BACKGROUND
specialized biotic interactions contribute to processes that structure communities
<cit>  and adapt populations to local partners and environments
<cit> .
but, why do specialists exist if a generalist organism can occupy more environments and partake in more beneficial interactions
<cit> ?
the “jack-of-all-trades is a master of none” hypothesis asserts that specialists persist in a heterogeneous environment because the fitness of a generalist utilizing a particular habitat is lower than that of a specialist adapted to that habitat
<cit> .
this hypothesis is based on the assumptions that adapting to new habitats involves fitness costs and that traits adaptive in one subset of environments are negatively genetically correlated with traits adaptive in other environments
<cit> , leading to local adaptation
<cit> .
when the habitat is a host or interaction partner, this trade-off could lead to co-adaptation
<cit> .
fitness feedbacks between plants and specialized soil-based microbes significantly affect plant productivity, community composition, and the distribution and abundance of plants
<cit> .
strong plant-soil feedbacks are often negative; due to antagonistic interactions between plants and pathogens
<cit> .
however, many plants engage in mutualistic interactions with soil-borne symbionts
<cit> .
is there sufficient specialization among plant hosts to soil-borne mutualistic symbionts to impact plant community structure and diversity via positive feedbacks?
multiple arguments predict that mutualists interacting with partners acquired from the environment are likely to be generalists
<cit> .
since mutualists have higher fitness when partnered than when not, any partner should be better than no partner.
if so, then selection should disfavor specialized mutualists  that resist associating with common partners
<cit> .
similarly, specialized mutualistic lineages should be vulnerable to perturbations in partner populations
<cit>  and the geographic distribution and spread of specialized mutualists could be limited by partner availability
<cit> .
generalized mutualists might also access a wider range of environmental conditions by utilizing partners with differing ecological tolerances
<cit> .
finally, evolutionary convergence of traits among mutualists within an interaction group
<cit> , but see
<cit> , could reduce variance in partner benefit and thereby weaken the effectiveness of selection to specialize
<cit> .
specialized mutualists nonetheless exist, e.g.
<cit> .
indeed, meta-analysis of bipartite interactions found that mutualistic webs are more specialized than antagonistic ones
<cit> .
further, mutualism theory predicts that, when available partners vary in quality, selection favors mechanisms by which mutualists can select partners
<cit> , which can lead to specialization.
however, if more-beneficial partners are rare or distributed unpredictably, then selection for specialization via partner choice might be weak
<cit> .
how frequently symbiotic mutualistic interactions are specialized is therefore an open question.
here, we examined specialization in a group of wild legumes that interact with bradyrhizobium genotypes in coastal sand dunes of sonoma county, california.
bradyrhizobium is a genus of rhizobia that can infect legume roots and fix nitrogen endosymbiotically within root nodules in an interaction that is generally mutualistic
<cit> .
dominant genes that either restrict nodulation with or alter the effectiveness of particular rhizobial genotypes have been described in both natural and managed plant populations
<cit> , but the effect of such genes on fitness feedbacks has been little studied.
moreover, little is known about whether legume-rhizobium interactions are sufficiently specialized to contribute to structuring communities via plant-soil feedbacks or coevolution.
we quantified the realized richness and phylogenetic breadth of rhizobial symbionts collected from plants growing in situ, and measured the frequency with which each host species associated with each of the identified bradyrhizobium genotypes.
association frequencies quantify the interaction
<cit>  or link
<cit>  strength of each pairwise interaction and were used to calculate the paired differences index pdi
<cit> , for each host.
this index summarizes variation in a partner’s link strengths
<cit>  with all available partners without making any assumptions about their statistical distribution.
realized association specificity
<cit>  is strongly influenced by the local sampling environment
<cit> .
therefore, we experimentally paired partners under controlled greenhouse conditions to examine fundamental association frequency
<cit>  of two host species.
we also used the greenhouse experiment to quantify the fitness effect of each host on the inoculated bradyrhizobium genotypes, which is a measure of the host’s potential impact on the rhizobium population and therefore its functional role within the community
<cit> .
finally, we used these data to test whether this system supports the “jack-of-all-trades is a master of none” hypothesis, as well as its underlying assumption that, as frequently interacting partners become co-adapted, they become less adapted to other partners and consequently exchange weaker benefits.
RESULTS
isolate collection and sequence datasets
we isolated bradyrhizobium bacteria from nodules of four native california legumes growing on the bodega dunes at bodega marine reserve and sonoma coast state park, sonoma county.
two species, acmispon strigosus and lupinus bicolor, are fast-growing annuals that are patchily distributed across the bodega dunes.
in contrast, a. heermannii is a decumbent suffrutescent perennial and l. arboreus is a large upright perennial shrub, both widely distributed across the dunes.
two dna regions  were pcr amplified and sequenced from two to eight isolates from each plant, which yielded  <dig> sequenced isolates, including  <dig> nifd amplicons and  <dig> its amplicons .
sequences that differed by one nucleotide were considered distinct, resulting in  <dig> its genotypes, of which  <dig> occurred in multiple nodules, and  <dig> nifd genotypes, of which  <dig> occurred in multiple nodules.
there were  <dig> unique genotype combinations.
realized in situ association specificity
the observed pdis of the four hosts  all differed significantly from the expected joint pdi  and from each other, which indicates that each of the four hosts specialized on a subset of the available nifd types, but differed in degree of specialization; l. bicolor was most specialized.
genetic breadth of symbiotic partners
in both gene networks, lupinus hosts associated with a genetically broader range of bacteria than did acmispon hosts.
isolates from within a host genus tended to cluster together  and exhibited little overlap between host genera.
host genera shared no genotypes in the nifd network .
a single common its genotype  was shared between host genera and one genotype from lupinus  clustered closely to most genotypes from acmispon .
