Reproductive bacteroids pay a direct fitness opportunity cost when they respire carbon to support nitrogen fixation rather than using it to support their own immediate reproduction, or hoarding it as PHB to support future reproduction (Ratcliff et al., 2008). Neither of these options is available to nonreproductive bacteroids, so we might expect rhizobial cheaters to be less common when bacteroids are nonreproductive. If this is true, are host sanctions still needed in these legume species? Are sanctions still possible, if bacteroids will have no descendants anyway?
1. Should hosts with nonreproductive bacteroids invest in sanctions?
We define sanctions as some action by the host plant that reduces the relative fitness of less beneficial rhizobia based on their low rate or low efficiency of nitrogen fixation. Reduced nodulation, perhaps as a result of incorrect recognition signals, would be an example of partner choice rather than sanctions (Kiers & Denison, 2008). Also, sanctions are defined by their effects on rhizobial fitness, not host fitness. However, selection among legumes will only maintain sanctions if individual plants that impose sanctions thereby increase their fitness. This legume fitness benefit need not depend on how a given legume response affects rhizobial evolution. Note that rhizobial cheaters may mimic recognition signals of mutualists or retain signals from mutualistic ancestors.
We previously showed theoretically that individual plants would benefit from reducing resource consumption in nodules containing less mutualistic rhizobia (West et al., 2002a). From the plant's point of view, this is simply an efficient use of resources. From the point of view of rhizobia, however, this preferential resource allocation could function as a sanction, reducing the relative fitness of less mutualistic strains.
If nonreproductive bacteroids have few cheating options, then legumes hosting dimorphic rhizobia may encounter less beneficial rhizobia less often than legumes that host reproductive bacteroids. For example, < 13% of Sinorhizobium meliloti and Rhizobium leguminosarum strains tested had rhizopine genes (Wexler et al., 1995). However, given the other possible cheating options discussed above, it might be premature to conclude that cheating is rare when bacteroids are nonreproductive.
Furthermore, even if rhizobia with nonreproductive bacteroids are rarely cheaters, that is, strains that potentially benefit from fixing less nitrogen, legumes may still face considerable variation in symbiont quality. Variation in symbiont quality could occur if rhizobia that are less beneficial to particular hosts arise as a result of conflicting selection imposed by different host genotypes (Heath & Tiffin, 2007). There could also be significant numbers of mutants that retain the ability to nodulate but are defective in nitrogen fixation, without necessarily benefiting from fixing less nitrogen (Denison & Kiers, 2004). Given such variation, legumes would benefit from preferential allocation of resources to the most effective rhizobia (West et al., 2002a), whether or not less effective bacteroids are actually cheating.
2. Among-bacteroid versus among-nodule sanctions
If even those legumes that suppress bacteroid reproduction have encountered less beneficial rhizobia often enough to select for some host response, what sort of response should we expect? The response favored by natural selection among legumes is not necessarily the one that most favors the evolution of rhizobial mutualism.
If mixed-strain nodules are rare, a plant could increase its nitrogen return per carbon investment by preferentially allocating more carbon to the best performing bacteroids, regardless of whether this allocation is among bacteroids within a nodule or among nodules. If mixed nodules are common, then legumes would benefit more from among-bacteroid allocation than from among-nodule allocation (Denison, 2000). With among-nodule allocation, some resources could be wasted on less beneficial bacteroids within better performing nodules. This is true whether or not bacteroids are reproductive.
Reduced carbon allocation to less mutualistic rhizobia might be achieved by a host-imposed limitation on some other resource, such as amino acids (Lodwig et al., 2003) or oxygen (Kiers et al., 2003), limiting carbon use indirectly. We expect most legume species to have at least one mechanism for preferentially allocating resources to the subset of rhizobia in their nodules that provide the most nitrogen, relative to their carbon consumption.
We referred to ‘allocation’ above to leave open the question of whether a given legume response constitutes a ‘sanction’, that is, whether it imposes selection for greater rhizobial mutualism. Differences in allocation by legumes among nonreproductive bacteroids will have no direct effect on rhizobial evolution, although it is conceivable that there could be an indirect effect. For example, a nonreproductive bacteroid that receives more carbon because it fixes more nitrogen might then send more carbon to its reproductive clonemates via rhizopines. This assumes that making fewer rhizopines was not the reason for its superior nitrogen fixation.
We do not know whether among-bacteroid allocation is physiologically possible, but this could be tested by comparing fitness of marked effective vs ineffective isogenic strains in mixed nodules. Among-nodule allocation would provide some efficiency benefit to the plant even if some nodules were mixed. Differential allocation among nodules would presumably affect whichever rhizobia are reproductive in those nodules. Whether or not it is the bacteroids that are reproductive, differential allocation to better performing nodules would favor rhizobial mutualism most if mixed nodules were rare. As discussed above, natural selection among legumes would not usually depend on how their responses to differences among bacteroids or nodules affect rhizobial evolution.
