For Trachymyrmex p38 kinase assay species with predominantly serine proteinase activity we plotted the average profile for Trachymyrmex sp3 (Trsp3-3) and Trachymyrmex cf. zeteki (Trzet3), which were very similar. As representatives of the basal

higher attine and leaf-cutting ant symbionts with predominantly metalloproteinase activity we plotted gardens of colonies Trcor10 and Acech322 as gardens of other colonies with this symbiont displayed very similar profiles. The phylogenetic tree is based on the LSU rRNA and Elongation Factor 1-alpha genes, except for samples 20 and 23 for which only the LSU gene could be sequenced. Only aLRT (approximate likelihood ratio test) support values > 0.5 are given. The pH optimum of total proteinase activity in the gardens reared by lower attines averaged 6.0 ± 0.11, while the peak of proteinase activity in basal higher attines and leaf-cutting ants colonies was closer to the pH levels (ca. VS-4718 5) in the fungus gardens (Figure 3; Table 1) (t36 = 9.3, p < 0.001), as one would expect when the higher attine fungus gardens would have become adapted to growing under more find more acidic conditions. However, in three of the four clades of higher attine and leaf-cutting symbionts, the total proteinases activity profiles between pH 5 and 7 were remarkably flat, as serine

proteinases became increasingly active at higher pHs (Figures 2 and 3). For example, the serine proteinases in Sericomyrmex amabilis colonies were most active at pH conditions of 7.0 ± 0.05, similar to lower attine gardens (7.0 ± 0.09) where serine proteinase activity is rarely seen, whereas an additional peak of serine proteinase activity at pH 5.2 ± 0.11 (different from the 7.0 mean above: t6 = 17.0, p < 0.001) was observed for the symbionts of Trachymyrmex

sp3 and T. cf. zeteki, but not for Sericomyrmex symbionts (Figures 2 and 3 and Table 1). Similar patterns were observed for metalloproteinases. The relatively low amounts produced in the symbionts of lower attine ants were most active under slightly acidic pH conditions (6.0 ± 0.11) and shifts towards more acidic optima were detected for the symbionts of Trachymyrmex cornetzi (5.6 ± 0.09) (t4 = 3.45, p = 0.026) and the leaf-cutting ants mutualists (5.2 ± 0.08) (t6 = 10.0, p < 0.001) (Figures 2 and 17-DMAG (Alvespimycin) HCl 3 and Table 1). Figure 3 The class-specific pH optima for serine (vertical axis) and metalloproteinases (horizontal axis) for the fungus gardens in Table 1 for which both pH optima could be measured from the same samples. The vertical axis is interrupted to allow the pH-optimum to be plotted for metalloproteinase activity in gardens where serine proteinase activity could not be measured. The overall pattern indicates that pH optima for metalloproteinases are always between ca. 5 and 6, whereas serine proteinase pH optima tend to fall between 7 and 8. All values are means ± SEMs. Dotted lines connect observations for the same species. See Table 1 for details.