Endosymbiotic bacteria nodulating a new endemic lupine Lupinus mariae-josephi from alkaline soils in Eastern Spain represent a new lineage within the Bradyrhizobium genus

Lupinus mariae-josephi is a recently described endemic Lupinus species from a small area in Eastern Spain where it thrives in soils with active lime and high pH. The L. mariae-josephi root symbionts were shown to be very slow-growing bacteria with different phenotypic and symbiotic characteristics from those of Bradyrhizobium strains nodulating other Lupinus. Their phylogenetic status was examined by multilocus sequence analyses of four housekeeping genes (16S rRNA, glnII, recA, and atpD) and showed the existence of a distinct evolutionary lineage for L. mariae-josephi that also included Bradyrhizobium jicamae. Within this lineage, the tested isolates clustered in three different sub-groups that might correspond to novel sister Bradyrhizobium species. These core gene analyses consistently showed that all the endosymbiotic bacteria isolated from other Lupinus species of the Iberian Peninsula were related to strains of the B. canariense or B. japonicum lineages and were separate from the L. mariae-josephi isolates. Phylogenetic analysis based on nodC symbiotic gene sequences showed that L. mariae-josephi bacteria also constituted a new symbiotic lineage distant from those previously defined in the genus Bradyrhizobium. In contrast, the nodC genes of isolates from other Lupinus spp. from the Iberian Peninsula were again clearly related to the B. canariense and B. japonicum bv. genistearum lineages. Speciation of L. mariae-josephi bradyrhizobia may result from the colonization of a singular habitat by their unique legume host.

Lupinus mariae-josphi, a new lupin endemic of soils with active lime and high pH in South Eastern Spain, is nodulated by a new bacterial lineage within Bradyrhizobium genus

Lupinus mariae-josephi is a recently described species (Pascual, 2004) able to grow in soils with high pH and active lime content in the Valencia province (Spain). L. mariae-josephi endosymbionts are extremely slowgrowing bacteria with genetic and symbiotic characteristics that differentiate them from Bradyrhizobium strains nodulating Lupinus spp. native of the Iberian Peninsula and adapted to grow in acid soils. Cross-inoculation experiments revealed that all the endosymbiotic isolates from L. mariae-josephi tested are legume-host selective and are unable to nodulate species such as L. angustifolius, and L. luteus. In contrast, Bradyrhizobium strains from Lupinus spp. tested were able to nodulate L. mariae-josephi, although the nodules fixed nitrogen inefficiently. Phylogenetic analysis was performed with housekeeping genes (rrn, glnII, recA, atpD) and nodulation gene nodC. Housekeeping gene phylogeny revealed that L. mariae-josephi rhizobia form a strongly supported monophyletic group within Bradyrhizobium genus. This cluster also includes B. jicamae and certain strains of B. elkanii. Contrarily, isolates from other Lupinus spp. native of the Iberian Peninsula were grouped mainly within B. canariense and two B. japonicum lineages. Phylogenetic analysis of L. mariae-josephi isolates based on the nodC symbiotic gene defined a solid clade close to isolates from Algerian Retama spp. and to fast-growing rhizobia.