Across their species ranges, native legumes Acmispon wrangelianus and A. brachycarpus share a diverse species pool of nitrogen-fixing Mesorhizobium symbionts

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Hanna Kehlet-Delgado and Maren Friesen, Department of Plant Pathology, Washington State University, Pullman, WA, Angeliqua Montoya, School of Biological Sciences, Washington State University Vancouver, Vancouver, WA, Camille Wendlandt, Crop & Soil Sciences, Washington State University, Vancouver, WA, Joel Griffitts, Microbiology and Molecular Biology, Brigham Young University, Provo, UT, Stephanie Porter, School of Biological Sciences, Washington State University, Vancouver, Vancouver, WA

Background/Question/Methods
In many host-microbe symbiotic mutualisms, the diversity of microbial symbionts infecting a host species across the host range remains cryptic. In the legume-rhizobium mutualism, where rhizobia fix nitrogen in host root nodules, symbiont diversity ranges from low, in cases where a single rhizobium species infects a single legume species, to high, in which multiple rhizobium genera infect a single legume species. In this study, we examine the genomes of Mesorhizobium strains isolated across the species range of Acmispon wrangelianus and Acmispon brachycarpus, legumes native to the western US across contrasting soils. Plant nodules were obtained from 15 California and Oregon sites with serpentine soil, containing unusual ion rations and high concentrations of Ni, and non-serpentine soil in order to obtain isolates with a range presumed Ni tolerance. Over 1000 Mesorhizobium strains were isolated from these nodules. The genomes of sixteen strains were sequenced with Pacbio technology and 286 with Illumina Miseq. Reads were de novo assembled and genomes were annotated using the Prokka tool. Pairwise average nucleotide identity (ANI) distances were calculated with the de novo assembled genomes along with reference Mesorhizobium genomes from type strains. A phylogeny was constructed with RAxML from a set of single-copy conserved genes. The pan-genome was mined for genes putatively related to Ni tolerance to examine the distribution of adaptation to Ni stress.
Results/Conclusions
We find that both native Acmispon legume host species associate with a similar and overlapping broad diversity of Mesorhizobium species. We present three complete Mesorhizobium genomes and 299 draft genomes. We find the majority of the newly sequenced Mesorhizobium cluster into groups delineated by >95% ANI and represent novel taxa as they are not closely related to any reference genomes. Since >95-96% ANI is often used as a criteria for bacterial species delineation, these groups could represent up to 13 new species units strongly associated with Acmispon. One exception was a group that shares >95% ANI with Mesorhizobium australicum WSM2073. Interestingly, the phylogeny does not strongly segregate by reserve, host, or soil type, indicating a range of microdiversity found among habitats. A putative Ni efflux operon is present in 129 genomes and correlates with serpentine soil strains, rather than to a specific clade, suggesting gain/loss through lateral gene transfer. This considers the relative roles of edaphic variation, host plant species, and biogeographic factors on the diversity of Mesorhizobium symbionts of Acmispon.