<cit> .
its region, based on the distance calculated by modeltest using a hky + i + g model of sequence evolution
<cit> .
dotted lines represent the position of a genotype on the network.
each box represents one genotype, each symbol represents a host species , and numbers indicate the number of strains representing a particular genotype; labels t <dig> to t <dig> in italics represent the bradyrhizobium genotypes used in the greenhouse experiment .
one genotype, t <dig>  appears only in the its network, because we were unable to sequence its nifd locus.
genotypes t <dig> and t <dig> do not figure in either network because they were addenda from another study, used for reference, but not part of the original sample.
genetic distance matrices among bacterial communities
bacterial populations differed significantly between the two host genera but not between species within a genus.
for both dna regions, significantly large Φst values and corrected between-population mismatch rates occurred between bradyrhizobium populations isolated from different host genera, but not between populations isolated from different host species within a genus .
however, bacteria isolated from lupinus were more genetically variable than those isolated from acmispon.
for both dna regions, the average pairwise sequence divergences between bradyrhizobium genotypes from lupinus were two- to five-fold greater than they were between genotypes from acmispon .
bradyrhizobium
sampled from
in situ
nodules
significance, obtained from  <dig> permutations: ns, not significant; *, p <  <dig> ; significant values in boldface.
sequence divergence , corrected between-population differences , and between-population Φst .
distribution of bacterial genetic variance
in a two-level hierarchical amova , neither its nor nifd sequence explained a significant component of variance between host species within genus.
most genetic variance occurred among plants within species  and between host genera .
permutations suggest that the variance component due to host genus was not significant ; even though genera harbored distinct genotypes in both networks  and all inter-genus values of pairwise genetic differences and Φst were statistically significant for both dna regions .
this discrepancy probably arises from a lack of power in the amova, which treats genus as a random effect, even though it is associated with only one degree of freedom.
its
and
nifd
sequences from
bradyrhizobium
from
in situ
nodules
significance: ns, not significant; ***, p <  <dig> .
sampled host species: a. strigosus, a. heermannii, l. bicolor and l. arboreus.
fundamental association specificity
in the greenhouse inoculation experiment, neither test host exhibited fundamental association specificity; each could nodulate all tested bradyrhizobium genotypes .
g × g interaction
on average, inoculation increased host shoot dry weight over that of uninoculated control plants.
the average increase was  <dig> -fold for a. strigosus  and  <dig> -fold for l. bicolor .
however, bacterial genotypes differed significantly in their effects on shoot dry weight .
notably, several genotypes failed to improve host growth beyond that of uninoculated controls .
<dig> and
<dig>
the conservative analysis presented above does not test for between-host differences in response specificity, which is typically detected as a significant interaction between test host species and bradyrhizobium genotype g × g interaction
<cit> , in an anova including data from both host species.
in a less conservative analysis, host by genotype  interactions were prominent in the best fitting model .
a significant g × g interaction of test host with bradyrhizobium groups isolated from different host species , occurs because variation in response to different bradyrhizobium genotypes was large in a. strigosus and almost absent in l. bicolor .
further, the g × g interaction due to test host species by bradyrhizobium genotype nested within species of origin  accounted for  <dig> % ±  <dig> % of the variance in plant dry weight.
this significant interaction indicates that even within a bradyrhizobium group isolated from the same host species, genotypes differed in the symbiotic benefits they provided to the two different test host species.
as expected from the conservative analysis, test host species differed significantly in dry weight  and groups of bradyrhizobium genotypes isolated from different host species differed significantly in average effect on test host shoot dry weight .
in particular, shoot dry weight produced by genotypes isolated from l. bicolor differed from that produced by genotypes isolated from other species .
when averaged across the test hosts, there was no significant variance in shoot dry weight due to inoculation with different bradyrhizobium genotypes isolated from the same host species  covariance parameter estimate ± 1 s. e. =  <dig>  ±  <dig> ; additional file
7: table s7).
the fold increase in growth of inoculated plants over uninoculated controls was strongly affected by the interaction of test host with source host .
further, the planned contrast comparing the fold increase in fitness obtained from bradyrhizobium genotypes isolated from conspecifics versus heterospecifics was highly significant .
both main effects were statistically significant , but the mean growth effect on each host species changed rank between conspecific and heterospecific pairings .
response specificity, as indicated by the paired difference indices calculated from within-block growth responses stimulated by the different bradyrhizobium genotypes, differed significantly between host species and was more than three-fold greater in a. strigosus  than in l. bicolor .
g × g interaction
bradyrhizobium fitness was estimated from nodule number, total nodule area, and area of the average nodule.
both nodule number and total nodule area differed significantly among bradyrhizobium genotypes on each host .
on a. strigosus, over 30% of variance in total nodule area was due to bradyrhizobium genotype: genotypes isolated from congeners produced significantly greater total nodule area than did those isolated from lupines.
ten percent of variance in nodule number was due to bradyrhizobium genotype: genotypes isolated from congeners produced slightly fewer nodules than did those isolated from lupines.
seventy percent of variance in average individual nodule area was due to bradyrhizobium genotype: genotypes isolated from congeners produced significantly larger nodules.
