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Research on the nitrogen-fixing symbiosis has been so far focused on two model legumes,  Medicago truncatula  and  Lotus japonicus , which use a sophisticated infection process involving infection thread formation. However, in 25% of the legumes, the bacterial entry occurs more simply in an intercellular fashion. Among them, some semi-aquatic  Aeschynomene  species present the distinctive feature to form nitrogen-fixing nodules on both roots and stems following elicitation by photosynthetic bradyrhizobia that do not produce Nod factors. This interaction is believed to represent a living testimony of the ancestral state of the rhizobium-legume symbiosis. To decipher the molecular mechanisms of this unique Nod-independent nitrogen-fixing symbiosis, we previously identified  A. evenia  C. Wright as an appropriate model legume, because it displays all the requisites for molecular and genetic approaches. To advance the use of this new model legume species, here we characterized the intraspecific diversity found in  A. evenia . For this, the accessions available in germplasm banks were collected and subjected to morphological investigations, genotyping with RAPD and SSR markers, molecular phylogenies using ITS and single nuclear gene sequences, and cross-compatibility tests. These combined analyses revealed an important intraspecific differentiation that led us to propose a new taxonomic classification for  A. evenia  comprising two subspecies and four varieties. The  A. evenia  ssp.  evenia  contains var.  evenia  and var.  pauciciliata  whereas  A. evenia  ssp.  serrulata  comprises var.  serrulata  and var.  major . This study provides information to exploit efficiently the diversity encountered in  A. evenia  and proposes subsp.  evenia  as the most appropriate subspecies for future projects aimed at identifying plant determinants of the Nod-independent symbiotic process.

Genotype Delimitation in the Nod-Independent Model Legume Aeschynomene evenia