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

Here, we describe a novel clade withinEnsifer melilotiand consider how geographic and ecological isolation contributed to the limited distribution of this group. Members of the genusEnsiferare best known for their ability to form nitrogen-fixing symbioses with forage legumes of three related genera,MedicagoL.,MelilotusMill., andTrigonellaL., which are members of the tribe Trifolieae. These legumes have a natural distribution extending from the Mediterranean Basin through western Asia, where there is an unsurpassed number of species belonging to these genera.Trigonella suavissimaL. is unusual in that it is the only species in the tribe Trifolieae that is native to Australia. We compared the genetic diversity and taxonomic placement of rhizobia nodulatingT. suavissimawith those of members of anEnsiferreference collection. Our goal was to determine if theT. suavissimarhizobial strains, like their plant host, are naturally limited to the Australian continent. We used multilocus sequence analysis to estimate the genetic relatedness of 56T. suavissimasymbionts to 28Ensiferreference strains. Sequence data were partitioned according to the replicons in which the loci are located. The results were used to construct replicon-specific phylogenetic trees. In both the chromosomal and chromid trees, the Australian strains formed a distinct clade withinE. meliloti. The strains also shared few alleles withEnsiferreference 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.IMPORTANCEIn 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 hostTrigonella suavissimais instead closely related to legumes native to the Mediterranean Basin and western Asia, suggesting that it may have been introduced in 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 either that theT. suavissimarhizobia 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.