Biogeography of a Novel Ensifer meliloti Clade Associated with the Australian Legume Trigonella suavissima

Here we describe a novel clade within Ensifer meliloti and consider how geographic and ecological isolation contributed to the limited distribution of this group. Members of the genus Ensifer are best known for their ability to form nitrogen-fixing symbioses with forage legumes of three related genera, Medicago L., Melilotus Mill., and Trigonella L., which are members of the tribe Trifoleae. They have a natural distribution extending from the Mediterranean basin through west Asia, where there is an unsurpassed number of species belonging to these genera. Trigonella suavissima L. is unusual in that it is the only species in the tribe Trifolieae that is native to Australia. We compare the genetic diversity and taxonomic placement of rhizobia nodulating T. suavissima to members of an Ensifer reference collection. Our goal was to determine if the T. suavissima strains, like their plant host, are naturally limited to the Australian continent. We used multilocus sequence analysis to estimate the genetic relatedness of 56 T. suavissima symbionts to 28 Ensifer reference strains. Sequence data was partitioned according to the replicons upon which the loci are located. Results were used to construct replicon-specific phylogenetic trees. In both the chromosomal and chromid trees the Australian strains formed a distinct clade within E. meliloti . The strains also shared few alleles with Ensifer reference strains from other continents. Carbon source utilization assays revealed that the strains are also unusual in their ability to utilize 2-oxoglutarate as a sole carbon source. A strategy was outlined for locating similar strains elsewhere. IMPORTANCE In this study we employed a biogeographical approach to investigate the origins of a symbiotic relationship between an Australian legume and its nitrogen-fixing rhizobia. The question of the ancestral origins of these symbionts is based on the observation that the legume host is not closely related to other native Australian legumes. Previous research has shown that the legume host Trigonella suavissima is instead closely related to legumes native to the Mediterranean basin and west Asia, suggesting that it may have been introduced into Australia from those regions. This led to the question of whether its rhizobia may have been introduced as well. In this study we were unable to find persuasive evidence supporting this hypothesis. Instead our results suggest that either the T. suavissima rhizobia are native to Australia, or that our methods for locating their close relatives elsewhere are inadequate. A strategy to investigate the latter alternative is proposed.