(a) Drawing of gongylidia and representative staphylae of different size. (b) Image of gongylidia (G) and staphylae (S) in the fungus garden of the leaf-cutting ant A. echinatior. (c) Phylogenetic reconstruction of the coevolution of ant symbiotic fungi (left) and fungus-growing ant genera (right) based on previously published work9,11,12,23, with the origin of gongylidia marked. Boxed genera highlight the phylogenetic position of the A. echinatior and C. longiscapus fungal cultivars used in the codon-based transcriptome comparison of the present study. Green: leaf-cutting ants cultivating L. gongylophorus, blue: higher attine ants cultivating a variety of Leucoagaricus spp. with gongylidia, light brown: lower-attine ants cultivating a diverse group of fungi without gongylidia from two clades (clade 1 and 2) in the family, Agaricaceae and Pterulaceae, cultivated by a subclade of ants in the genus Apterostigma. (d) Mean total area (mm2) covered by staphylae (±s.e.) per mm2 of fungus garden surface showing significantly higher cover proportions in Atta and (partially) Acromyrmex leaf-cutting ants (green) compared with Trachymyrmex and Sericomyrmex higher-attine ants (blue) (Kruskal–Wallis, H=27.4, df=12, P=0.007 **, mean values illustrated by horizontal dotted lines). (e) Gongylidia diameter (μm)±s.e. per fungus garden (Kruskal–Wallis, H=19.2, df=12, NS: Not Significant) and (f) Number of staphylae per mm2 fungus garden (Kruskal–Wallis, H=10.7, df=12, NS: Not Significant). The number of sampled colonies per species is given inside each column.