Origins and Diversification of T3SS Figure 2. Discrimination between NF-T3SSs and flagella based on Hmmer profile scores. Each T3SS core protein was scored using the protein profiles for flagella (Y axis) and for NF-T3SSs (X axis). One sees a clear separation around the main diagonal. The color indicates the system (blue for flagellar proteins, red for NF-T3SS proteins). A circle indicates a correct prediction, and a triangle a wrong prediction. The analysis is described in Materials and Methods, and the learning dataset is shown in Figure S1. doi:10.1371/journal.pgen.1002983.g002 distinct monophyletic groupings (Figure S4) that have probably been inherited vertically because the topology and branching structures of these phylogenies resemble those of the 16S rDNA tree (Figure 5). The conservation of these systems in sequence, gene composition and genetic organization is striking because the species harboring them diverged a long time ago. For example, the 16S rDNA subunits of Anaeromyxobacter and Myxococcus show lower sequence similarity than that between Escherichia coli and Vibrio cholerae. The strong conservation of these loci in sequence and organization over such long time scales suggests that they are functional. We argue that the Myxo NF-T3SSs probably derived from ancestors that were neither flagella nor protein delivery systems. Myxo systems lack proteins that are indispensable for flagellar function, such as FlgBCDEK, FliEG, FlgH and MotAB. They also lack NF-T3SS genes such as the secretin, the major needle subunit (SctF), the tip protein (LcrV) and the translocon (YopB/YopD). Homologs of the tip and translocon proteins may have been missed in our sequence similarity searches because of their rapid evolution. However, the flagellar proteins, the secretin and SctF are highly conserved and should have been found by our sequence similarity searches had they been present. Our profiles show significant sequence similarity between SctF, the major needle subunit of the NF-T3SS, and FliC/FlgL (the flagellin and a hook- associated protein), whose homology was previously suggested based on structural data [71,101]. Hence, the earliest NF-T3SS probably contained an ancestral SctF that was lost in Myxo systems. The lack of an outer membrane channel and of SctF suggests that Myxo systems are not able to deliver effectors to eukaryotic cells and possibly not even to secrete proteins to the extracellular space. Multiple origins of secretins We next analyzed the diversification of NF-T3SS within the main branch of its phylogeny, i.e. among loci including a secretin. This branch includes all NF-T3SSs shown experimentally to deliver effectors into the eukaryotic cytosol. The first split along this branch separates the Chlamy from the other systems with 100% bootstrap support (Figure 4, Figure S3, Table S4). Subsequent diversification was very rapid within the other taxa as shown by a succession of short branches with weak bootstrap support (Figure 4 and Figures S3, S4, S5). To investigate the early NF-T3SS diversification we made a phylogenetic analysis of the NF-T3SS secretin together with secretins from T2SS, T4P, Tad system and filamentous phages [102]. Surprisingly, the phylogeny shows that secretins have been independently recruited to the NF-T3SS on at least three occasions (Figure 6A). The Rhizo secretin RhcC2 branches together with secretins from the Tad loci (e.g. RcpA and CpaC) [103]. The secretin domain of Chlamy NF-T3SSs (excluding a large unique N- ter region, see Figure 6B and [81,92]) clusters with the gene IV secretin of filamentous phages within T2SS secretins. The secretins from the remaining NF-T3SSs cluster together in a third group PLOS Genetics | www.plosgenetics.org 5 September 2012 | Volume 8 | Issue 9 | e1002983