Convergent Evolution of Intron Structures not U2-type introns remains somewhat mysterious, as does the preponderance of U2 introns relative to U12. problems with annotations, such problems are very unlikely to be driving the large qualitative differences observed. Evidence that polyT Tails and Weak Branch Point Sites Are Ancestral We find different patterns of polyT motif distribution along the introns in different lineages, which likely reflect differences in polyT functionality and in how polyT-binding factors regulate splicing in these lineages. Indeed, the differential conservation and evolution of spliceosomal proteins binding polyT tracts (PTB, SXL, TIA1, Nam8, etc.) in each species is likely to determine the position of polyT motifs along introns and what role these motifs play in splicing regulation. Among these locations, we find that the so-called ‘‘polyT tail’’, spanning from a position likely corresponding to the branch point to the 39 intron terminus, is common to a wide variety of groups ranging from plants to animals to various, widely diverged, protists, strongly suggesting that the existence of a polyT tail may be ancestral. Furthermore, we show that, as in the case of 59 splice sites, strong branch point site consensus have evolved indepen- dently in only intron-poor species, whereas all intron-rich species have weak branch point sites. Since a wide variety of studies have shown that eukaryotic ancestors have harbored relatively high intron numbers [3,35–38], our results suggest that the eukaryotic ancestors also had weak branch point site consensus, as with most modern eukaryotic groups. These conclusions extend an excellent recent study from Schwartz et al. [27], who studied BP and polyY motifs in 19 opisthokonts and 3 other eukaryotic species, and reached similar conclusions about ancestral intron structures. Our inclusion of a more diverse set of species spanning all of known eukaryotic species allows us to reach deeper into eukaryotic evolution, potentially getting much closer to the initial origin of spliceosomal introns. In particular, we find that sequences from the first characterized rhizarian, as well as for various heterokonts, follow the patterns found in other kingdoms. This striking similarity over very broad evolutionary distances significantly strengthens our conclusions about ancestral eukaryotic splicing, rendering them independent of the placement of the root of the eukaryotic phylogeny. The presence of these intronic features, polyT tail and weak branch point sites, in the eukaryotic ancestors adds to the developing picture of the spliceosomal system in early eukaryotes – with highly developed spliceosome, weak 59 and branch point sequences, a polyT tail and complex splicing patterns [2,9,39,40]. Quality of Predictions One limitation of the data deserves comment. We analyze annotated intron sequences, making our analysis subject to the quality of available annotations. Problems with the annotations may very directly influence the characteristics studied here, since for instance more consensus-like sequences are more likely to be identified as introns. Such concerns may also affect comparative analysis if the annotation efforts for different species are differentially sensitive to different kinds of introns. However, it is very unlikely that such limitations are likely to drive the qualitative differences we see here. For a ‘‘weak’’ species with for instance 30% of its true introns exhibiting the same branch point to be incorrectly identified as a ‘‘strong’’ species (with, say, 80% of predicted introns with the same branch point) would require that the vast majority of its non-consensus introns have gone unannotated. For the reverse to occur, it would be required that there were so many falsely predicted introns that it had drowned out the signal almost entirely. Thus, while it is important to point out that our results are likely not accurate to the second decimal place due to Strong 59 Splice Sites in E. cuniculi Possibly, the most important impact of annotation errors could be to reduce the signal in very intron-poor species. For instance, further scrutiny suggested that 2 of the 15 predicted introns in E. cuniculi may not in fact be introns at all: both are a multiple of 3 basepairs and lack inframe stop codons; these two introns have the weakest 59 boundaries (matching the consensus (GT)AAGT at 1 and 2 out of 4 positions, compared to at least 3 matches for the other 13 introns); and one intron has similar sequences at the two boundaries, suggesting that this intron prediction could reflect a reverse transcriptase artifact in EST preparation [17]. Notably, in our previous work on donor splice sites [9], E. cuniculi represented the only intron-poor species lacking very clear strong boundaries. Excluding these two questionable introns, E. cuniculi has a donor site information content of 6.2 bits, comparable to the other intron-poor lineages. Conclusion These results attest to plasticity of spliceosomal intron structures through the history of eukaryotes. The availability of large numbers of eukaryotic genomes now allows comparative analysis of an increasing diversity of genomic structures. Present and previous works have provided an increasingly detailed picture of the patterns and determinants of intron-exon structures, one of the hallmarks of eukaryotic genome organization. Definitive identifi- cation of the causes of highly regular 39 intron structures awaits the identification of additional lineages exhibiting this pattern. Methods Genome Sequences and Data Sources GenBank fully-sequence genome annotations were downloaded from NCBI webpage (http://www.ncbi.nlm.nih.gov) or Ensembl database (http://www.ensembl.org) for 7 metazoa: human (Homo sapiens (NCBI 36 Ensembl 38.36)), zebra fish (Danio rerio (Zv5 Ensembl 38.35e)), Strongylocentrotus purpuratus (AAGJ00000000.2), Drosophila melanogaster (release 4.1), mosquito (Anopheles gambiae (AgamP3 Vectorbase 38.3a)), Caenorhabditis elegans (WS150 Worm- base 38.150a), Ciona intestinalis (CINT1.95); 16 fungi: Aspergillus fumigatus Af293 (AAHF00000000.1), Aspergillus nidulans FGSC A4 (AACD00000000.1), Neurospora crassa OR74 A (AABX00000000.1), Gibberella zeae PH-1 (AACM00000000.1), Yarrowia lipolytica CLIB122 (CR382127-31.1), Saccharomyces cerevisiae YJM789 (AAFW00000000.1), Candida glabrata CBS138 (provided by J. E. Stajich), Candida albicans SC5314 (provided by J. E. Stajich), Kluyveromyces lactis NCYC 2644 (AADM00000000.1), Eremothecium gossypii ATCC 10895 (AE016814-20.1), Debaryomyces hansenii CBS767 (CR382133-9.1), Schizosaccharomyces pombe 972h (AL672256-8.1), Ustilago maydis 521 (AACP00000000.1), Cryptococ- cus neoformans B3501-A (NC_006670, NC_006679-87, NC006691- 4), Encephalitozoon cuniculi GB-M1 (AL391737.1, AL590442-50.1), Rhizopus oryzae RA 99-880 (AACW00000000.2); 2 amoebae: Dictyostelium discoideum AX4 (AAFI00000000.1), Entamoeba histolytica HM-1:IMSS (AAFB00000000.1); 2 plants: Arabidopsis thaliana (NC_003070.5, NC_003071.3, NC_003074.4, NC_003075.3, NC_003076.4), Oryza sativa (Build 2.1); 3 green algae: Chlamydo- monas reinhardtii (JGI Chlamy 3.0), Ostreococcus lucimarinus CCE9901 (GenBank version 1, CP000581- CP000601), Ostreococcus tauri OTH95 (GenBank version 1, CR954201- CR954220); 6 apli- complexans: Plasmodium falciparum HB3 (AANS00000000.1), Plas- modium yoelii yoelii (AABL00000000.1), Plasmodium chabaudi PLoS Genetics | www.plosgenetics.org 12 August 2008 | Volume 4 | Issue 8 | e1000148