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In the legume–rhizobia symbiosis, one such ancient system, we find two different rhizobial life histories, which depend on the species of legume host. In nodules of some hosts, including alfalfa (Medicago sativa) and peanut (Arachis hypogaea), rhizobia that differentiate into nitrogen-fixing bacteroids undergo major transformations, including swelling or branching and sometimes amplification of the bacterial genome (Mergaert et al., 2006). As discussed later, this extreme differentiation most likely prevents bacteroids from resuming normal cell division (free-living states), even if they are released from nodules during senescence (Sutton & Paterson, 1983). The next generation of symbiotic rhizobia for these hosts is presumably descended from rhizobia that had reproduced within the same nodules but not yet differentiated into bacteroids. By contrast, in hosts such as cowpea (Vigna unguiculata) and birdsfoot trefoil (Lotus corniculatus), the rhizobia still undergo differentiation into bacteroids but this process is not as irreversible. Bacteroids in these hosts are less swollen and have no genome amplification and, therefore, continue to reproduce after leaving the nodules (Mergaert et al., 2006).
The effect of legume host species on bacteroid differentiation was studied extensively by Sutton & Paterson (1980, 1983), but specific plant mechanisms were unknown and only a handful of closely related species were investigated. Currently, it is widely accepted that the size and shape of nitrogen-fixing bacteroids vary widely and are controlled by the legume host rather than the genotype of the rhizobia (Oke & Long, 1999). For example, a single rhizobial strain will differentiate into spherical swollen bacteroids in peanut but remain rod-shaped in cowpea (Sen et al., 1986). Similarly, Mergaert et al. (2006) has shown that recombinant rhizobial strains will transform into different bacteroid morphologies depending on the host species; transgenic rhizobia, which never evolved with one host, nonetheless showed the same level of bacteroid differentiation as the host’s wild type rhizobia.
Extreme bacteroid differentiation has recently been shown in Medicago truncatula to be imposed by nodule-specific cysteine rich (NCR) plant peptides (Van de Velde et al., 2010). These compounds have properties similar to antimicrobial defensins that block bacterial cell division, often causing genomic endoreduplication and alteration of cell shape (Latch & Margolin, 1997). Differentiation may also involve extreme alteration of the synthesis of peptidoglycan, an elastic polymer of bacterial cell walls known to regulate osmotic pressure and cell shape (Lam et al., 2009). The NCR peptide-coding sequences are also present in closely related genera of Medicago that host swollen bacteroids, but not in Lotus japonicus, Phaseolus vulgaris or Glycine max (Alunni et al., 2007 and references therein), those species hosting nonswollen bacteroids.
Similarly, the correlation between swelling and loss of bacteroid reproductive viability has been consistent among many tested species, such as G. max (Gresshoff & Rolfe, 1978; Zhou et al., 1985), P. vulgaris (W. C. Ratcliff unpublished), Macroptilium atropurpureum (W. C. Ratcliff unpublished), L. japonicus (Müller et al., 2001), Trifolium repens (Zhou et al., 1985) and M. sativa (McRae et al., 1989; Vasse et al., 1990; Ratcliff et al., 2008). Although Khetmalas & Bal (2005) characterized rod-shaped rhizobia inside senescing Arachis pintoi nodules as dedifferentiated formerly spherical bacteroids, the preponderance of evidence suggests that swollen bacteroids rarely dedifferentiate back into free-living forms and that it is the undifferentiated cells that repopulate the soil for future symbiosis. Bacteroid morphology is therefore considered a reasonable proxy for reproductive viability until we find a more effective means of observing bacteroids in their natural states to see how they either dedifferentiate and reproduce or are broken down (by plant, rhizobial, or exogenous enzymes).