on l. bicolor, only 15% of variance in total nodule area was explained by bradyrhizobium genotype.
although genotypes isolated from congeners produced significantly greater nodule area, the difference was slight.
bradyrhizobium genotype explained almost 15% of variance in nodule number, but there was no significant difference between genotypes isolated from congeners versus acmispon species.
less than 20% of variance in the average individual nodule area on l. bicolor was due to bradyrhizobium genotype.
genotypes isolated from congeners produce marginally significantly larger nodules than did those isolated from acmispon hosts.
paired difference indices  calculated from host effect on bradyrhizobium fitness under non-competitive conditions of single inoculations is presented in table
<dig>  when measured in terms of nodule number, a. strigosus was significantly more specialized than l. bicolor, whereas pdi of the two hosts did not differ when calculated in terms of total nodule area.
bradyrhizobium
fitness components
l. bicolor
a. strigosus
*natural log transformed.
†square-root transformed.
for each statistic, lcl = lower 95% confidence limit and ucl = upper 95% confidence limit; diff pdi = pdi difference between species.
variables that differed significantly between hosts are in bold face.
association between bacterial genotypes and phenotypes
the matrices of pairwise phenotypic differences in a. strigosus shoot dry weight and average nodule area were each significantly associated with pairwise genetic differences at the nifd locus; association with the its region was not significant for shoot dry weight and marginal for average nodule area.
traits measured on l. bicolor exhibited no significant associations .
r
p
significance, estimated from  <dig>  permutations: ns, not significant; *, p <  <dig> ; **, p <  <dig> ; ***, p <  <dig> .
fitness feedback between host and symbiont
for a. strigosus, there was an overall positive relationship between total nodule area and host dry weight  <  <dig> ).
bradyrhizobium genotypes that were more beneficial for a. strigosus also produced on that host fewer nodules  that were larger .
a weak positive relationship between total nodule area and host dry weight  <  <dig> ) suggests a weaker but still positive fitness feedback between l. bicolor and bradyrhizobium symbionts.
in l. bicolor, there was no relationship between host dry weight and nodule number  >  <dig> ).
missing samples and cross-contamination
nodule data were lost from 14 l. bicolor plants.
seven out of  <dig> control plants had nodules, indicating cross-contamination .
these plants  were removed from analyses.
cross-contamination probably occurred late in the experiment because the nodulated control plants were still very small and had formed only a few small nodules.
we are confident that these potential cross contaminations do not undermine our conclusions, since any broader cross contamination would only reduce phenotypic differences observed between bradyrhizobium genotypes.
discussion
association richness and phylogenetic niche breadth
when growing wild, all four legume species that we examined specialized on subsets of the nodulating community of bradyrhizobium genotypes.
however, accounting for genetic breadth of symbionts in wild-collected nodules shifted the ranking of host specialization.
when bradyrhizobium genotype was ignored , l. bicolor ranked as most specialized and l. arboreus as most generalized.
however, bradyrhizobium isolates from wild-collected nodules were genetically clustered by host genus and each lupinus species hosted a phylogenetically broader group of symbionts than did either acmispon species.
clustering was strongest when isolates were categorized by nifd genotypes, which suggests that hosts respond to trait loci located on the bradyrhizobium symbiosis island.
the observation that species in acmispon exhibit greater realized association specificity than do those in lupinus agrees with previous surveys of wild-collected nodules from plants in these genera
<cit> .
however, neither l. bicolor nor a. strigosus exhibited fundamental association specificity in greenhouse inoculation tests: both hosts could nodulate all tested bradyrhizobium strains.
a gap between fundamental and realized association frequency is not uncommon
<cit>  and indicates that environmental context strongly influences nodulation.
the complex biotic community within natural soil could influence a genotype’s nodulation success
<cit> .
for example, soil pathogens could trigger systemic acquired resistance in the host, which might affect nodulation by some rhizobial genotypes but not others
<cit> .
the availability in soil of multiple rhizobial genotypes also provides scope for plants to actively prefer certain genotypes.
host-symbiont recognition signaling is a well-studied feature of legume-rhizobium interactions
<cit> .
in particular, it has been hypothesized that, during nodule formation and prior to nitrogen fixation, plants can recognize and prefer more-beneficial genotypes via pre-infection partner choice; reviewed in
<cit> .
it is unclear however, whether this host ability would be evolutionarily durable, as it requires genetic linkage between at least three sets of genes in two different organisms .
such linkage could be stable under very specific conditions, but could also be broken by any incident of recombination
<cit> .
in several legume-rhizobium systems, including a. strigosus <cit> , multiple-genotype inoculation experiments have found that more-beneficial and/or co-evolved genotypes are more likely to occupy nodules
<cit> .
however, none of these studies could definitively distinguish plant choice from interactions among rhizobia, which could be due to varying abilities to compete for soil resources
<cit> , withstand direct interference by other genotypes
<cit> , colonize the rhizosphere
<cit> , and utilize a range of rhizosphere resources
<cit> .
in experiments with bradyrhizobium strains collected from our site, neither in vitro growth rate nor interstrain interference was correlated with nodulation rate on a. strigosus <cit> , but these and other hypotheses should be further tested in more complex conditions where bacterial traits not directly related to symbiont effectiveness might influence nodulation ability.
realized association frequency might also be restricted by patchily distributed plant and symbiont genotypes
<cit> , which could be caused by coevolution
<cit>  and/or soil heterogeneity.
