Frequency of perithecia without neck in crosses homozygous for the Δ<i>pah</i> mutations.

a<p>Each cross was performed in duplicate on two separate Petri dishes inoculated with <i>mat+</i> and <i>mat−</i> strains. When the mycelia were confluent, sterile water was poured to disperse microconidia over the surface of the cultures and promote fertilization of female organs of the compatible mating-type. 5 to 9 independent counts were made seven days after fertilization on 150 to 400 perithecia formed on different sectors of the two dishes. Data from one*, two** or three*** assays were pooled. Δ<i>pah2</i> Δ<i>pah4</i>, Δ<i>pah2</i> Δ<i>pah6</i> and Δ<i>pah2</i> Δ<i>pah7</i> had the same % of perithecia without neck as Δ<i>pah2</i>. All the perithecia from Δ<i>pah1</i> Δ<i>pah5</i>, Δ<i>pah2</i> Δ<i>pah5</i>, Δ<i>pah3</i> Δ<i>pah5</i>, Δ<i>pah4</i> Δ<i>pah5</i> and Δ<i>pah6</i> Δ<i>pah5</i> had no neck. All the perithecia from the other double mutants had a beak.</p

Fertility of the homeobox gene mutants.

<p>Repartition of fruiting bodies in homeobox mutants (top). Eight-centimeter M2 plates were inoculated in the center with mat+/mat− heterokaryotic cultures and incubated for seven days. Perithecia are visible as small black dots. Morphology of fruiting bodies in the Δ<i>pah</i> mutants (bottom). Images are from representative fruiting bodies from the plates in the top panel. For Δ<i>pah2</i>, arrows indicate perithecia with neck and arrowheads indicate perithecia without neck. WT, wild type.</p

Phylogenetic tree of fungi and the presence of croziers and clamps.

<p>The tree is based on the latest phylogenetic data including those of James et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037488#pone.0037488-James1" target="_blank">[3]</a>. In the Ascomycota, a smiling face indicates that croziers are present and a sad face indicates that they are absent. In the Basidiomycota, a smiling face indicates that clamps are present and a sad face indicates that they appear absent. No face is shown when we were not able to find data on the presence of croziers/clamps in the literature or that species from the group grow only as yeast.</p

Germination of WT and Δ<i>pah5</i> ascospores.

<p>Ascospores were incubated for 6 h at 27°C on germination medium and observed by light microscopy. <b>A.</b> WT ascospores from a WT <i>mat+</i> × WT <i>mat−</i> cross are melanized and germ tubes originate from a germination peg (*) located at the pole opposite to the primary appendage (arrowhead). <b>B.</b> Δ<i>pah5</i> ascospores from a trikaryotic culture (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037488#s2" target="_blank">Results</a>) are not fully melanized and germ tubes appear at both poles (arrow points at the germ tube originating from the primary appendage (*)). Numerous Δ<i>pah5</i> ascospores did not germinate even after incubation for several days.</p

Development of clamps and croziers shares several features.

<p>In both cases, division starts with the formation of a peg, lateral in Basidiomycota and apical in Ascomycota. Conjugate nuclear division then follows, resulting in one cell with two nuclei and two cells with one nucleus. Uninucleate cells undergo anastomosis to recreate the dikaryotic conditions. In Basidiomycota, the apical cell continues its vegetative growth and undergoes further cell division. In contrast, in most Ascomycota, the apical cell usually differentiates into a meiocyte and undergoes karyogamy and meiosis, although in a few species it may continue to divide as a dikaryon.</p

Evolution of HD transcription factors in Sordariomycetes.

<p>Top, alignment of the HD domains of the <i>P. anserina</i> HD transcription factors. The alignment was obtained with ClustalW2 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037488#pone.0037488-Larkin1" target="_blank">[49]</a> and colored according to the ClustalX color scheme provided by Jalview <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037488#pone.0037488-Waterhouse1" target="_blank">[50]</a>. Numbers indicate the position in the protein of the first amino acids of the homeodomains. The bottom consensus line and the secondary structure are according to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037488#pone.0037488-Brglin1" target="_blank">[9]</a>. Bottom, PhyML tree of the HD factors of <i>P. anserina</i>, <i>N. crassa</i> and <i>M. grisea</i>. Black dots indicate statistically supported branches (>80%, 200 bootstraps). Non-TALE HD in red, TALE HD in blue.</p

Crozier formation in the homeobox gene mutants.

<p>Perithecium centra were fixed, labeled with DAPI, and observed for the presence of croziers. Croziers were observed in all homeobox mutants, as shown here for Δ<i>pah1</i>. Some combinations of multiple mutants were also examined and found to differentiate croziers, as exemplified here by the Δ<i>pah2</i>Δ<i>pah3</i>Δ<i>pah4</i> triple mutant and the Δ<i>pah1</i>Δ<i>pah2</i>Δ<i>pah3</i>Δ<i>pah4</i>Δ<i>pah5</i>Δ<i>pah6</i> sextuple mutant.</p