Scoring the overall diversity of nos and pgc germ granule content. (A) Changes in germ granule mRNA content are scored using a pipeline called the GCTAnalysis. The pipeline begins by acquiring 3D confocal images and transforming them into quantified 2D Granule Censuses. Next, a new census that represents a change between two Granule Censuses is generated by subtracting two censuses. To score the overall magnitude of change, the Euclidean norm of the resulting census is calculated. (B–G) Changes in nos and pgc germ granule mRNA content between different pairs of Drosophila species in stage 13 oocytes with the overall score noted. The heatmap represents a change in quantity for a given nos and pgc combination found in the Granule Census; the darker the matrix element, the larger the change between the paired species, whereas white represents no change in a particular composition between the different species. (H) Undirected graph where nodes are represented by species and edges are represented by the score calculated in (B–G): melanogaster (m), pseudoobscura (p), virilis (v), and nebulosa (n). (I) Venn diagram that displays the number of nos and pgc mRNA compositions that are unique or overlapping between different Drosophila species.