(a) Germarium cartoon showing Terminal Filament (TF) cells, Germline Stem Cells (GSCs), GSC daughters developing into 16-cell germline cysts (grey), Escort Cells (EC, yellow), Follicle Stem Cells (FSC) and Follicle Cells (FC) from anterior (left) to the newest egg chamber. Two potential FSC lineages are shown (blue, green) together with the Fas3 staining pattern (red). (b) Dividing FCs marked by heat-shock induced genetic recombination (red) will exit the ovariole (late egg chambers not shown) within 5 days after heat-shock (AHS). Any marked FCs present 5 or more days AHS derive from an FSC, whether the FSC still remains (blue lineage) or not (green lineage). (c,d) Ovariole from multicolour lineage analysis 9d AHS with (c) three FSC phenotypes (B, GR, BR) anterior to Fas3 staining (pink overlaid by dotted white line) in this z-section and (d) patches of all six possible FC colours. Letters indicate the presence of a given transgene (B- Blue lacZ, G-Green GFP, R- Red, RFP; see Supplementary Fig. 1a–c). (e) Radial location of single candidate FSC nuclei was scored according to z-section location as falling within central (5 cases), upper (5 cases) or lower thirds (6 cases) of the germarial circumference. (f) Cross-section of radially distributed FSC nuclei of the indicated colours surrounding central germline cells. (g,h) Germaria 9d AHS. (g) The only BR FSC candidate is in layer 2 (“BR”, cyan arrow), anterior to another FSC (“G”, white arrow). (h) The only BR FSC candidate is in layer 3 (“BR”, yellow arrow) with two FSCs (cyan and white arrows) that are more posterior (right) but still anterior to Fas3 staining (enhanced as dotted white line). (i) Frequency of germaria with the indicated numbers of labeled FSCs (summing all lineages) in layers 1, 2, 3 and in total. (j) Two surfaces (combined z-sections 1–3 and 11–14) of an egg chamber populated by four different FC colours. (k) Frequency of egg chambers with the indicated number of distinct FC colours among all egg chambers from ovarioles with at least four distinct FSC lineages. Scale bar in (f) 10μm; all others 20μm. (d,j) Diagonal white lines indicate an edge of the original image (shown in Supplementary Fig. 9).