Rising vapor-pressure deficit increases nitrogen fixation in a legume crop.

Atmospheric vapor-pressure deficit (VPD) is increasing in many regions and has a large impact on plant productivity. A VPD increase leads to raising transpiration rate (TR) and soil-water demand, risking productivity penalties. Like water, nitrogen is critical to productivity, but the effect of VPD on legume nitrogen fixation is undocumented. To address this, we developed a portable system for quantifying nitrogen fixation noninvasively and at a high temporal resolution by tracking the rate of hydrogen gas evolution by root nodules. Combining field and controlled-environment experiments where we measured leaf gas exchange and H2 production by nodules, we confirmed the ability of the system to track nitrogen fixation dynamics. Raising VPD from 0.5 to 3 kPa within c. 2.5 h under well-watered conditions increased nitrogen fixation by up to 25% in addition to TR, consistent with the hypothesis that raising VPD in that range might have alleviated nitrogenase feedback inhibition. Genotypic differences were found in this response, indicating a potential for breeding. Our study provides evidence for an important environmental effect on nitrogen fixation that is not taken into account in current crop and vegetation models, pointing to untapped avenues for better understanding climate change effects on legumes and nitrogen cycling.

The century experiment: the first twenty years of UC Davis' Mediterranean agroecological experiment.

The Century Experiment at the Russell Ranch Sustainable Agriculture Facility at the University of California, Davis provides long-term agroecological data from row crop systems in California's Central Valley starting in 1993. The Century Experiment was initially designed to study the effects of a gradient of water and nitrogen availability on soil properties and crop performance in ten different cropping systems to measure tradeoffs and synergies between agricultural productivity and sustainability. Currently systems include 11 different cropping systems-consisting of four different crops and a cover crop mixture-and one native grass system. This paper describes the long-term core data from the Century Experiment from 1993-2014, including crop yields and biomass, crop elemental contents, aerial-photo-based Normalized Difference Vegetation Index data, soil properties, weather, chemical constituents in irrigation water, winter weed populations, and operational data including fertilizer and pesticide application amounts and dates, planting dates, planting quantity and crop variety, and harvest dates. This data set represents the only known long-term set of data characterizing food production and sustainability in irrigated and rainfed Mediterranean annual cropping systems. There are no copyright restrictions associated with the use of this dataset.

Experimental evolution of multicellularity.

Multicellularity was one of the most significant innovations in the history of life, but its initial evolution remains poorly understood. Using experimental evolution, we show that key steps in this transition could have occurred quickly. We subjected the unicellular yeast Saccharomyces cerevisiae to an environment in which we expected multicellularity to be adaptive. We observed the rapid evolution of clustering genotypes that display a novel multicellular life history characterized by reproduction via multicellular propagules, a juvenile phase, and determinate growth. The multicellular clusters are uniclonal, minimizing within-cluster genetic conflicts of interest. Simple among-cell division of labor rapidly evolved. Early multicellular strains were composed of physiologically similar cells, but these subsequently evolved higher rates of programmed cell death (apoptosis), an adaptation that increases propagule production. These results show that key aspects of multicellular complexity, a subject of central importance to biology, can readily evolve from unicellular eukaryotes.

Disentangling direct and indirect fitness effects of microbial dormancy.

Disentangling individual selection from kin selection is one of the greatest challenges of evolutionary biology. Even solitary organisms that do not interact directly with conspecifics may interact indirectly with them through competition for resources. As a result, traits that appear to affect individual fitness alone can also modify the fitness of relatives nearby and thus may evolve partially through these cryptic indirect fitness effects. Here we develop a method to quantitatively separate direct and indirect fitness consequences when some microbes become dormant, while neighbors of the same genotype remain active. Dormant microbes typically survive stresses that kill metabolically active cells, but dormancy also has a social side effect, sparing resources that may be used by nondormant individuals for growth. In structured populations, spared resources may be preferentially consumed by nondormant clonemates, providing an indirect benefit. Without population structure, however, exploitation by a never-dormant competitor imposes an indirect fitness cost on dormant cells. Cryptic indirect fitness effects may play a significant role in the evolution of many ostensibly asocial traits.

An evolutionary perspective on increasing net benefits to crops from symbiotic microbes.

