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

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

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

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