Some previous studies also correlated bacteroid type to nodule type, although it has not been shown that this correlation is universal. Medicago, Pisum, Vicia and Trifolium (closely related species in the Inverse-Repeat Legume Clade (IRLC)) all have indeterminate nodule types (those with persistent meristems) and swollen bacteroids, whereas Glycine, Phaseolus, Macroptilium and Lotus all have determinate nodule types (transient meristems) and nonswollen bacteroids. It has been observed in many determinate nodules (within Phaseoloid and Dalbergioid clades) that the rhizobia-infected cells divide and enlarge along with the rhizobia inside them (Chandler et al., 1982; Sprent & Thomas, 1984), whereas host cells of indeterminate nodules (IRLC) are each infected with rhizobia from a branch of the infection thread and do not divide further, and neither do the infecting rhizobia (Sprent & Thomas, 1984).
While we have begun to understand the relationship between bacteroid swelling and loss of reproductive viability, we have little understanding of how widely distributed swollen bacteroids are in the legume family. We also know little about the evolutionary effects of swollen bacteroids on the legume–rhizobia interaction. Selection pressures for symbiotic strategies may differ between bacteroid morphologies with implications for rhizobial evolution. For example, when bacteroids themselves are reproductive, hoarding high-energy lipid polymers, such as polyhydroxybutyrate (PHB), inside their cells at the expense of nitrogen fixation may have a direct benefit to the bacteroid fitness. Conversely, when bacteroids are nonreproductive, it is the undifferentiated rhizobia that must reap the benefits of the mutualism in ways that may interact with nitrogen fixation by the bacteroids. Rhizopines, for example, are simple sugar-like compounds produced (possibly by diverting resources from nitrogen fixation) by some swollen bacteroids and catabolized by the undifferentiated rhizobia (Murphy et al., 1995). Only rhizobial strains commonly found in legume species hosting swollen bacteroids are known to possess rhizopine-synthesis genes (Wexler et al., 1995), suggesting that rhizobia with evolutionary dead-end bacteroids may have evolved this alternative strategy for cheating.
Furthermore, if rhizobial cheating strategies or intensities vary, the optimal level of host sanctions (Kiers et al., 2003) may also vary among legume species. Without host-imposed selection, rhizobia could lose the ability to fix nitrogen (West et al., 2002). However, host sanctions have not been adequately demonstrated in legume species with nonreproductive bacteroids and other mutualistic systems have shown that levels of sanction can vary among species (Jander & Herre, 2010). In the legume–rhizobia system, the exact mechanisms of host sanctions are still unknown, although limiting oxygen diffusion into nodules may play some role (Kiers et al., 2003) and reduce the fitness of the undifferentiated reproductive rhizobia inside (Oono et al., 2009). However, at present, we have no evidence that sanctions are universal or that sanctions always work at the whole-nodule level. One could conceive of symbiosome-level sanctions that only target bacteroids or that sanctions differ physiologically between species with nonreproductive and reproductive bacteroids. Given that host-imposed swelling of bacteroids may affect the coevolution of legume–rhizobium symbiosis, we explore here the taxonomic distribution of this trait and its possible evolutionary history in the legume phylogeny. By mapping the life-history characteristics of the rhizobia onto the host phylogeny, ecophylogenetic hypotheses can be tested to understand how the interaction evolved (Armbruster, 1992). This has been previously done in studies pertaining to the evolution of nonmutualists among mutualist lineages (Pellmyr et al., 1996; Hibbett et al., 2000) as well as new life-history adaptations among partner lineages, such as fragrance-collecting vs resin-collecting bee pollination (Armbruster, 1992) or dioecy vs monoecy in figs pollinated by fig-wasps (Weiblen, 2000; Greef & Compton, 2002; Harrison & Yamamura, 2003). In our study, we characterized bacteroid swelling in various legume hosts, to test whether host traits leading to swollen bacteroids were gained or lost among different lineages of legumes. This could indicate whether there are benefits or costs to legume hosts with this trait. Our specific questions are: are host traits that cause swollen bacteroids ancestral or derived? How common is bacteroid swelling beyond the well-studied model legume species and can we assess a broader phylogenetic pattern? We also tested whether there is correlated evolution between nodule type and bacteroid type over a wider set of legume species.