WUL Cell Count

Images were processed using ImageJ (National Institute of Health, Bethesda, MD). First, images were mean filtered with a radius of 15 pixels and subtracted from the original image to reduce noise. Gametophytes were selected by outlining the thallus with a Wacom Intuos tablet (Wacom, Saitama, Japan). Processed images were then passed through an object identification pipeline in CellProfiler v3.1.9 (Broad Institute, Cambridge, MA). The pipeline used a global Otsu threshold with a smoothing scale of 1.3488, distinguished clumped objects by shape, and used a propagation method of drawing dividing lines between objects. The typical diameter of objects allowed was adjusted for each gametophyte timepoint, with a Min-Max range of 6-25 pixels and narrowed until the output no longer identified background cell wall fluorescence. The number of accepted objects was collected for each image.