(A) A metazoan phylogenetic tree. Bilatarians are classified as either protostomes (green) or deuterostomes (red) based on differences in embryo development. Non-bilatarian phyla (blue) include cnidarians, ctenophores, and sponges. Tetrahymena is a ciliated protist that also expresses Piwi proteins. (B) Phylogenetic analysis of Argonaute proteins demonstrates a clear distinction between the Piwi (yellow) and Argonuate (purple) subfamilies. Phylogram generated using Phylogeny.fr (29). Numbers on the tree demonstrate branch supports. NCBI accession numbers from top to bottom are as follows: Prg-1 (NM_059720), Prg-2 (NM_068593), Piwi (NM_057527), Aubergine (NM_001103674), Ago3 (NM_001043162), Smedwi-2 (EU586258), Zili (NM001080199), Mili (NM_021308), Hili (NM_001135721), EfPiwiA (AB533505), BpPiwi1 (AB455103), Miwi2 (NM_177905), Hiwi2 (NM_152431), Ziwi (NM_18338), Miwi (NM_021311), Hiwi (NM_004764.4), ClytiaPiwi (EU199802), EfPiwiB (AB533506.1), Smedwi1 (DQ186985), Smedwi2 (DQ186986), Twi11 (EU183125), Twi1 (AB084111), EfAgoA (AB533507), Drosophila Ago1 (NM_166020), Zebrafish Ago2 (XM_694134), Mouse Ago2 (AY135688), Human Ago2 (NM_001164623), Mouse Ago3 (AY135689), Zebrafish Ago3 (XM_690923), Mouse Ago4 (AY135690), Mouse Ago1 (AY135687), Drosophila Ago2 (NM_140518).