in the bodega dunes, two episodes of holocene dune advance
<cit>  left contrasting soils that might structure both plant and bradyrhizobium populations: l. bicolor is restricted to mid-holocene dunes whereas a. strigosus and a. heermannii occur only on poorly stabilized late-holocene dunes .
in contrast, l. arboreus occurs across dunes of both ages
<cit> .
we specifically sampled nodules from l. arboreus across its habitat range to control for potential confounding of host and bradyrhizobium distribution; indeed, l. arboreus nodules harbored genotypes from across each gene network.
however, the genotypic composition of isolates from l. bicolor did not differ significantly from those isolated from l. arboreus, which suggests that soil habitat is not the only determinant of genotype in wild-collected nodules of lupinus.
response and effect specificity, fitness trade-offs, and fitness feedback
the “jack-of-all-trades is a master of none” hypothesis was supported by evidence that specialist a. strigosus obtained more fitness benefit from its genetically narrower group of beneficial symbionts than generalist l. bicolor obtained from its genetically broader group of compatible symbionts.
as predicted by the co-adaptation hypothesis, each host received greater fitness benefit from genotypes isolated from congeners or conspecifics .
although the sample sizes used to estimate fitness are not large, this pattern of response specificity has been detected in several studies of unmanaged legumes
<cit>  and suggests that symbiont effectiveness may be increased by host-symbiont coevolution
<cit> .
a. strigosus plants were highly specialized in their fitness effect on the tested bradyrhizobium genotypes, which is consistent with previous evidence that this host imposes absolute sanctions
<cit>  on less effective genotypes
<cit> .
there was much less variance among bradyrhizobium genotypes in the benefits they obtained from l. bicolor.
also supporting the co-adaptation hypothesis, genotypes of bradyrhizobium were better adapted to hosts related to those from which their ancestors were isolated.
this pattern was strongest on a. strigosus: genotypes isolated from congeneric hosts produced greater total nodule area and therefore likely yielded more progeny than did genotypes isolated from lupines.
as a consequence, symbiont benefits to a. strigosus fitness positively fed back to symbiont fitness via increased allocation to nodules: genotypes beneficial to this host realized greater fitness benefits than did those that were not.
an overall positive correlation between nifd genotypic mean values of total nodule area and host shoot dry weight on a. strigosus suggests that strong positive fitness feedback between mutualists in this partnership
<cit>  is driven by traits encoded in the symbiosis island.
this feedback pattern was much weaker on the generalist, l. bicolor, and was not significantly correlated with its genotype.
positive partner feedback can produce positive frequency dependence and drive the most beneficial partners to local fixation
<cit> , a process that has been predicted for other legumes that exhibit symbiont specificity
<cit> .
much stronger positive fitness feedback might allow specialist a. strigosus to outcompete generalist l. bicolor in local patches
<cit> .
however, if l. bicolor were a better colonizer, which is expected of generalists
<cit> , then it might persist in a spatially patchy environment through a competition-colonization trade-off
<cit> .
in that case, a. strigosus and l. bicolor would not coexist at a local scale, but both might persist at a larger spatial scale via patch dynamics.
this process could also spatially structure bradyrhizobium populations
<cit> .
indeed, wild a. strigosus plants were found in association with a genetically narrow range of bradyrhizobium genotypes that were most beneficial to their growth, suggesting that these genotypes dominate bradyrhizobium populations in areas inhabited by this host.
in contrast, as predicted from the weaker correlations between plant and rhizobial fitness components on l. bicolor, wild l. bicolor plants associated with a genetically wider range of bradyrhizobium genotypes that were not necessarily the most beneficial.
CONCLUSIONS
a field survey detected differences in partner specificity among four sampled host species and greenhouse experiments revealed that at this site a. strigosus clearly specializes on a genetically narrower range of symbionts than does l. bicolor.
the fitness benefits that these two hosts received from symbiotic partners affirmed two important tenets of evolutionary theory.
first, as predicted by the jack-of-all-trades is a master of none hypothesis, specialist a. strigosus obtained greater benefit from its genetically narrow group of beneficial symbionts than generalist l. bicolor obtained from a genetically more diverse group.
second, as predicted by the co-adaptation hypothesis
<cit> , each test species performed better with partner genotypes isolated from conspecifics.
further, host fitness benefit translated directly into symbiont fitness via increased allocation to nodules: nodules occupied by genotypes isolated from conspecific hosts received the greatest benefit.
these patterns were strongest in a. strigosus.
positive fitness feedback between this specialized host and its co-adapted symbionts could drive positive plant-soil feedback and contribute to natural distribution patterns observed in the field.