3. Testing for sanctions by comparing rhizobial strains
When we ask whether a legume host imposes sanctions, we are asking whether they respond to differences among strains in actual symbiotic performance, for example nitrogen return on carbon investment, in ways that reduce the fitness of less beneficial strains. We know that legumes also respond to recognition signals during initial infection and perhaps later. In this paper, we are not considering the extent to which such responses to signals, as opposed to responses to actual performance, are adaptive for legumes.
There are two different types of evidence suggesting that legume species that suppress bacteroid reproduction may also impose sanctions on poorly performing strains. So far, however, each approach has been implemented in ways that limit our ability to draw firm conclusions about these species. One approach is to compare rhizobial strains, to see whether those that provide substantially less benefit to a given host also obtain less benefit from that host, as would be expected if that host imposes sanctions. A second approach is to manipulate a strain's nitrogen fixation rate by adjusting nitrogen concentration.
One problem with comparing strains is that differences in rhizobial fitness could be a result of some host-strain interaction other than performance-based sanctions. For example, a strain that fixes nitrogen very efficiently (i.e. nitrogen fixed per carbon respired) in a given host might also have signal molecules or surface antigens that limit nodulation or nodule growth in that host. A comparison with another strain that fixes nitrogen less efficiently but forms more nodules or reproduces better in nodules (as a result of signaling interactions, not performance-based sanctions) could incorrectly be interpreted as evidence against host sanctions. Comparing a larger number of strains would reduce the chance of drawing incorrect conclusions from idiosyncratic associations between signals and symbiotic performance.
Comparing benefits provided and obtained by different strains is also more difficult than generally recognized. It is easy to compare the growth of plants each inoculated with a single strain of rhizobia. However, growth with single-strain inoculation is an imperfect proxy for differences in mutualism among strains under field conditions, where each plant is infected by multiple strains. With single-strain inoculation, a poorly nodulating strain with high nitrogen-fixation efficiency might result in less plant growth than a less efficient strain that forms more nodules. Plant growth with single-strain inoculation would incorrectly identify this as a less beneficial strain. In the field, however, the poorly nodulating strain might only occupy 10% of the nodules on a plant, while providing 20% of the nitrogen. A better method might be to compare the growth of plants with two different strains in their nodules, in different proportions. The more beneficial strain is the one that increases plant growth as its nodule representation increases. A benefit:cost ratio would also be a better measurement to assess a strain's symbiotic qualities. For example, the ratio of nitrogen fixation rate to respiration rate of a single nodule or the ratio of host shoot biomass to total nodule mass would be a more accurate evaluation of the strain's symbiotic quality, independent of differences in nodulation efficiency. After all, in the real world, no one strain makes the majority of nodules on a single host.
Measuring differences among strains in rhizobial fitness benefits from symbiosis is also nontrivial. Nodule number from single-strain inoculation is not an appropriate proxy for rhizobial fitness. With single-strain inoculation, the number of nodules formed may depend on whether a strain can suppress the plant's autoregulation of nodule number, for example by producing rhizobitoxine, which blocks ethylene signaling (Sugawara et al., 2006). A rhizobitoxine producer may increase the total number of nodules but not its own proportional representation (Ratcliff & Denison, 2009). Competitiveness for nodulation in the field depends on survival under field conditions and potentially on hostile interactions among rhizobia, such as through production of bacteriocins (Schwinghamer & Brockwell, 1978), neither of which is measured in single-strain inoculation assays. However, the number of nodules per plant in single-strain inoculation can be used to estimate a plant benefit per nodule, somewhat alleviating the problem discussed in the previous paragraph.
Nodule size is widely measured and more promising as a proxy for rhizobial fitness. In soybean, which hosts reproductive bacteroids, much of the difference in rhizobia per nodule with sanctions was explained by nodule weight (Kiers et al., 2003). Similarly, Heath & Tiffin (2007) reported a positive correlation (r = 0.59) between reproductive rhizobia (determined by plate counts) and nodule length in Medicago truncatula. However, the relationship between nodule size and the number of reproductive rhizobia inside may not always be comparable among different strains. Therefore, even if nodules containing cheaters have lower average sizes, this does not guarantee that they have fewer reproductive cells per nodule. Given this uncertainty, reliance on nodule size as a proxy for rhizobial fitness seems unnecessarily risky. We suggest actually counting viable rhizobia per nodule, which can be done using agar plates with an appropriate growth medium. We also recommend measuring PHB per cell, using flow cytometry, because rhizobial cells can accumulate enough PHB to support doubling or tripling of their numbers (Ratcliff et al., 2008). In light of our concerns about single-strain inoculation and nodule size, we hesitate to draw firm conclusions from most of the published data, which are somewhat contradictory.
