High Diversity of Bradyrhizobial Species Fix Nitrogen with Woody Legume Spartocytisus supranubius in a High Mountain Ecosystem.

The symbiosis between rhizobia and legumes is of pivotal importance in nitrogen-poor ecosystems. Furthermore, as it is a specific process (most legumes only establish a symbiosis with certain rhizobia), it is of great interest to know which rhizobia are able to nodulate key legumes in a specific habitat. This study describes the diversity of the rhizobia that are able to nodulate the shrub legume Spartocytisus supranubius in the harsh environmental conditions of the high mountain ecosystem of Teide National Park (Tenerife). The diversity of microsymbionts nodulating S. supranubius was estimated from a phylogenetic analysis of root nodule bacteria isolated from soils at three selected locations in the park. The results showed that a high diversity of species of Bradyrhizobium and two symbiovars can nodulate this legume. Phylogenies of ribosomal and housekeeping genes showed these strains distributed into three main clusters and a few isolates on separate branches. These clusters consist of strains representing three new phylogenetic lineages of the genus Bradyrhizobium. Two of these lineages belong to the B. japonicum superclade, which we refer to as B. canariense-like and B. hipponense-like, as the type strains of these species are the closest species to our isolates. The third main group was clustered within the B. elkanii superclade and is referred to as B. algeriense-like as B. algeriense is its closest species. This is the first time that bradyrhizobia of the B. elkanii superclade have been reported for the canarian genista. Furthermore, our results suggest that these three main groups might belong to potential new species of the genus Bradyrhizobium. Analysis of the soil physicochemical properties of the three study sites showed some significant differences in several parameters, which, however, did not have a major influence on the distribution of bradyrhizobial genotypes at the different locations. The B. algeriense-like group had a more restrictive distribution pattern, while the other two lineages were detected in all of the soils. This suggests that the microsymbionts are well adapted to the harsh environmental conditions of Teide National Park.

Endophytic Bosea spartocytisi sp. nov. Coexists with rhizobia in root nodules of Spartocytisus supranubius growing in soils of Teide National Park (Canary Islands).

Two rod-shaped Gram negative strains, SSUT16T and SSUT22, were isolated from root nodules of Spartocytisus supranubius in soils of the Teide National Park (Tenerife, Spain). The 16S rRNA gene sequences of these two novel strains classified them within genus Bosea with similarity values ranging from 97.65 % to 99.54 % with respect to the other species of this genus. The MLSA analysis from a concatenation of the two housekeeping- genes, recA and gyrB, showed that Bosea thiooxidans LMG 26210T and B. robiniae LMG 26381T are the two closest relative species with which they share similarity sequences values of 94.42 % and 94.27 %, respectively. The genome sequence analysis of strain SSUT16T showed average nucleotide identity percentages (ANIb) and digital DNA-DNA hybridization (dDDH) below 84 % and 33 %, respectively, with the type strains of all sequenced species of genus Bosea. These values are much lower than the currently accepted cut-off values for these two parameters to delineate bacterial species, confirming that the novel strains constitute a novel Bosea species. In addition, they are also distinguished from the other closest species in their fatty acid composition and in other phenotypic characteristics. Genome sequence analysis showed the absence of the common nodulation and nitrogen fixation genes in the novel strains. Therefore, based on the results of phylogenetic, genomic, chemotaxonomic and phenotypic characterization, we propose a new species named Bosea spartocytisi sp. nov., with type strain SSUT16T (=LMG 32510T = CECT 30526T = HAMBI 3759T).

Definition of the novel symbiovar canariense within Mesorhizobium neociceri sp. nov., a new species of genus Mesorhizobium nodulating Cicer canariense in the "Caldera de Taburiente" National Park (La Palma, Canary Islands).

Cicer canariense is a highly promiscuous wild chickpea nodulated by Mesorhizobium strains in La Palma Island located at Canary archipelago. Four of these strains, CCANP34, CCANP35T, CCANP38 and CCANP95 belong to a group phylogenetically close to Mesorhizobium caraganae with 100% similarity values in the 16S rRNA gene. However, the genomes of the strains CCANP35T and M. caraganae LMG 24397T obtained in this work showed ANIb and dDDH values of 90.02% and 44.1%, respectively. These values are lower than those currently accepted for different bacterial species showing that the Canarian strains do not belong to the species M. caraganae. The Canarian strains also differ from M. caraganae in the amounts of several fatty acids and in several phenotypic traits. Based on the obtained results the Canarian strains belong to a novel species for which we propose the name Mesorhizobium neociceri sp. nov. and whose type strain is CCANP35T. The results of the phylogenetic analyses of nodC and nifH symbiotic genes showed that the Canarian strains represent a novel symbiovar within genus Mesorhizobium phylogenetically divergent to that encompassing M. caraganae. We propose the names canariense and caraganae for the symbiovars encompassing the strains of M. neociceri and M. caraganae, respectively.