Hypothesized evolution of CalS5 gene expression in seed plant pollen. CalS gene relationships from Figure 2 and Additional file 3 are superimposed on a Moss (Physcomitrella) + Seed plant species tree . Filled boxes indicate aniline blue staining of the inner wall of mature pollen (lower) or pollen tube walls/callose plugs (upper) and have been associated with CalS5 or CalS13 expression in all but Amborella (see text). Unfilled boxes represent lack of staining for callose. Branches are coloured to reflect inferred changes in CalS5 gene expression patterns, based on data from this study. Nymphaeaceae includes Nymphaea and Nuphar.