K/Kis directly correlated with codon bias, here measured using the ENC' statistic (49; lower ENC' prime values indicate higher codon bias) in () , () , and ()

<p><b>Copyright information:</b></p><p>Taken from "Positive selection for unpreferred codon usage in eukaryotic genomes"</p><p>http://www.biomedcentral.com/1471-2148/7/119</p><p>BMC Evolutionary Biology 2007;7():119-119.</p><p>Published online 18 Jul 2007</p><p>PMCID:PMC1936986.</p><p></p> Error bars indicate standard error. This relationship suggests that recent selective pressures on codon usage in these groups generally reinforce historic selective pressures

Frequencies of different forms of splice variation, arranged by phylogenetic group

The two bar charts show the relative frequencies of each type of splice variation. The ratio of CEs to RIs is shown in the chart on the left, while the one on the right displays competing 5' and competing 3' splice sites. Note that the CE/RI ratio shows wide variation among kingdoms while the ratio of competing 5' to competing 3' splice sites is remarkably consistent. A high-level overview of the phylogenetic tree is shown on the far left, and the organisms' names are colored according to their phylogenetic grouping. To see all four forms of splice variation on a single bar plot, see Additional data file 1. The data for was taken from a previous study [8].<p><b>Copyright information:</b></p><p>Taken from "Cross-kingdom patterns of alternative splicing and splice recognition"</p><p>http://genomebiology.com/2008/9/3/R50</p><p>Genome Biology 2008;9(3):R50-R50.</p><p>Published online 5 Mar 2008</p><p>PMCID:PMC2397502.</p><p></p

Average lengths of CEs compared to average lengths of internal constitutive exons

Species are sorted by the fractional difference between these two lengths. In organisms where CEs are common (animals and plants) CEs are almost identical in length to constitutive exons, while in species where CEs are rare (fungi and protists) CEs tend to be significantly shorter than constitutive exons. In animals and plants, where ED is common, CEs are spliced by the same process as constitutive exons and these two groups are thus subject to the same length constraints. In organisms that splice primarily by ID, including fungi and protists, the lengths of constitutive exons are not constrained by ED. However, CEs in these organisms are still recognized by ED. Thus, in these species, constitutive exons can grow longer than CEs.<p><b>Copyright information:</b></p><p>Taken from "Cross-kingdom patterns of alternative splicing and splice recognition"</p><p>http://genomebiology.com/2008/9/3/R50</p><p>Genome Biology 2008;9(3):R50-R50.</p><p>Published online 5 Mar 2008</p><p>PMCID:PMC2397502.</p><p></p

Average lengths of RIs compared with lengths of constitutive introns and introns adjacent to CEs

RI length, which is constrained by ID, is fairly constant across all organisms. In protists and fungi, average RI length is close to that of constitutive introns, because ID is the primary mode of splice site recognition for both groups. In animals, constitutive intron length differs substantially from RI length because most constitutive introns are recognized by ED and are not subject to the same length constraints as RIs, which are recognized by ID. Plants fall between unicellular organisms and animals. Data are shown only for organisms with at least five RIs. For introns next to CEs, data are shown only for organisms with at least eight CEs with unambiguous adjacent intron lengths on both sides. Introns next to CEs are usually longer than constitutive introns, because these introns are recognized by ED and are free from ID length constraints.<p><b>Copyright information:</b></p><p>Taken from "Cross-kingdom patterns of alternative splicing and splice recognition"</p><p>http://genomebiology.com/2008/9/3/R50</p><p>Genome Biology 2008;9(3):R50-R50.</p><p>Published online 5 Mar 2008</p><p>PMCID:PMC2397502.</p><p></p

Intron Conservation in the PRPP Synthetase Gene

<div><p>(A) Alignment of PRPP synthetase putative orthologs MG07148, NCU06970, FG09299, and AN1965. A black-edged rectangle indicates an intron position passing our quality filters, whereas an unedged gray rectangle indicates an intron position that was removed by our filter. Blue boxes mark raw intron gains, red boxes indicate raw intron losses, and gray boxes within black-edged rectangles highlight all other introns. We manually corrected an annotation error in the first intron of the last row of the alignment.</p> <p>(B) Phylogenetic conservation pattern of introns in the PRPP sythetase gene. Each passing intron position was categorized as being present in A. nidulans (A), F. graminearum (F), M. grisea (M), N. crassa (N), A. nidulans and N. crassa (AN), F. graminearum and M. grisea (FM), or all four organisms (AFMN). There are no passing cases of conservation in three or four species. The number of introns in each category is shown with a purple line. The black error bar plot shows the mean and standard deviation for each category for all 2,008 ortholog sets after fitting to a Poisson distribution (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0020422#s4" target="_blank">see Materials and Methods</a>). The number of introns in M. grisea and N. crassa is significantly higher, at the <i>p</i> < 1 × 10⁻⁹ level.</p></div

