AtEAF1 and PIE1 represent two distinct protein families that share highly similar HSA and SANT domains and do not coexist outside plants. (a) Protein alignment of domain relatives of Arabidopsis thaliana (At) AtEAF1 and PIE1, found in Saccharomyces cerevisiae (Sc), Schizosaccharomyces pombe (Sp), Brachypodium distachyon (Bd), Selaginella moellendorffii (Sm), Physcomitrella patens (Pp) and Homo sapiens (Hs). A dotted rectangle depicts the region of yeast EAF1 that recruits ARP4 and actin, according to Szerlong et al. . Columns containing over 75% gaps were removed from the alignment for clarity. Similarity shading of the alignments in (a) and (b) was done using the Blosum62 matrix. White indicates < 60% similarity, light grey 60 to 80%, dark grey 80 to 100% and black 100%. (b) A close-up of the conserved fragments of the HSA domain and the SANT domain. (c) Protein domains HSA, ATPase and SANT form three different domain architectures, which occur in various combinations across eukaryotes. All sequences and alignments used in this figure can be found in the Additional file 3. * Mean pairwise identity over all pairs in the column. A sliding window of 5 was used in the histogram.