 specialized biotic interactions contribute to processes that structure communities and adapt populations to local partners and environments. but, why do specialists exist if a generalist organism can occupy more environments and partake in more beneficial interactions? the “jack-of-all-trades is a master of none” hypothesis asserts that specialists persist in a heterogeneous environment because the fitness of a generalist utilizing a particular habitat is lower than that of a specialist adapted to that habitat. this hypothesis is based on the assumptions that adapting to new habitats involves fitness costs and that traits adaptive in one subset of environments are negatively genetically correlated with traits adaptive in other environments, leading to local adaptation. when the habitat is a host or interaction partner, this trade-off could lead to co-adaptation. fitness feedbacks between plants and specialized soil-based microbes significantly affect plant productivity, community composition, and the distribution and abundance of plants. strong plant-soil feedbacks are often negative; due to antagonistic interactions between plants and pathogens. however, many plants engage in mutualistic interactions with soil-borne symbionts. is there sufficient specialization among plant hosts to soil-borne mutualistic symbionts to impact plant community structure and diversity via positive feedbacks? multiple arguments predict that mutualists interacting with partners acquired from the environment are likely to be generalists. since mutualists have higher fitness when partnered than when not, any partner should be better than no partner. if so, then selection should disfavor specialized mutualists that resist associating with common partners. similarly, specialized mutualistic lineages should be vulnerable to perturbations in partner populations and the geographic distribution and spread of specialized mutualists could be limited by partner availability. generalized mutualists might also access a wider range of environmental conditions by utilizing partners with differing ecological tolerances. finally, evolutionary convergence of traits among mutualists within an interaction group, but see, could reduce variance in partner benefit and thereby weaken the effectiveness of selection to specialize. specialized mutualists nonetheless exist, e.g.. indeed, meta-analysis of bipartite interactions found that mutualistic webs are more specialized than antagonistic ones. further, mutualism theory predicts that, when available partners vary in quality, selection favors mechanisms by which mutualists can select partners, which can lead to specialization. however, if more-beneficial partners are rare or distributed unpredictably, then selection for specialization via partner choice might be weak. how frequently symbiotic mutualistic interactions are specialized is therefore an open question. here, we examined specialization in a group of wild legumes that interact with bradyrhizobium genotypes in coastal sand dunes of sonoma county, california. bradyrhizobium is a genus of rhizobia that can infect legume roots and fix nitrogen endosymbiotically within root nodules in an interaction that is generally mutualistic. dominant genes that either restrict nodulation with or alter the effectiveness of particular rhizobial genotypes have been described in both natural and managed plant populations, but the effect of such genes on fitness feedbacks has been little studied. moreover, little is known about whether legume-rhizobium interactions are sufficiently specialized to contribute to structuring communities via plant-soil feedbacks or coevolution. we quantified the realized richness and phylogenetic breadth of rhizobial symbionts collected from plants growing in situ, and measured the frequency with which each host species associated with each of the identified bradyrhizobium genotypes. association frequencies quantify the interaction or link strength of each pairwise interaction and were used to calculate the paired differences index pdi, for each host. this index summarizes variation in a partner’s link strengths with all available partners without making any assumptions about their statistical distribution. realized association specificity is strongly influenced by the local sampling environment. therefore, we experimentally paired partners under controlled greenhouse conditions to examine fundamental association frequency of two host species. we also used the greenhouse experiment to quantify the fitness effect of each host on the inoculated bradyrhizobium genotypes, which is a measure of the host’s potential impact on the rhizobium population and therefore its functional role within the community. finally, we used these data to test whether this system supports the “jack-of-all-trades is a master of none” hypothesis, as well as its underlying assumption that, as frequently interacting partners become co-adapted, they become less adapted to other partners and consequently exchange weaker benefits.  isolate collection and sequence datasets we isolated bradyrhizobium bacteria from nodules of four native california legumes growing on the bodega dunes at bodega marine reserve and sonoma coast state park, sonoma county. two species, acmispon strigosus and lupinus bicolor, are fast-growing annuals that are patchily distributed across the bodega dunes. in contrast, a. heermannii is a decumbent suffrutescent perennial and l. arboreus is a large upright perennial shrub, both widely distributed across the dunes. two dna regions were pcr amplified and sequenced from two to eight isolates from each plant, which yielded sequenced isolates, including nifd amplicons and its amplicons. sequences that differed by one nucleotide were considered distinct, resulting in its genotypes, of which occurred in multiple nodules, and nifd genotypes, of which occurred in multiple nodules. there were unique genotype combinations. realized in situ association specificity the observed pdis of the four hosts all differed significantly from the expected joint pdi and from each other, which indicates that each of the four hosts specialized on a subset of the available nifd types, but differed in degree of specialization; l. bicolor was most specialized. genetic breadth of symbiotic partners in both gene networks, lupinus hosts associated with a genetically broader range of bacteria than did acmispon hosts. isolates from within a host genus tended to cluster together and exhibited little overlap between host genera. host genera shared no genotypes in the nifd network. a single common its genotype was shared between host genera and one genotype from lupinus clustered closely to most genotypes from acmispon.. its region, based on the distance calculated by modeltest using a hky + i + g model of sequence evolution. dotted lines represent the position of a genotype on the network. each box represents one genotype, each symbol represents a host species, and numbers indicate the number of strains representing a particular genotype; labels t to t in italics represent the bradyrhizobium genotypes used in the greenhouse experiment. one genotype, t appears only in the its network, because we were unable to sequence its nifd locus. genotypes t and t do not figure in either network because