Horizontal gene transfer and homologous recombination drive the evolution of the nitrogen-fixing symbionts of Medicago species

Abstract

Using nitrogen-fixing Sinorhizobium species that interact with Medicago plants as a model system, we aimed at clarifying how sex has shaped the diversity of bacteria associated with the genus Medicago on the interspecific and intraspecific scales. To gain insights into the diversification of these symbionts, we inferred a topology that includes the different specificity groups which interact with Medicago species, based on sequences of the nodulation gene cluster. Furthermore, 126 bacterial isolates were obtained from two soil samples, using Medicago truncatula and Medicago laciniata as host plants, to study the differentiation between populations of Sinorhizobium medicae, Sinorhizobium meliloti bv. meliloti, and S. meliloti bv. medicaginis. The former two can be associated with M. truncatula (among other species of Medicago), whereas the last organism is the specific symbiont of M. laciniata. These bacteria were characterized using a multilocus sequence analysis of four loci, located on the chromosome and on the two megaplasmids of S. meliloti. The phylogenetic results reveal that several interspecific horizontal gene transfers occurred during the diversification of Medicago symbionts. Within S. meliloti, the analyses show that nod genes specific to different host plants have spread to different genetic backgrounds through homologous recombination, preventing further divergence of the different ecotypes. Thus, specialization to different host plant species does not prevent the occurrence of gene flow among host-specific biovars of S. meliloti, whereas reproductive isolation between S. meliloti bv. meliloti and S. medicae is maintained even though these bacteria can cooccur in sympatry on the same individual host plants.

FIG. 1.

Locations of the genetic markers used in this study on the genome of Sinorhizobium meliloti strain 1021. The four loci used to perform the MLSA study are framed by dotted lines, and their locations are indicated by dotted lines. Gene clusters located near each genetic marker are indicated by black boxes. It is noteworthy that the IGS_NOD marker is located near genes involved in symbiotic specificity (nod genes), symbiotic efficiency (nif/fix genes), secretion (virB gene), and conjugation (tra genes). The locations of the partial sequences of the nodABC and nodEG genes used to obtain a supertree are indicated above the schematic representation of the symbiotic gene area.

FIG. 2.

Bayesian supertree obtained from nodABC-nodEG gene cluster. Bipartition support was assessed using the posterior probabilities of the different clades. Letters refer to the allelic diversity described for the IGS_NOD marker in both this study and that of Bailly et al. (1). The topology indicates that symbionts of the genus Medicago cluster in a monophyletic group. Within this group, four clades, which are indicated by black lines, include the following: (i) R. mongolense/R. gallicum bv. orientale isolates; (ii) S. meliloti bv. medicaginis isolates; (iii) isolates which cluster in a group called S. meliloti ecotype NRR, i.e., bacteria associated with Medicago noeana, M. rigiduloides, and M. radiata; and (iv) S. medicae isolates. Conversely, S. meliloti bv. meliloti, which is indicated by a gray line, is paraphyletic due to the position of the S. medicae clade.

FIG. 3.

Distributions of several measures illustrating the dissimilarity between population pairs belonging to either S. meliloti bv. meliloti, S. meliloti bv. medicaginis, or S. medicae. Data are provided independently for each of the four loci used for the MLSA. (A) Averages of the genetic distances among individuals belonging to two different bacterial groups (i.e., π_s), with 95% confidence intervals. (B) Ratios of the average genetic distances among individuals within and between S. meliloti biovars (i.e., π_d/π_s). (C) Percentages of allelic richness specific to S. meliloti bv. meliloti and S. meliloti bv. medicaginis, indicated by black and white areas, respectively. Striped areas illustrate the percentages of allelic richness shared by pairs of taxa. (D) Distribution of pairwise F_ST values between S. meliloti biovars. NS, **, and ***, differentiation test P values above a 5% threshold, below a 1% threshold, and below a 0.1% threshold, respectively.

FIG. 4.

Maximum likelihood trees obtained for each of the four markers used in the MLSA scheme. The number of occurrences of each allele of the data set is indicated in parentheses. Alleles belonging to either S. medicae, S. meliloti bv. medicaginis, or S. meliloti bv. meliloti isolates are framed by solid lines, a dotted line, and a dashed-dotted line, respectively, if they group in a clade. A double-dash symbol on a branch indicates that the branch is not drawn to scale (see Fig. 3 for more information about genetic divergence between bacterial groups). S. medicae alleles cluster in a clade on all phylogenies. This is consistent with the hypothesis of sexual isolation between S. meliloti and S. medicae. Conversely, isolates belonging to either S. meliloti bv. meliloti or S. meliloti bv. medicaginis do not cluster in monophyletic groups in all phylogenies except that for IGS_NOD. The incongruence among the four topologies suggests that recombination occurred among S. meliloti biovars.