(CAAJ00000000.1), Theileria parva Muguga (AAGK00000000.1), Cryptosporidium parvum Iowa (AAEE00000000.1) and Toxoplasma gondii RH (AAQM00000000.1); and 2 heterokonts: Phytophthora ramorum strain Pr102 (AAQX00000000.1) and Phytophthora sojae strain P6497 (AAQY00000000.1). All reported intron sequences were obtained from published supplementary material for: Guillardia theta NM [41], Cyanidioschyzon merolae 10D [42], Trichomonas vaginalis [5,16] and Bigellowiella natans NM [8] and Giardia lamblia ATCC 50803 strain WB C6 [43] (plus 2 introns we have identified), the mesozoan Dicyema acuticephalum [44], and the rhizarian Plasmodiophora brassicae [45]. Available gene sequences for unpublished or incomplete genomes were downloaded one by one as CDS annotations from NCBI web page (http://www.ncbi.nlm.nih.gov/): Brassica oleracea (1096 introns in 314 genes). Introns for Paramecium tetraurelia were extracted from nucleotide links of NCBI taxonomy (1082 introns in 401 genes). Introns for Candida guilliermondii (13 introns), Candida lusitaniae (10 introns) and Candida tropicalis (34 introns) were provided by J. E. Stajich. Study of Branch Point Consensus Strength Studying the clear branch point consensus from a wide variety of intron-poor species, we first define an extended branch point consensus, WCTRAYN, consistent with the minimal consensus NYYNAN described for a wide variety of eukaryotic groups [19,23,24,46]. For each 50 species, we next studied the percentage of introns showing the most common hexamer matching this extended consensus, allowing two-fold degeneracy at zero, one, two and three sites of the putative branch point hexamer. The four measures are complementary. We did not aim to identify and study branch points in all introns. Instead, the percentage of use of the most common motifs gives a straight-forward measure of the strength of the signal for a given species. The use of a similar approach to measure the strength of the 59 splice site (whose definition is trivial) shows a clear correspondence between the measure of strength as the percentage of introns with the most common sequence motif (i.e. GTAAGT, GTATGT, etc.) and as information content, used broadly in the literature, with a coefficient of correlation between both variables for the species included in this study is r = 0.96. Study of 59 and 39 Splice Site Consensus and BP-AG Distance We aligned the final 20 nt of each intron for each species using WebLogo (http://weblogo.berkeley.edu/logo.cgi). To better char- acterize the evolution of BP-AG constraint in Y. lipolytica, we further studied BP-AG distance in the 9 hemiascomycetes species. In all hemiascomycetes species, the vast majority of introns contained a single TACTAAC sequence, used as the branch point. The BP-AG distance was defined as the number of base pairs (Nn) for 59TACTAAC|Nn|AG-exon39. For 59 splice sites we used a similar methodology as described in [9]. The first 6 bases of each intron were extracted, and References 1. Vanacova S, Yan W, Carlton JM, Johnson PJ (2005) Spliceosomal introns in the deep-branching eukaryote Trichomonas vaginalis. Proc Natl Acad Sci USA 102: 4430–4435. 2. Collins L, Penny D (2005) Complex spliceosomal organization ancestral to extant eukaryotes. Mol Biol Evol 22: 1053–1066. 3. Slamovits CH, Keeling PJ (2006) A high density of ancient spliceosomal introns in oxymonad excavates. BMC Evol Biol 6: 34. 4. Nixon J, Wang A, Morrison H, McArthur A, Sogin M, et al. (2002) A spliceosomal intron in Giardia lamblia. Proc Natl Acad Sci USA 99: 3701–3705. Convergent Evolution of Intron Structures information content in bits for positions +3 to +6 was calculated using PICTOGRAM software online (http://genes.mit.edu/ pictogram.html). Study of Ostreococcus Introns We downloaded available EST sequences for O. lucimarinus from NCBI on April 4th, and performed standalone BLASTN searches for each predicted intron-containing O. lucimarinus gene against the ESTs. Preliminary confirmed introns were identified as those in which an EST hit with .60 bits and with .90% sequence identity spanned the intron position (reaching at least 3 nt on each side of the intron position). Each of these introns was then analyzed by eye to exclude non-canonical intron positions as well as those for which sequence similarity between the regions spanning the 59 and 39 predicted splice boundaries were consistent with a template- switching artifact during the reverse transcription step of the EST library preparation [16,17]. Orthologous Intron Analysis For each species considered, databases of intron/exon structures of predicted gene transcripts were prepared from the genome annotations. Homologs were identified by one-way BLASTP searches of intron-containing genes from intron-poor species against predicted proteomes from intron-rich species. Putatively orthologous introns were identified as those present at identical alignment positions (position and phase) in both species. Related species were defined as: C. parvum for T. gondii; Y. lipolytica (chosen as the representative hemiascomycete due to its greater intron density (3 times higher than S. cerevisiae), in order to increase sample size) for S. pombe and A. fumigatus; and the C. merolae and the G. theta nuclemorph for A. thaliana. Due to the lack of related intron-poor species, H. sapiens introns were divided into only two groups. Search for polyT Motif Distributions We used the minimal definition for polyT motif, defined as six consecutive nucleotides containing at least 3 T’s and no A’s [19,21,22]. The study of introns for each species was performed using custom Perl scripts. The last 2 and the first 10 base pairs of each intron were excluded. Acknowledgments We thank Jason Stajich for providing introns for Candida species, and Tony Russell for helpful comments on the manuscript. We thank Eugene Koonin and Jordi Garcia-Ferna`ndez and their groups for intellectual support and stimulation, for financial support, and for fostering environments of open intellectual exploration in their respective groups. Author Contributions Conceived and designed the experiments: MI SWR. Performed the experiments: MI SWR. Analyzed the data: MI SWR. Contributed reagents/materials/analysis tools: MI SWR. Wrote the paper: MI SWR. 5. Carlton JM, Hirt RP, Silva JC, Delcher AL, Schatz M, et al. (2007) Draft Genome Sequence of the Sexually Transmitted Pathogen Trichomonas vaginalis. Science 315: 207–212. 6. Roy SW (2006) Intron-rich ancestors. Trends Genet 22: 468–471. 7. Jeffares DC, Mourier T, Penny D (2006) The biology of intron gain and loss. Trends Genet 22: 16–22. 8. Gilson PR, Su V, Slamovits CH, Reith ME, Keeling PJ, et al. (2006) Complete nucleotide sequence of the chlorarachniophyte nucleomorph: Nature’s smallest nucleus. Proc Natl Acad Sci 103: 9566–9571. PLoS Genetics | www.plosgenetics.org 13 August 2008 | Volume 4 | Issue 8 | e1000148