Taxonomy of endosymbiotic bacteria from a novel Lupinus sp. (Lupinus mariae-josephi) endemic of a limed-alkaline soil habitat in Southeastern Spain

Abstract

Lupinus mariae-josephi is a recently described Lupinus species (Pascual 2004) endemic of a Southeastern area of Spain with soils singularly of high pH and active lime content where it is endangered due to the reduced size of its habitat. Ten isolates of L. mariae-josephi endosymbiotic bacteria were obtained using trap-plants and soils from five sampling points within a native plant population area in Llombai (Valencia, Spain). The microsymbionts are extra-slow (ultrabradytrophic) growing bacteria with phenotypic and symbiotic characteristics singularly different from Bradyrhizobium strains nodulating other Lupinus spp. thriving in the Iberian Peninsula and adapted to growth in acidic soils. Cross-inoculation experiments revealed that these L. mariae-josephi endosymbiotic bacteria isolates are unable to nodulate or efficiently fix nitrogen with other Lupinus spp. Their phylogenetic status was examined by a multilocus sequence analysis of four housekeeping genes (16S rDNA, glnII, recA, atpD) and the symbiotic nodC gene. The 16S rDNA phylogenetic analysis showed that L. mariae-josephi isolates are related to strains nodulating Retama spp. in northeastern Algeria (Boulila et al., 2009), Phaseolus lunatus from Peru (Ormeño-Orrillo et al., 2006), as well as to B. elkanii, B. jicamae and B. pachyrhizi species, forming a new clade (Clade I) within the Bradyrhizobium genus. All the single and concatenated glnII+recA and glnII+recA+atpD analyses consistently support the existence of Clade I, and also revealed that, within this clade, the L. mariae-josephi endosymbiotic bacteria belong to a single evolutionary lineage that also includes strains nodulating Retama spp. from northeastern Algeria. Within this new Bradyrhizobium lineage, the phylogenetic analyses performed showed essentially convergent results indicating that the tested L. mariae-josephi isolates nested in three sub-groups that might correspond to novel sister Bradyrhizobium species. Bradyrhizobium Clade I is highly differentiated from the Bradyrhizobium clade (Clade II) that includes currently named Bradyrhizobium species and well-delineated unnamed genospecies. Singularly, all the endosymbiotic bacteria from Lupinus species adapted to acid soils in the Iberian Peninsula and tested in this study are included in Clade II. They are related either to strains of the B. canariense or B. japonicum lineages. The phylogenetic analysis based on the symbiotic nodC gene showed that L. mariae-josephi endosymbiotic bacteria define a novel branch in the nodC Bradyrhizobium tree. This branch groups together with a branch that gathers isolates from recently studied legume symbiosis such as isolates from Retama spp., which suggests the existence of a common unique ancestor for the symbiotic genes of these two groups of bradyrhizobia. In contrast, the symbiotic genes of isolates from other Lupinus spp. from the Iberian Peninsula are clearly related to the B. canariense lineage. The allopatric (geographic) speciation of the L. mariae-josephi bradyrhizobia may result from the colonization of a singular habitat, such as the basic and high calcium carbonate soils of the Valencia area, by its unique legume host