Plant-imposed, fitness-reducing sanctions against less-beneficial symbionts have been documented for rhizobia, mycorrhizal fungi, and fig wasps. Although most of our examples are for rhizobia, we argue that the evolutionary persistence of mutualism in any symbiosis would require such sanctions, if there are multiple symbiont genotypes per host plant. We therefore discuss methods that could be used to develop and assess crops with stricter sanctions. These include methods to screen strains for greater mutualism as resources to identify crop genotypes that impose stronger selection for mutualism. Single-strain experiments that measure costs as well as benefits have shown that diversion of resources by rhizobia can reduce nitrogen-fixation efficiency (N per C) and that some legumes can increase this efficiency by manipulating their symbionts. Plants in the field always host multiple strains with possible synergistic interactions, so benefits from different strains might best be compared by regressing plant growth or yield on each strain's abundance in a mixture. However, results from this approach have not yet been published. To measure legacy effects of stronger sanctions on future crops, single-genotype test crops could be planted in a field that recently had replicated plots with different genotypes of the sanction-imposing crop. Enhancing agricultural benefits from symbiosis may require accepting tradeoffs that constrained past natural selection, including tradeoffs between current and future benefits.

Failure to fix nitrogen by non-reproductive symbiotic rhizobia triggers host sanctions that reduce fitness of their reproductive clonemates.

The legume-rhizobia symbiosis is a classical mutualism where fixed carbon and nitrogen are exchanged between the species. Nonetheless, the plant carbon that fuels nitrogen (N(2)) fixation could be diverted to rhizobial reproduction by 'cheaters'--rhizobial strains that fix less N(2) but potentially gain the benefit of fixation by other rhizobia. Host sanctions can decrease the relative fitness of less-beneficial reproductive bacteroids and prevent cheaters from breaking down the mutualism. However, in certain legume species, only undifferentiated rhizobia reproduce, while only terminally differentiated rhizobial bacteroids fix nitrogen. Sanctions were, therefore, tested in two legume species that host non-reproductive bacteroids. We demonstrate that even legume species that host non-reproductive bacteroids, specifically pea and alfalfa, can severely sanction undifferentiated rhizobia when bacteroids within the same nodule fail to fix N(2). Hence, host sanctions by a diverse set of legumes play a role in maintaining N(2) fixation.

Comparing symbiotic efficiency between swollen versus nonswollen rhizobial bacteroids.

Symbiotic rhizobia differentiate physiologically and morphologically into nitrogen-fixing bacteroids inside legume host nodules. The differentiation is apparently terminal in some legume species, such as peas (Pisum sativum) and peanuts (Arachis hypogaea), likely due to extreme cell swelling induced by the host. In other legume species, such as beans (Phaseolus vulgaris) and cowpeas (Vigna unguiculata), differentiation into bacteroids, which are similar in size and shape to free-living rhizobia, is reversible. Bacteroid modification by plants may affect the effectiveness of the symbiosis. Here, we compare symbiotic efficiency of rhizobia in two different hosts where the rhizobia differentiate into swollen nonreproductive bacteroids in one host and remain nonswollen and reproductive in the other. Two such dual-host strains were tested: Rhizobium leguminosarum A34 in peas and beans and Bradyrhizobium sp. 32H1 in peanuts and cowpeas. In both comparisons, swollen bacteroids conferred more net host benefit by two measures: return on nodule construction cost (plant growth per gram nodule growth) and nitrogen fixation efficiency (H(2) production by nitrogenase per CO(2) respired). Terminal bacteroid differentiation among legume species has evolved independently multiple times, perhaps due to the increased host fitness benefits observed in this study.

Multiple evolutionary origins of legume traits leading to extreme rhizobial differentiation.

SUMMARY: *When rhizobia differentiate inside legume host nodules to become nitrogen-fixing bacteroids, they undergo a physiological as well as a morphological transformation. These transformations are more extreme in some legume species than others, leading to fundamental differences in rhizobial life history and evolution. Here, we analysed the distribution of different bacteroid morphologies over a legume phylogeny to understand the evolutionary history of this host-influenced differentiation. *Using existing electron micrographs and new flow cytometric analyses, bacteroid morphologies were categorized as swollen or nonswollen for 40 legume species in the subfamily Papilionoideae. Maximum likelihood and Bayesian frameworks were used to reconstruct ancestral states at the bases of all major subclades within the papilionoids. *Extreme bacteroid differentiation leading to swelling was found in five out of the six major papilionoid subclades. The inferred ancestral state for the Papilionoideae was hosting nonswollen bacteroids, indicating at least five independent origins of host traits leading to swollen bacteroids. *Repeated evolution of host traits causing bacteroid swelling indicates a possible fitness benefit to the plant. Furthermore, as bacteroid swelling is often correlated with loss of reproductive viability, the evolution of bacteroid cooperation or cheating strategies could be fundamentally different between the two bacteroid morphologies.