Example Ortholog Alignment

<div><p>(A) Alignment of protein sequences for orthologs MG04228, NCU05623, FG06415, and AN1892 with intron characters inserted. “0,” “1,” and “2” indicate the phase of an intron. A black-edged rectangle indicates an intron position passing our quality filters; an unedged gray rectangle indicates an intron position that was removed by our filter. The green rectangle indicates conserved introns, the blue box marks a raw intron gain, and the gray boxes within black-edged rectangles highlight all other introns. The consensus (bottom) line characters are as follows: asterisk, identical residue in all four sequences; colon, similar residue; and period, neutral residue.</p> <p>(B) Nucleotide alignment of the region flanking the gained intron in (A). Putative 5′ and 3′ splice sites and a branch point sequence are highlighted in blue.</p></div

Positional Biases in Intron Gain and Loss

<div><p>Relative intron positions were defined as the number of bases in the coding sequence upstream of the intron divided by the total length of the coding sequence. These relative positions were binned into five categories (quintiles), each representing one-fifth of the coding sequence length (quintiles numbered 1–5 on the x-axis).</p> <p>(A) Introns passing quality filter (light blue, back) and introns adjacent to gaps in the protein alignment that were removed by our quality filter (orange, front).</p> <p>(B) Raw and inferred gains. Raw gains (green, back) are those introns present in exactly one organism (excluding the outgroup, A. nidulans). Inferred gains (blue, front) are corrected for the estimated number of cases that arose by other combinations of gain and loss events. Inferred gains are thus slightly lower than raw gains.</p> <p>(C) Raw and inferred losses. Raw losses (green, front) are those introns absent in the organism in question but present in at least one of its siblings (descendants of its parent in the phylogenetic tree) and one of its cousins (non-descendants of its parent). Inferred losses (red, back) are corrected for the estimated number of introns lost along multiple lineages, or gained and then lost. Inferred losses are thus slightly higher than raw losses.</p> <p>(D) Number of introns gained (blue) and lost (red) since last common ancestor (losses shown as negative numbers).</p> <p>(E) Intron loss rate at each position since last common ancestor (introns lost per ancestral intron). Error bars represent binomial standard deviation.</p></div

Phylogenetic Tree and Intron Conservation Patterns

<p>(A) Phylogenetic tree of the four fungal organisms studied <i>(M. grisea, N. crassa, F. graminearum,</i> and <i>A. nidulans)</i> with estimated time scale in millions of years ago. The rooted organismal tree was constructed using an unweighted pair group method using arithmetic averages based on a concatenated alignment of 2,073 orthologous gene sets. Estimated dates of divergence from <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0020422#pbio-0020422-Taylor1" target="_blank">Taylor et al. (1999)</a>, <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0020422#pbio-0020422-Berbee1" target="_blank">Berbee and Taylor (2000)</a>, and <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0020422#pbio-0020422-Heckman1" target="_blank">Heckman et al. (2001)</a>.(B) Classification of intron presence (+) and absence (−) patterns across the four fungal species. A blue “+” indicates a raw intron gain in the corresponding organism, a red “−” indicates a raw intron loss in the corresponding organism, a green “+” indicates a conserved intron, and all other introns are indicated in black.</p

Alignment Filtering Protocol

<div><p>(A) Schematic of filtering protocol applied to a ten-residue window on each side of every intron position. If either side failed the filter, the position was discarded.</p> <p>(B) Distributions of minimum percent identity and similarity in ten-residue windows around 181 randomly selected intron positions, for three manual classifications. The minima were taken between the left and right windows. The yellow lines indicate the chosen thresholds of at least 50% similarity and 30% identity, and bars are colored yellow if they fall above the thresholds (pass) or orange if they fall below the thresholds (fail). Parentheses indicate the number of introns in each class that pass the cutoff and the total number of introns in that class. The five lowest-percent identity and similarity bars, containing 77 positions, in the “non-homologous” plot are omitted so as to not obscure the rest of the histogram.</p></div