Origins and Diversification of T3SS Figure 4. Concatenate phylogeny of the core NF-T3SS proteins with flagellar homologs. The eight individual gene trees are displayed in Figure S3. The large monophyletic clades highlighted by different colors correspond to the NF-T3SS families (names in bold, see Text S2). Colored circles indicate the bootstrap support of these families and of the relationships between them. The ‘‘Ecology/Host’’ panel indicates the predominant bacterium/eukaryote associations for each family (see main text and Figure S4). The external branch indicates the position of the flagellum sequences and was inferred with the ‘‘rooted’’ dataset (‘‘rooted’’ tree is in Figure S5). ‘‘Chlamy’’ stands for Chlamydiales, ‘‘Desulfo’’ for Desulfovibrionales, ‘‘Myxo’’ for Myxococcales, and ‘‘Rhizo’’ for Rhizobiales. The scale of the tree is given in substitutions per site. doi:10.1371/journal.pgen.1002983.g004 proposed to serve as a protein secretion system [116,117]. Interestingly, flagella-like loci have been detected in the genomes of a dozen species that are not thought to have flagella [74]. These observations suggest that flagellar T3SSs have been exapted on multiple occasions for secretion of proteins unrelated to the flagellum. However, NF-T3SSs are monophyletic, indicating that their exaptation from the flagel- lum only occurred once. During experimental evolution, mutations that induce a loss of function are among the most frequent adaptive events detected [118]. We would then anticipate that exaptation to the NF-T3SS would have been accompanied by deletions of flagellum-specific genes. Yet the core function of the T3SS as a facilitator of protein translocation was probably never lost: (i) the secretion apparatus is highly conserved; (ii) the needle protein, which is translocated by the T3SS, is homologous to flagellar proteins; (iii) experimental evidence shows that extant NF-T3SS effectors can be secreted by the flagellum T3SS [119]. Our results contradict the proposal that NF-T3SS evolved in Chlamydiales before being transferred to Proteobacteria [62,93]. Transfer of the multiple loci within the Chlamy system would require multiple events, whereas the unique loci of other NF- T3SSs can be transferred in one single event. All Chlamy NF- T3SSs include a T2SS-like secretin whereas other NF-T3SSs share a secretin from a distinct origin (Figure 6). Finally, key elements between these systems have no homologs, e.g. the proposed tip protein for Chlamy (CT584) cannot be aligned to tip proteins from other systems [120]. Hence, we propose that the last common ancestor of the NF-T3SSs derived from a flagellum, lacked a secretin and included a periplasmic or extracellular structure based on SctF. Interestingly, Spirochetes have periplasmic flagella, and in some cases lack the outer membrane protein FlgH [121]. Inactivation of the secretin in S. enterica SPI1 NF-T3SS prevents the formation of extracellular appendages but does not prevent the secretion of the main needle filament subunit (SctF) to the periplasm, where it remains associated with the inner ring proteins PLOS Genetics | www.plosgenetics.org 7 September 2012 | Volume 8 | Issue 9 | e1002983