Host sanctions and the legume-rhizobium mutualism.

Explaining mutualistic cooperation between species remains one of the greatest problems for evolutionary biology. Why do symbionts provide costly services to a host, indirectly benefiting competitors sharing the same individual host? Host monitoring of symbiont performance and the imposition of sanctions on 'cheats' could stabilize mutualism. Here we show that soybeans penalize rhizobia that fail to fix N(2) inside their root nodules. We prevented a normally mutualistic rhizobium strain from cooperating (fixing N(2)) by replacing air with an N(2)-free atmosphere (Ar:O(2)). A series of experiments at three spatial scales (whole plants, half root systems and individual nodules) demonstrated that forcing non-cooperation (analogous to cheating) decreased the reproductive success of rhizobia by about 50%. Non-invasive monitoring implicated decreased O(2) supply as a possible mechanism for sanctions against cheating rhizobia. More generally, such sanctions by one or both partners may be important in stabilizing a wide range of mutualistic symbioses.

Do plant parts compete for resources? An evolutionary viewpoint.

Simultaneously growing sinks are thought to compete for plant resources. Negative correlations, for example between grain number and stem mass in cereals, indeed resemble competition; but is the notion of intra-plant competition evolutionarily justified? Here we review intra-plant competition in light of two aspects of evolutionary biology: (a) major transitions that led to the reorganization of evolutionary individuals (e.g. isolated DNA molecules and independent cells) into new units of adaptation (e.g. chromosomes and multicellular organisms) with associated constraints to intra-individual conflict; and (b) genomic conflicts within individual plants with implications for resource allocation. Against this background, we look at apparent competition among genetically identical plant parts, and conclude that plants might use competition-like mechanisms to allocate resources, but only to the extent that these proximate mechanisms enhance overall plant fitness. In dealing with apparent competition among genetically different plant structures, we emphasize developing seeds attached to the same maternal plant, and the determination of yield components in annual crops. We propose that competition-like mechanisms among genetically different plant parts have been strongly shaped by the evolution of genomic conflict between parent and offspring, between female and male parents, and among siblings. By defining the number and potential size of grain simultaneously and before fertilization, a strong maternal control of resource allocation is exerted that favours uniform offspring size and partially counteracts genomic conflict.

Controlling the reproductive fate of rhizobia: how universal are legume sanctions?

When a single host plant is infected by more than one strain of rhizobia, they face a tragedy of the commons. Although these rhizobia benefit collectively from nitrogen fixation, which increases host-plant photosynthesis, each strain might nonetheless increase its own reproduction, relative to competing strains, by diverting resources away from nitrogen fixation. Host sanctions can limit the evolutionary success of such rhizobial cheaters (strains that would otherwise benefit by fixing less nitrogen). Host sanctions have been shown in soybean (Glycine max) nodules, where the next generation of symbiotic rhizobia is descended from bacteroids (the differentiated cells that can fix nitrogen). Evidence for sanctions is less clear in legume species that induce rhizobial dimorphism inside their nodules. There, bacteroids are swollen and cannot reproduce regardless of how much nitrogen they fix, but sanctions could reduce reproduction of their undifferentiated clonemates within the same nodule. This rhizobial dimorphism can affect rhizobial evolution, including cheating options, in ways that may affect future generations of legumes. Both the importance of sanctions to hosts and possible physiological mechanisms for sanctions may depend on whether bacteroids are potentially reproductive.

Resource acquisition and allocation traits in symbiotic rhizobia with implications for life-history outside of legume hosts.

Resources that microbial symbionts obtain from hosts may enhance fitness during free-living stages when resources are comparatively scarce. For rhizobia in legume root nodules, diverting resources from nitrogen fixation to polyhydroxybutyrate (PHB) has been discussed as a source of host-symbiont conflict. Yet, little is known about natural variation in PHB storage and its implications for rhizobial evolution. We therefore measured phenotypic variation in natural rhizobia populations and investigated how PHB might contribute to fitness in the free-living stage. We found that natural populations of rhizobia from Glycine max and Chamaecrista fasciculata had substantial, heritable variation in PHB acquisition during symbiosis. A model simulating temperature-dependent metabolic activity showed that the observed range of stored PHB per cell could support survival for a few days, for active cells, or over a century for sufficiently dormant cells. Experiments with field-isolated Bradyrhizobium in starvation culture suggest PHB is partitioned asymmetrically in dividing cells, consistent with individual-level bet-hedging previously demonstrated in E. meliloti. High-PHB isolates used more PHB over the first month, yet still retained more PHB for potential long-term survival in a dormant state. These results suggest that stored resources like PHB may support both short-term and long-term functions that contribute to fitness in the free-living stage.