they were addenda from another study, used for reference, but not part of the original sample. genetic distance matrices among bacterial communities bacterial populations differed significantly between the two host genera but not between species within a genus. for both dna regions, significantly large Φst values and corrected between-population mismatch rates occurred between bradyrhizobium populations isolated from different host genera, but not between populations isolated from different host species within a genus. however, bacteria isolated from lupinus were more genetically variable than those isolated from acmispon. for both dna regions, the average pairwise sequence divergences between bradyrhizobium genotypes from lupinus were two- to five-fold greater than they were between genotypes from acmispon. bradyrhizobium sampled from in situ nodules significance, obtained from permutations: ns, not significant; *, p < ; significant values in boldface. sequence divergence, corrected between-population differences, and between-population Φst. distribution of bacterial genetic variance in a two-level hierarchical amova, neither its nor nifd sequence explained a significant component of variance between host species within genus. most genetic variance occurred among plants within species and between host genera. permutations suggest that the variance component due to host genus was not significant; even though genera harbored distinct genotypes in both networks and all inter-genus values of pairwise genetic differences and Φst were statistically significant for both dna regions. this discrepancy probably arises from a lack of power in the amova, which treats genus as a random effect, even though it is associated with only one degree of freedom. its and nifd sequences from bradyrhizobium from in situ nodules significance: ns, not significant; ***, p < . sampled host species: a. strigosus, a. heermannii, l. bicolor and l. arboreus. fundamental association specificity in the greenhouse inoculation experiment, neither test host exhibited fundamental association specificity; each could nodulate all tested bradyrhizobium genotypes. g × g interaction on average, inoculation increased host shoot dry weight over that of uninoculated control plants. the average increase was -fold for a. strigosus and -fold for l. bicolor. however, bacterial genotypes differed significantly in their effects on shoot dry weight. notably, several genotypes failed to improve host growth beyond that of uninoculated controls. and the conservative analysis presented above does not test for between-host differences in response specificity, which is typically detected as a significant interaction between test host species and bradyrhizobium genotype g × g interaction, in an anova including data from both host species. in a less conservative analysis, host by genotype interactions were prominent in the best fitting model. a significant g × g interaction of test host with bradyrhizobium groups isolated from different host species, occurs because variation in response to different bradyrhizobium genotypes was large in a. strigosus and almost absent in l. bicolor. further, the g × g interaction due to test host species by bradyrhizobium genotype nested within species of origin accounted for % ±  % of the variance in plant dry weight. this significant interaction indicates that even within a bradyrhizobium group isolated from the same host species, genotypes differed in the symbiotic benefits they provided to the two different test host species. as expected from the conservative analysis, test host species differed significantly in dry weight and groups of bradyrhizobium genotypes isolated from different host species differed significantly in average effect on test host shoot dry weight. in particular, shoot dry weight produced by genotypes isolated from l. bicolor differed from that produced by genotypes isolated from other species. when averaged across the test hosts, there was no significant variance in shoot dry weight due to inoculation with different bradyrhizobium genotypes isolated from the same host species covariance parameter estimate ± 1 s. e. =  ± ; additional file 7: table s7). the fold increase in growth of inoculated plants over uninoculated controls was strongly affected by the interaction of test host with source host. further, the planned contrast comparing the fold increase in fitness obtained from bradyrhizobium genotypes isolated from conspecifics versus heterospecifics was highly significant. both main effects were statistically significant, but the mean growth effect on each host species changed rank between conspecific and heterospecific pairings. response specificity, as indicated by the paired difference indices calculated from within-block growth responses stimulated by the different bradyrhizobium genotypes, differed significantly between host species and was more than three-fold greater in a. strigosus than in l. bicolor. g × g interaction bradyrhizobium fitness was estimated from nodule number, total nodule area, and area of the average nodule. both nodule number and total nodule area differed significantly among bradyrhizobium genotypes on each host. on a. strigosus, over 30% of variance in total nodule area was due to bradyrhizobium genotype: genotypes isolated from congeners produced significantly greater total nodule area than did those isolated from lupines. ten percent of variance in nodule number was due to bradyrhizobium genotype: genotypes isolated from congeners produced slightly fewer nodules than did those isolated from lupines. seventy percent of variance in average individual nodule area was due to bradyrhizobium genotype: genotypes isolated from congeners produced significantly larger nodules. on l. bicolor, only 15% of variance in total nodule area was explained by bradyrhizobium genotype. although genotypes isolated from congeners produced significantly greater nodule area, the difference was slight. bradyrhizobium genotype explained almost 15% of variance in nodule number, but there was no significant difference between genotypes isolated from congeners versus acmispon species. less than 20% of variance in the average individual nodule area on l. bicolor was due to bradyrhizobium genotype. genotypes isolated from congeners produce marginally significantly larger nodules than did those isolated from acmispon hosts. paired difference indices calculated from host effect on bradyrhizobium fitness under non-competitive conditions of single inoculations is presented in table  when measured in terms of nodule number, a. strigosus was significantly more specialized than l. bicolor, whereas pdi of the two hosts did not differ when calculated in terms of total nodule area. bradyrhizobium fitness components l. bicolor a. strigosus *natural log transformed. †square-root transformed. for each statistic, lcl = lower 95% confidence limit and ucl = upper 95% confidence limit; diff pdi = pdi difference between species. variables that differed significantly between hosts are in bold face. association between bacterial genotypes and phenotypes the matrices of pairwise phenotypic differences in a. strigosus shoot dry weight and average nodule area were each significantly associated with pairwise genetic differences at the nifd