Burkholderia species are the most common and preferred nodulating symbionts of the Piptadenia group (tribe Mimoseae)

Abstract

Burkholderia legume symbionts (also called α-rhizobia) are ancient in origin and are the main nitrogen-fixing symbionts of species belonging to the large genus Mimosa in Brazil. We investigated the extent of the affinity between Burkholderia and species in the tribe Mimoseae by studying symbionts of the genera Piptadenia (P.), Parapiptadenia (Pp.), Pseudopiptadenia (Ps.), Pityrocarpa (Py.), Anadenanthera (A.) and Microlobius (Mi.), all of which are native to Brazil and are phylogenetically close to Mimosa, and which together with Mimosa comprise the "Piptadenia group". We characterized 196 strains sampled from 18 species from 17 locations in Brazil using two neutral markers and two symbiotic genes in order to assess their species affiliations and the evolution of their symbiosis genes. We found that Burkholderia are common and highly diversified symbionts of species in the Piptadenia group, comprising nine Burkholderia species, of which three are new ones and one was never reported as symbiotic (B. phenoliruptrix). However, α-rhizobia were also detected and were occasionally dominant on a few species. A strong sampling site effect on the rhizobial nature of symbionts was detected, with the symbiont pattern of the same legume species changing drastically from location to location, even switching from β to α-rhizobia. Coinoculation assays showed a strong affinity of all the Piptadenia group species towards Burkholderia genotypes, with the exception of Mi. foetidus. Phylogenetic analyses of neutral and symbiotic markers showed that symbiosis genes in Burkholderia from the Piptadenia group have evolved mainly through vertical transfer, but also by horizontal transfer in two species.

Figure 1. Comparison between Piptadenia’s group plant phylogeny and the occurrence of alpha and beta-rhizobia as nodule symbionts in the field or during coinoculation experiments.

The plant phylogeny (A) is based on a trnL-F/trnK-matK combined dataset, and was built by parsimony with TNT1.1 (default parameters, on www.phylogeny.fr) using the Jobson & Luckow dataset (downloaded from Treebase, study number S1763, and amended with the P. trisperma from this study). The % of α and β-rhizobia per legume host (in bold) from field sampling (B) or from the coinoculation experiment (C) are represented as white (Burkholderia) and black (α-rhizobia) squares, with the number of strains sampled within each square. *: statistics of symbionts from and . The grey colored square for Stryphnodendron indicates that % of α-rhizobia originates from a trapping experiment on soil (see Material & Methods section).

Figure 2. Comparison of phylogenies between neutral and symbiotic markers in beta-rhizobia.

The neutral marker phylogeny (A) is based on a partition of 800 bp of 16 S rDNA and 600 bp of recA genes, and was built by a Bayesian analysis with priors estimated by ML (see Experimental procedure section). Numbers at ach nodes indicates posterior probabilities (upper number) and bootstraps values % (lower number) from a ML analyses built in parallel (with a GTR+I+G model, 1000 bootstrap replicates). Bootstraps are only indicated when >50%. Node values in bold indicates supported nodes (both by posterior probabilities and bootstrap) retained for clades delineation. The nodC phylogeny is based on 437 bp alignments, and was built by ML using a GTR+I+G model, with 1000 bootstraps replicates (% indicated at tree nodes if >50%). Abbreviations used: B.: Burkholderia, C.: Cupriavidus, BPL: Burkholderia phenoliruptrix, BT: B. tuberum, BSa: B. sabiae, BD: B. diazotrophica, BSy: B. symbiotica, BM: B. mimosarum, BN: B. nodosa, BSP1,2,3,4: Burkholderia sp. 1 to 4, ^T indicates species type strains. Accession numbers are listed in Table S3.

Figure 3. Distribution of rhizobial species according to host plant and sampling regions.

The % of each species per legume host was calculated according to sampled isolates listed in Table 1. Sampling region is listed below each histogram while on top are indicated the number of isolates. Abbreviations: SER, RJ: Seropédica, Rio de Janeiro; MGS: Mato Grosso do Sul; PAR: Paranà; SP: Sao Paulo; CB, RJ: Cabo Frio, Rio de Janeiro; BU, RJ: Buzios, Rio de Janeiro; ES: Espirito Santo; MG: Minas Geraïs; PHF, SP: Paraibuna horto florestal, Sao Paulo; BA: Xique-Xique, Bahia; IFSP, SP: IFSP, Sao Paulo; Ac: Anadenanthera colubrina, Ap: A. peregrina, Mf: Microlobius foetidus; Pg: Piptadenia gonoacantha; Ppan: P. paniculata; Pt: P. trisperma; Pv: P. viridiflora; Pppt: Parapiptadenia pterosperma; Ppr: Parapiptadenia rigida.