Human selection and the relaxation of legume defences against ineffective rhizobia.

Enforcement mechanisms are thought to be important in maintaining mutualistic cooperation between species. A clear example of an enforcement mechanism is how legumes impose sanctions on rhizobial symbionts that fail to provide sufficient fixed N2. However, with domestication and breeding in high-soil-N environments, humans may have altered these natural legume defences and reduced the agricultural benefits of the symbiosis. Using six genotypes of soya beans, representing 60 years of breeding, we show that, as a group, older cultivars were better able to maintain fitness than newer cultivars (seed production) when infected with a mixture of effective and ineffective rhizobial strains. Additionally, we found small differences among cultivars in the ratio of effective:ineffective rhizobia released from their nodules, an indicator of future rhizobial strain fitness. When infected by symbionts varying in quality, legume defences against poor-quality partners have apparently worsened under decades of artificial selection.

Ten-year comparison of the influence of organic and conventional crop management practices on the content of flavonoids in tomatoes.

Understanding how environment, crop management, and other factors, particularly soil fertility, influence the composition and quality of food crops is necessary for the production of high-quality nutritious foods. The flavonoid aglycones quercetin and kaempferol were measured in dried tomato samples (Lycopersicon esculentum L. cv. Halley 3155) that had been archived over the period from 1994 to 2004 from the Long-Term Research on Agricultural Systems project (LTRAS) at the University of California-Davis, which began in 1993. Conventional and organic processing tomato production systems are part of the set of systems compared at LTRAS. Comparisons of analyses of archived samples from conventional and organic production systems demonstrated statistically higher levels (P < 0.05) of quercetin and kaempferol aglycones in organic tomatoes. Ten-year mean levels of quercetin and kaempferol in organic tomatoes [115.5 and 63.3 mg g(-1) of dry matter (DM)] were 79 and 97% higher than those in conventional tomatoes (64.6 and 32.06 mg g(-1) of DM), respectively. The levels of flavonoids increased over time in samples from organic treatments, whereas the levels of flavonoids did not vary significantly in conventional treatments. This increase corresponds not only with increasing amounts of soil organic matter accumulating in organic plots but also with reduced manure application rates once soils in the organic systems had reached equilibrium levels of organic matter. Well-quantified changes in tomato nutrients over years in organic farming systems have not been reported previously.

Why are most rhizobia beneficial to their plant hosts, rather than parasitic?

Multiple strains per plant and root-to-root (not seed-borne) transmission should favor rhizobia that invest in their own reproduction, rather than symbiotic N2 fixation, as analogous factors may favor pathogen virulence. But legumes can select for greater mutualism, controlling nodule O2 supply and reducing reproduction of rhizobia that fix less N2.

Legume Sanctions and the Evolution of Symbiotic Cooperation by Rhizobia.

The legume-rhizobium symbiosis is an ideal model for studying the factors that limit the evolution of microbial mutualists into parasites. Legumes are unable to consistently recognize parasitic rhizobia that, once established inside plant cells, use plant resources for their own reproduction rather than for N2 fixation. Evolution of parasitism in rhizobia, driven partly by competition among multiple rhizobial strains infecting the same plant, may be countered by postinfection legume sanctions. Both the biochemical options for rhizobial cheating and the evolutionary effect of legume sanctions depend on differences in rhizobial life history associated with nodule type. In legumes with determinate nodule growth, rhizobia typically retain the ability to reproduce after differentiating into N2-fixing bacteroids. Sanctions against individual bacteroids (e.g., acid hydrolases) would therefore select for cooperative rhizobia. In nodules with indeterminate growth, bacteroids generally lose the ability to reproduce, so legume sanctions against bacteroids would have no effect on rhizobial evolution. Whole-nodule sanctions (e.g., decreased nodule O2 permeability) could be effective, via kin selection of undifferentiated rhizobia that persist in indeterminate nodules and replenish soil populations after nodule senescence. Mixed nodules could reduce the effectiveness of whole-nodule sanctions. The frequency of mixed nodules under field conditions is unknown.