locus; association with the its region was not significant for shoot dry weight and marginal for average nodule area. traits measured on l. bicolor exhibited no significant associations. r p significance, estimated from permutations: ns, not significant; *, p < ; **, p < ; ***, p < . fitness feedback between host and symbiont for a. strigosus, there was an overall positive relationship between total nodule area and host dry weight  <  ). bradyrhizobium genotypes that were more beneficial for a. strigosus also produced on that host fewer nodules that were larger. a weak positive relationship between total nodule area and host dry weight  <  ) suggests a weaker but still positive fitness feedback between l. bicolor and bradyrhizobium symbionts. in l. bicolor, there was no relationship between host dry weight and nodule number  >  ). missing samples and cross-contamination nodule data were lost from 14 l. bicolor plants. seven out of control plants had nodules, indicating cross-contamination. these plants were removed from analyses. cross-contamination probably occurred late in the experiment because the nodulated control plants were still very small and had formed only a few small nodules. we are confident that these potential cross contaminations do not undermine our conclusions, since any broader cross contamination would only reduce phenotypic differences observed between bradyrhizobium genotypes. discussion association richness and phylogenetic niche breadth when growing wild, all four legume species that we examined specialized on subsets of the nodulating community of bradyrhizobium genotypes. however, accounting for genetic breadth of symbionts in wild-collected nodules shifted the ranking of host specialization. when bradyrhizobium genotype was ignored, l. bicolor ranked as most specialized and l. arboreus as most generalized. however, bradyrhizobium isolates from wild-collected nodules were genetically clustered by host genus and each lupinus species hosted a phylogenetically broader group of symbionts than did either acmispon species. clustering was strongest when isolates were categorized by nifd genotypes, which suggests that hosts respond to trait loci located on the bradyrhizobium symbiosis island. the observation that species in acmispon exhibit greater realized association specificity than do those in lupinus agrees with previous surveys of wild-collected nodules from plants in these genera. however, neither l. bicolor nor a. strigosus exhibited fundamental association specificity in greenhouse inoculation tests: both hosts could nodulate all tested bradyrhizobium strains. a gap between fundamental and realized association frequency is not uncommon and indicates that environmental context strongly influences nodulation. the complex biotic community within natural soil could influence a genotype’s nodulation success. for example, soil pathogens could trigger systemic acquired resistance in the host, which might affect nodulation by some rhizobial genotypes but not others. the availability in soil of multiple rhizobial genotypes also provides scope for plants to actively prefer certain genotypes. host-symbiont recognition signaling is a well-studied feature of legume-rhizobium interactions. in particular, it has been hypothesized that, during nodule formation and prior to nitrogen fixation, plants can recognize and prefer more-beneficial genotypes via pre-infection partner choice; reviewed in. it is unclear however, whether this host ability would be evolutionarily durable, as it requires genetic linkage between at least three sets of genes in two different organisms. such linkage could be stable under very specific conditions, but could also be broken by any incident of recombination. in several legume-rhizobium systems, including a. strigosus, multiple-genotype inoculation experiments have found that more-beneficial and/or co-evolved genotypes are more likely to occupy nodules. however, none of these studies could definitively distinguish plant choice from interactions among rhizobia, which could be due to varying abilities to compete for soil resources, withstand direct interference by other genotypes, colonize the rhizosphere, and utilize a range of rhizosphere resources. in experiments with bradyrhizobium strains collected from our site, neither in vitro growth rate nor interstrain interference was correlated with nodulation rate on a. strigosus, but these and other hypotheses should be further tested in more complex conditions where bacterial traits not directly related to symbiont effectiveness might influence nodulation ability. realized association frequency might also be restricted by patchily distributed plant and symbiont genotypes, which could be caused by coevolution and/or soil heterogeneity. in the bodega dunes, two episodes of holocene dune advance left contrasting soils that might structure both plant and bradyrhizobium populations: l. bicolor is restricted to mid-holocene dunes whereas a. strigosus and a. heermannii occur only on poorly stabilized late-holocene dunes. in contrast, l. arboreus occurs across dunes of both ages. we specifically sampled nodules from l. arboreus across its habitat range to control for potential confounding of host and bradyrhizobium distribution; indeed, l. arboreus nodules harbored genotypes from across each gene network. however, the genotypic composition of isolates from l. bicolor did not differ significantly from those isolated from l. arboreus, which suggests that soil habitat is not the only determinant of genotype in wild-collected nodules of lupinus. response and effect specificity, fitness trade-offs, and fitness feedback the “jack-of-all-trades is a master of none” hypothesis was supported by evidence that specialist a. strigosus obtained more fitness benefit from its genetically narrower group of beneficial symbionts than generalist l. bicolor obtained from its genetically broader group of compatible symbionts. as predicted by the co-adaptation hypothesis, each host received greater fitness benefit from genotypes isolated from congeners or conspecifics. although the sample sizes used to estimate fitness are not large, this pattern of response specificity has been detected in several studies of unmanaged legumes and suggests that symbiont effectiveness may be increased by host-symbiont coevolution. a. strigosus plants were highly specialized in their fitness effect on the tested bradyrhizobium genotypes, which is consistent with previous evidence that this host imposes absolute sanctions on less effective genotypes. there was much less variance among bradyrhizobium genotypes in the benefits they obtained from l. bicolor. also supporting the co-adaptation hypothesis, genotypes of bradyrhizobium were better adapted to hosts related to those from which their ancestors were isolated. this pattern was strongest on a. strigosus: genotypes isolated from congeneric hosts produced greater total nodule area and therefore likely yielded more progeny than did genotypes isolated from lupines. as a consequence, symbiont benefits to a. strigosus fitness positively fed back to symbiont fitness via increased allocation to nodules: genotypes beneficial to this host realized greater fitness benefits than did those that were not. an overall positive correlation