Burkholderia species are ancient symbionts of legumes

Abstract Burkholderia has only recently been recognized as a potential nitrogen-fixing symbiontof legumes, but we find that the origins of symbiosis in Burkholderia are much deeperthan previously suspected. We sampled 143 symbionts from 47 native species of Mimosaacross 1800 km in central Brazil and found that 98% were Burkholderia. Gene sequencesdefined seven distinct and divergent species complexes within the genus Burkholderia.The symbiosis-related genes formed deep Burkholderia-specific clades, each specific to aspecies complex, implying that these genes diverged over a long period withinBurkholderia without substantial horizontal gene transfer between species complexes.Keywords: biodiversity, Brazil, Mimosa, nitrogen fixation, rhizobiaReceived 27 August 2009; revision received 19 October 2009; accepted 25 October 2009 Introduction The genus Burkholderia belongs to the b-proteobacteriaclass and currently includes more than 50 species thatcolonize a wide diversity of niches ranging from soiland water to plants and animals. Nevertheless, Burk-holderia remains mostly known and studied through itsvarious pathogenic representatives. For this reason, thedescription in 2001 of legume-nodulating Burkholderia(Moulin et al. 2001) and their subsequent characteriza-tion as real symbionts (Chen et al. 2003, 2005a,b; Barrett& Parker 2005, 2006, 2006; Andam et al. 2007) was adouble shock, changing our perceptions of both the eco-logical capabilities of Burkholderia and the taxonomicdiversity of legume symbionts.Legume-nodulating bacteria, collectively called rhizo-bia, live as saprophytes in the soil and in a facultativesymbiosis with plants. They induce the formation ofroot nodules, within which they fix atmospheric nitro-gen and provide it to the plant in exchange for carboncompounds. This mutualistic association occurs in themajority of legumes and constitutes the main terrestrialsystem of biological nitrogen fixation.Rhizobia were already known to be taxonomicallyheterogeneous and polyphyletic, but the knownexamples were confined to the Alphaproteobacteria class(‘a-rhizobia’) until the identification of nodulating Burk-holderia and Cupriavidus established that there were also‘b-rhizobia’ (Chen et al. 2001; Moulin et al. 2001). So far,five nodulating Burkholderia species have been described:Burkholderia tuberum (Vandamme et al. 2002), B. phyma-tum (Vandamme et al. 2002), B. mimosarum (Chen et al.2006), B. nodosa (Chen et al. 2007) and B. sabiae (Chenet al. 2008). Symbiotic strains were also identified in B.caribensis (Chen et al. 2003) and B. cepacia (Rasolomam-pianina et al. 2005), previously described as nonsymbi-otic species, and some isolates represent other species,which are yet unnamed (Barrett & Parker 2006; Parketet al. 2007; Garau et al. 2009). The only b-rhizobium so