The increases of CDC48 and STE20 expressions up to stages 5 and 6 are in line with the role that the proteins coded by these genes have in the control of cell division and the regulation of sexual differentiation, respectively, during the FB ripening, when mitosis and meiosis occur during asci maturation and sporogenesis. The expression of aif1, FIS1, and DNM1, which steadily decreases from the developmental stage 3 up to stage 6, as found by histochemical and transmission electron microscopy (TEM) investigations (terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL), Fig. 3 and TEM, Fig. 4), signs of PCD, would suggest that TUNEL positivity, in some cells of the T. melanosporum FB, is not due to the apoptotic subroutine of PCD but another one (autophagy or necrosis). However a large genetic tool-kit related to PCD is present in the T. melanosporum genome; some of the PCD genes are homologous to PCD genes of the human genome and of species other than Ascomycetes, such as mouse, Aves, Amphibia, Zebrafish, Ascidiacea, Mollusca, etc. thus vertebrates and invertebrates (Figure S2). In Figure S2, the deduced AA sequences identities of PCD involved genes of T. melanosporum genome versus the human homologous sequences are reported. Figure S2 shows also the percentual identities of the deduced AA sequences of various invertebrates and vertebrates versus the human homologous sequences.Fig. 3In situ cell death detection by means of TUNEL reaction on cryostat sections of T. melanosporum fruit bodies at different developmental stages,Stage 3 (a–c); stage 4 (d–h); stage 5 (i–k); stage 6 (l–n). The three columns illustrate representative images of, respectively, Giemsa staining (for structural analysis and stage assessment), DAPI staining (for total nuclei visualization), and TUNEL reaction. a asci, as ascospores, p peridium, pa paraphyses, sv sterile vein. Bars = 50 µm in b, c, g, h, j, and k; 100 µm in e, f, l, m, and n; 200 µm in a, d, and iFig. 4TEM analysis of cell death in T. melanosporum (stage 4) fruiting bodies.a Toluidine blue-stained semithin section, showing the paraphyses region at the interface between sterile and fertile veins, where significant TUNEL positivity has been detected. b, c Examples of intact sterile hyphae, with normal nuclei; d sterile hyphae showing cytoplasmic emptying and vacuolization. e, f Altered nuclei with irregular condensed chromatin masses and dilated nuclear envelope lumen (arrow). a asci, n nuclei, pa paraphyses, sv sterile vein. Bars = 50 µm in a; 4 µm in b, d; 2 µm in c; 1 µm in e, f. TEM transmission electron microscopy