(A) Flow chart of filtering steps to identify candidate synaptonemal complex proteins in Pristionchus, with the number of remaining proteins after each step shown to the right. See Materials and methods for details. (B) 3D scatter plot comparing amino acid substitutions per site, coefficient of variation of protein length, and coiled-coil conservation score of all Pristionchus proteins. Blue dots, candidate SC proteins; other, gray dots. (C, D) Number of DAPI bodies (C) and total viable self-progeny (D) from wild-type P. pacificus, Ppa-syp-1::OLLAS, HA::Ppa-syp-1, and Ppa-syp-1 knockout hermaphrodites. In (C), representative DAPI body images are shown for wild-type and Ppa-syp-1 knockout. (E) Left: image of a prophase region of a gonad from a HA::Ppa-syp-1 hermaphrodite where meiosis progresses from left to right (white arrow), stained with antibodies against HA (magenta). Scale bar = 5 μm, scale bar in inset = 2 μm. (F, G) STED images of a representative chromosome from Ppa-syp-1::OLLAS (F, C-terminus) and HA::Ppa-syp-1 (G, N-terminus) hermaphrodites stained with antibodies against OLLAS or HA tags, respectively (colored according to pixel intensity). Insets show higher magnification of sections in the dashed boxes. Note the ‘railroad tracks’ configuration in (F). Scale bars in (F, G) = 1 μm. (H) Top: model depicting Ppa-SYP-1 as a transverse filament protein, with antibodies targeting the C- and N-termini. Expected distance between C-terminal epitopes is ~100 nm. N-terminal epitopes are expected to be too close to be resolved. Bottom: line scans of normalized pixel intensity across the SC in Ppa-SYP-1::OLLAS (green) and HA::Ppa-SYP-1 (magenta). Bold green and magenta lines represent the average of multiple line scans. (I) Average pairwise percent amino acid identity (top) and coiled-coil score (bottom) for Ppa-SYP-1 from eight Pristionchus species. Sliding window average of percent identity is shown (blue line). Observable steps in the percent identity bar graphs are attributed to fewer species in the Pristionchus proteome dataset (8 Pristionchus species vs. 25 Caenorhabditis species in Figure 2). Magenta and yellow lines in coiled-coil plot, average score at each position; gray lines, individual species scores. Gene model depicting conserved coiled-coil domains (gray-filled ovals) is shown above the coiled-coil plot. Black arrowheads point to two Polo-Box Domains in Ppa-SYP-1, which are conserved in Pristionchus.