motifs reﬂect the evolutionary relationship between the corresponding splice sites in the ﬁve species. The clustering of the 50ss motifs by splice-site subtype strongly suggests that these subtypes were present in a common ancestor of Nucleic Acids Research, 2006, Vol. 34, No. 14 3965 these ﬁve eukaryotes, and have evolved separately. In con- trast, 30ss subtypes tend to cluster by species, suggesting a more recent origin for many of the 30ss. Since U2-type 50ss is initially recognized by its base pair- ing to the U1 snRNA which is a more speciﬁc interaction, while the 30ss is deﬁned by a more non-speciﬁc binding of the U2AF heterodimer to the PPT and AG element, which in turn stabilizes base pairing between U2 snRNA and the BPS. The different properties of RNA–RNA interactions at the 50ss versus RNA–protein interactions at the 30ss imply a greater ﬂexibility in 30ss selection. In support of this view we note that even within a species, there is more leeway within the 30ss for speciﬁcation of the PPT, the AG and the upstream BPS (10,26–28). CONCLUSIONS We have created a reliable dataset of genomic splice sites from ﬁve model organisms, based on RefSeq genes and their mappings. We have classiﬁed these splice sites into various categories, based on carefully curated PWMs for each species and splice-site type. Non-canonical splice sites, that do not ﬁt into the four major categories, get a short shrift in databases that use large-scale automated annotation, but we have con- sidered these cases carefully. This comprehensive set of splice sites allows robust statistical analyses, while minimiz- ing errors. We have implemented a variety of tools, which can be used to further analyze the splice sites in our collection. Experiments can never reproduce the entire range of phe- nomena that occur in nature, nor can they easily detect certain subtle features that can only be revealed by statistical analysis of a large dataset. For these reasons, the genomic dataset pro- vides an ideal ‘dry-bench’ to study global patterns in splice sites. In addition, by using the information content of the splice-site motifs and the phylogenetic distance derived from them, we draw inferences on the evolution of the splice sites, which are only possible with large and reliable datasets. We believe this dataset and its analyses will spur further experimental work. ACKNOWLEDGEMENTS We thank Dhruv Pant for help with GObar and phylogenetic trees, Andrew Olson for help in linking GeneSeer to SpliceRack and James Gurtowski for developing the logo program used to depict the splice-site motifs. We also thank the anonymous reviewers for suggestions that improved the paper. X.R., M.L.H. and A.R.K. acknowledge support from NIH grants CA13106 and GM42699. Funding to pay the Open Access publication charges for this article was provided by Cancer Center, CSHL. Conflict of interest statement. None declared. REFERENCES 1. 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