Miller & Sirois (1982) measured growth of four alfalfa cultivars with each of five rhizobial strains. They found a high correlation (r = 0.78) between average nodule weight (perhaps representative of rhizobial benefits) and average yield per plant with single-strain inoculation (perhaps representative of host benefits) (Fig. 3). For this data set, the correlation was similar if plant benefit from rhizobia was corrected for nodules per plant. This is the same sort of correlation seen in soybean (Kiers & Denison, 2008), for which we have direct experimental evidence for sanctions (Kiers et al., 2003). However, we cannot exclude the possibility that healthier alfalfa plants simply support larger nodules (in addition to or instead of more nodules per plant). This could be true even if alfalfa has no ability to selectively support the best nodules within a plant.
Preferential host support of the best performing nodules can be detected using mixed-inoculation experiments. For example, Singleton & Stockinger (1983) found that soybean nodules containing nitrogen-fixing rhizobia were 2.5 times as big as nodules on the same plant that contained an ineffective strain. Heath & Tiffin (2009) also used mixed inoculation, testing three rhizobial strains on four M. truncatula families. Nodule length varied fourfold, but was not correlated with estimates of genotype-specific host benefits from each strain. This was interpreted as evidence against sanctions. However, host benefits of each strain were measured in a previous experiment that used single-strain inoculation. With that approach, differences in total nitrogen fixation per plant among strains may be attributable to differences in nodulation, rather than differences in nitrogen fixation efficiency or rate per rhizobial cell.
Another important point is that a given set of strains may not include any whose nitrogen fixation rate is low enough to trigger sanctions, even in hosts that do impose sanctions on the worst strains. Therefore, to use comparisons among rhizobia to test whether a given legume genotype can impose sanctions, one should include a nodulating but nonfixing strain. Comparing this nonfixing control with an isogenic fixing strain could reduce the chance of drawing incorrect conclusions about sanctions because of idiosyncratic signaling interactions. Whether rhizobia that are ineffective enough to trigger sanctions are common (or rare, as suggested by Heath and Tiffin for their field locations) is a separate question. Plant alleles for sanctions that are never used would tend to disappear, however, unless they serve some other function.
Given uncertainties about using nodule size as a proxy for rhizobial fitness and plant growth with single-strain inoculation as a proxy for rhizobial mutualism, as discussed above, we consider the results of Miller & Sirois (1982) to be weak evidence for sanctions in alfalfa and the results of Heath & Tiffin (2009) to be weak evidence against sanctions in M. truncatula. It seems likely that the apparent discrepancy between these two studies is a consequence of deficiencies in methods, rather than a fundamental difference between these related legume species.
These same methodological concerns also apply to studies of two other legume species that host dimorphic rhizobia. Nutman (1946) reported that some rhizobia that are ineffective on clover nonetheless produce large nodules. More recently, Laguerre et al. (2007) found rhizobia that produce very large nodules on pea, but result in less plant growth than other strains. Neither report determined whether these large nodules actually contained large numbers of reproductive rhizobia.
4. Manipulating nitrogen fixation to test for sanctions
Given the problems discussed in the previous section, we favor testing for sanctions by manipulating nitrogen fixation rate rather than comparing rhizobial genotypes that may differ in various ways. We do this by reducing the nitrogen concentration around one or more nodules, thereby limiting nitrogen fixation. Our approach also allows us to vary nitrogen fixation quantitatively and over time.
In soybean, which hosts reproductive bacteroids, nonfixing nodules (in Ar:O2) grew less and contained fewer viable rhizobia per nodule (Kiers et al., 2003). Intermediate nitrogen concentrations, allowing intermediate rates of nitrogen fixation, led to intermediate sanctions (Kiers et al., 2006). We have used both single-nodule chambers and split-root chambers, with similar results.
We are currently conducting similar experiments using alfalfa and also plan to test other species hosting nonreproductive bacteroids. Meanwhile, there is some indirect evidence that host sanctions may be widespread. Minchin et al. (1983) found a physiologically similar response to decreasing nitrogen fixation in a variety of legume species, including some that host nonreproductive bacteroids. Blocking nitrogen fixation in nodulated roots (using either an nitrogen-free Ar:O2 atmosphere or acetylene, a competitive inhibitor of nitrogenase activity) triggered a decrease in nodule respiration rate, subsequently linked to a decrease in nodule O2 permeability. Although the fitness effects of decreased respiration for dimorphic rhizobia have not been measured, the decreased nodule O2 permeability is the same response we saw in soybean (Kiers et al., 2003). It therefore seems plausible, although far from certain, that sanctions may operate in a wide variety of legume species, by similar mechanisms.