between nifd genotypic mean values of total nodule area and host shoot dry weight on a. strigosus suggests that strong positive fitness feedback between mutualists in this partnership is driven by traits encoded in the symbiosis island. this feedback pattern was much weaker on the generalist, l. bicolor, and was not significantly correlated with its genotype. positive partner feedback can produce positive frequency dependence and drive the most beneficial partners to local fixation, a process that has been predicted for other legumes that exhibit symbiont specificity. much stronger positive fitness feedback might allow specialist a. strigosus to outcompete generalist l. bicolor in local patches. however, if l. bicolor were a better colonizer, which is expected of generalists, then it might persist in a spatially patchy environment through a competition-colonization trade-off. in that case, a. strigosus and l. bicolor would not coexist at a local scale, but both might persist at a larger spatial scale via patch dynamics. this process could also spatially structure bradyrhizobium populations. indeed, wild a. strigosus plants were found in association with a genetically narrow range of bradyrhizobium genotypes that were most beneficial to their growth, suggesting that these genotypes dominate bradyrhizobium populations in areas inhabited by this host. in contrast, as predicted from the weaker correlations between plant and rhizobial fitness components on l. bicolor, wild l. bicolor plants associated with a genetically wider range of bradyrhizobium genotypes that were not necessarily the most beneficial.  a field survey detected differences in partner specificity among four sampled host species and greenhouse experiments revealed that at this site a. strigosus clearly specializes on a genetically narrower range of symbionts than does l. bicolor. the fitness benefits that these two hosts received from symbiotic partners affirmed two important tenets of evolutionary theory. first, as predicted by the jack-of-all-trades is a master of none hypothesis, specialist a. strigosus obtained greater benefit from its genetically narrow group of beneficial symbionts than generalist l. bicolor obtained from a genetically more diverse group. second, as predicted by the co-adaptation hypothesis, each test species performed better with partner genotypes isolated from conspecifics. further, host fitness benefit translated directly into symbiont fitness via increased allocation to nodules: nodules occupied by genotypes isolated from conspecific hosts received the greatest benefit. these patterns were strongest in a. strigosus. positive fitness feedback between this specialized host and its co-adapted symbionts could drive positive plant-soil feedback and contribute to natural distribution patterns observed in the field. 
in the wild, each lupinus species hosted a broader genetic range of bradyrhizobium than did either acmispon species, suggesting that acmispon species are more specialized. lupinus bicolor benefited from a broader range of genotypes but averaged less benefit from each. second, as predicted by coevolutionary theory, each test species performed better with partner genotypes isolated from conspecifics. to address this question, we quantified realized partner richness and phylogenetic breadth of four wild-grown native legumes and performed inoculation trials to test the ability of two hosts to nodulate, benefit from, and provide benefit to bradyrhizobium genotypes. nevertheless, a. strigosus exhibited more specificity, i.e., greater variation in its response to, and effect on, bradyrhizobium genotypes. plant-soil feedbacks help to structure plant and microbial communities, but how frequently are soil-based symbiotic mutualistic interactions sufficiently specialized to influence species distributions and community composition? first, as predicted by the jack-of-all-trades is a master of none hypothesis, specialist a. strigosus obtained greater benefit from its beneficial symbionts than did generalist l. bicolor. in the greenhouse, however, l. bicolor and a. strigosus did not differ in fundamental association specificity: all inoculated genotypes nodulated both hosts. the “jack-of-all-trades is a master of none” hypothesis asserts that specialists persist because the fitness of a generalist utilizing a particular habitat is lower than that of a specialist adapted to that habitat. this study affirmed two important tenets of evolutionary theory. both hosts obtained more fitness benefit from symbionts isolated from conspecific hosts; those symbionts in turn gained greater fitness benefit from hosts of the same species from which they were isolated. finally, positive fitness feedback between the tested hosts and symbionts suggests that positive plant-soil feedback could contribute to their patchy distributions in this system.
 specialized interactions help structure communities, but persistence of specialized organisms is puzzling because a generalist can occupy more environments and partake in more beneficial interactions. the “jack-of-all-trades is a master of none” hypothesis asserts that specialists persist because the fitness of a generalist utilizing a particular habitat is lower than that of a specialist adapted to that habitat. yet, there are many reasons to expect that mutualists will generalize on partners. plant-soil feedbacks help to structure plant and microbial communities, but how frequently are soil-based symbiotic mutualistic interactions sufficiently specialized to influence species distributions and community composition? to address this question, we quantified realized partner richness and phylogenetic breadth of four wild-grown native legumes and performed inoculation trials to test the ability of two hosts to nodulate, benefit from, and provide benefit to bradyrhizobium genotypes.  in the wild, each lupinus species hosted a broader genetic range of bradyrhizobium than did either acmispon species, suggesting that acmispon species are more specialized. in the greenhouse, however, l. bicolor and a. strigosus did not differ in fundamental association specificity: all inoculated genotypes nodulated both hosts. nevertheless, a. strigosus exhibited more specificity, i.e., greater variation in its response to, and effect on, bradyrhizobium genotypes. lupinus bicolor benefited from a broader range of genotypes but averaged less benefit from each. both hosts obtained more fitness benefit from symbionts isolated from conspecific hosts; those symbionts in turn gained greater fitness benefit from hosts of the same species from which they were isolated.  this study affirmed two important tenets of evolutionary theory. first, as predicted by the jack-of-all-trades is a master of none hypothesis, specialist a. strigosus obtained greater benefit from its beneficial symbionts than did generalist l. bicolor. second, as predicted by coevolutionary theory, each test species performed better with partner genotypes isolated from conspecifics. finally, positive fitness feedback between the tested hosts and symbionts suggests that positive plant-soil feedback could contribute to their patchy distributions in this system. mutualismsymbiosisspecializationcoevolutionlupinus bicolor acmispon strigosus lotusalpha-proteobacterialegumesrhizobia 