South african papilionoid legumes are nodulated by diverse burkholderia with unique nodulation and nitrogen-fixation Loci

Abstract

The root-nodule bacteria of legumes endemic to the Cape Floristic Region are largely understudied, even though recent reports suggest the occurrence of nodulating Burkholderia species unique to the region. In this study, we considered the diversity and evolution of nodulating Burkholderia associated with the endemic papilionoid tribes Hypocalypteae and Podalyrieae. We identified distinct groups from verified rhizobial isolates by phylogenetic analyses of the 16S rRNA and recA housekeeping gene regions. In order to gain insight into the evolution of the nodulation and diazotrophy of these rhizobia we analysed the genes encoding NifH and NodA. The majority of these 69 isolates appeared to be unique, potentially representing novel species. Evidence of horizontal gene transfer determining the symbiotic ability of these Cape Floristic Region isolates indicate evolutionary origins distinct from those of nodulating Burkholderia from elsewhere in the world. Overall, our findings suggest that Burkholderia species associated with fynbos legumes are highly diverse and their symbiotic abilities have unique ancestries. It is therefore possible that the evolution of these bacteria is closely linked to the diversification and establishment of legumes characteristic of the Cape Floristic Region.

Figure 1. Cluster A (Fig. S2) of the 16S rRNA maximum-likelihood (ML) phylogeny for Burkholderia.

Similar groupings were recovered following analysis of the data using Bayesian Inference (BI). Statistical support of ≥ 60% for ML bootstrap (BS) and ≥ 0.60 BI posterior probability (PP) are indicated at the branches in the order PP/BS. Diazotrophic Burkholderia species appear in blue, nodulating isolates in red and all nodulating South African isolates in green, with those isolates from this study also appearing in bold. All strain/isolate designations are followed by the name of the legume host and country of geographic origin. Names of the indigenous legume hosts are abbreviated as in Table 1. The GenBank accession numbers for the 16S rRNA sequences are listed in Table S1. The scale bar indicates the number of nucleotide substitutions per site. There are four groups of isolates which could not be included in full (due to size constraints) the ‘missing’ isolates are indicated as follows: *A1– Burkholderia sp. STM3671 Mimosa pudica French Guiana, Burkholderia sp. STM6020 Mimosa pudica French Guiana; *A2 - Burkholderia sp. JPY-582 Mimosa hexandra Brazil, Burkholderia sp. Br3462 Mimosa flocculosa Brazil, Burkholderia sp. mpa4.1 Mimosa pigra Australia; *A3 - WK1.1d+WK1.1f+WK1.1g+WK1.1i+WK1.1j+WK1.1k+WK1.1m+HC1.1bh H. sophoroides and lastly *A4 - Kb13+ Kb14+ Kb16 V. oroboides, HC1.1bc H. sophoroides.

Figure 2. An ML phylogeny inferred from the nifH data for Burkholderia.

Similar groupings were generated following BI analysis of the data. Statistical support of ≥ 60% for ML bootstrap (BS) and ≥ 0.60 BI posterior probability (PP) are indicated at the branches in the order PP/BS. Isolate/strain designations are followed by legume host names (where applicable) and geographic origin. The names of the indigenous legume hosts are abbreviated as in Table 1. Diazotrophic isolates appear in blue, nodulating isolates in red, and nodulating South African isolates in green with those from this study in bold. The GenBank accession numbers for the nifH sequences are listed in Table S1. The scale bar indicates the number of nucleotide substitutions per site.

Figure 3. An ML phylogeny inferred from the nodA data for nodulating species and isolates of Burkholderia.

Similar groupings were generated following BI analysis of the data. Statistical support of ≥ 60% for ML bootstrap (BS) and ≥ 0.60 BI posterior probability (PP) are indicated at the branches in the order PP/BS. Isolate/strain designations are followed by host name and geographic origin. The names of the indigenous legumes focused on in this study are abbreviated as in Table 1. Beta-rhizobial isolates from South Africa are indicated in green (and those from this study is in bold), while those from other regions appear in red. The scale bar indicates the number of nucleotide substitutions per site.