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Ecological genomics of mutualism decline
in nitrogen-fixing bacteria
Christie R. Klinger1, Jennifer A. Lau2 and Katy D. Heath1
Research
Cite this article: Klinger CR, Lau JA, Heath
KD. 2016 Ecological genomics of mutualism
decline in nitrogen-fixing bacteria. Proc. R. Soc.
B 283: 20152563.
http://dx.doi.org/10.1098/rspb.2015.2563
Received: 2 December 2015
Accepted: 15 February 2016
Subject Areas:
evolution, genomics, ecology
Keywords:
symbiosis, mutualism, microbe,
deposition, adaptation, bacteria
1
Department of Plant Biology, University of Illinois Urbana-Champaign, 505 South Goodwin Avenue, Urbana,
IL 61801, USA
2
W.K. Kellogg Biological Station and Department of Plant Biology, Michigan State University, East Lansing,
MI, USA
KDH, 0000-0002-6368-744X
Anthropogenic changes can influence mutualism evolution; however,
the genomic regions underpinning mutualism that are most affected by
environmental change are generally unknown, even in well-studied model
mutualisms like the interaction between legumes and their nitrogen
(N)-fixing rhizobia. Such genomic information can shed light on the agents
and targets of selection maintaining cooperation in nature. We recently
demonstrated that N-fertilization has caused an evolutionary decline in
mutualistic partner quality in the rhizobia that form symbiosis with clover.
Here, population genomic analyses of N-fertilized versus control rhizobium
populations indicate that evolutionary differentiation at a key symbiosis
gene region on the symbiotic plasmid (pSym) contributes to partner quality
decline. Moreover, patterns of genetic variation at selected loci were consistent
with recent positive selection within N-fertilized environments, suggesting
that N-rich environments might select for less beneficial rhizobia. By studying
the molecular population genomics of a natural bacterial population within a
long-term ecological field experiment, we find that: (i) the N environment is
indeed a potent selective force mediating mutualism evolution in this symbiosis, (ii) natural variation in rhizobium partner quality is mediated in part by
key symbiosis genes on the symbiotic plasmid, and (iii) differentiation at
selected genes occurred in the context of otherwise recombining genomes,
resembling eukaryotic models of adaptation.
Author for correspondence:
Katy D. Heath
e-mail: kheath@illinois.edu
1. Introduction
Electronic supplementary material is available
at http://dx.doi.org/10.1098/rspb.2015.2563 or
via http://rspb.royalsocietypublishing.org.
Mutualisms play critical roles in natural and managed ecosystems. Recent work on
context-dependency in mutualisms has shown that changes in the economy of
benefits, such as a shift in the availability of an important traded resource in the
environment, can alter the symbiont community composition and/or the forms
of natural selection acting on mutualists, even causing the evolution of decreased
partner quality or abandonment of the mutualism [1–5]. Despite these recent
advances, however, we generally lack empirical evidence for the selective agents
that act on mutualist partner quality and thus how partner quality (co)evolves
in natural mutualist populations [6,7]. Moreover, few mutualist systems to date
have afforded both the ecological relevance and genomic resolution necessary to
study the mechanistic underpinnings of mutualism evolution.
The legume–rhizobium symbiosis, in which leguminous plants house
nitrogen (N)-fixing bacteria in root nodules, is responsible for the vast majority
of non-anthropogenically fixed N in terrestrial systems and thus plays a major
role in the N cycle [8,9]. This symbiosis has become a key model for mutualism
evolution, given the relative ease with which rhizobium genetic variants can be
isolated and subsequently manipulated in experiments to test key predictions
of evolutionary theory [10–14]. This recent boom in studies of phenotypic evolution in rhizobia, a long history of molecular genetic investigation resulting in
well-annotated reference genomes (e.g. [15,16]), and most recently the ability to
collect high-quality population genomic data with relative ease (e.g. [17–20]),
& 2016 The Author(s) Published by the Royal Society. All rights reserved.
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(a) Study system
Here, we studied genomic variation among 63 strains of
R. leguminosarum bv. trifolii, N-fixing rhizobial mutualists of
clover (Trifolium spp.), that were isolated and characterized
phenotypically by Weese et al. [5]; complete methods detailing
the long-term ecological experiment, strain isolation, and phenotypic experiments are contained there. Briefly, rhizobium strains
were isolated from both N-fertilized (22 years of fertilization with
(b) Whole genome sequencing and annotation
Strain isolates were grown on solid tryptone yeast (TY) media
plates, incubated at 308C until bacterial colonies became visible
(3– 4 days), and isolated strains were grown in 5 ml TY liquid
media [32] in a shaking incubator for 1 – 2 days at 308C. Genomic
DNA was extracted using the MasterPureTM DNA Purification
Kit (Epicentre). We used a NanoDropTM 2000c (Thermo Scientific, Waltham, MA, USA) to ensure that all samples had A260/
280 readings between 1.8 and 2.0. We generated multiplexed
libraries (no bead normalization step) using the Nextera XT
DNA Sample Prep Kit (Illumina, San Diego, CA, USA). Libraries
were quantified with a Qubit fluorometer 2.0 (Life Technologies,
Carlsbad, CA, USA) and then diluted with molecular grade
water for normalization. Normalized libraries were pooled for
paired end sequencing in a single lane on a HiSeq2000 sequencer
(Illumina) at the W.M. Keck Center for Comparative and
Functional Genomics at the University of Illinois at UrbanaChampaign. The lane produced 467 million reads, resulting in
an average of 720 megabase (MB) and 97 coverage per genome.
We used the A5 pipeline [33] with default parameters to automate sequence data cleaning, error correction, de novo assembly,
and quality control. Genomes were assembled de novo rather
than using reference-based assembly to avoid potential biases in
synteny and to better assess highly variable (i.e. flexible) genomic
content often found in bacteria [20,34,35]. A single control strain
(155_C) could not be assembled properly due to poor sequence
quality and was excluded from subsequent analyses. A portion
of gaps were filled in silico with GAPFILLER v. 1.11, which re-uses
paired reads to fill gaps left in draft assemblies [36]. We annotated
the genomes by uploading A5 assemblies to the ‘Rapid Annotation
using Subsystem Technology’ server [37]. Genes from annotated
assemblies were grouped into recognized homologues using the
ITEP toolkit [38], which uses the MCL program (Markov Chain
Clustering) to cluster BLASTp results (0.4 cut-off, 2.0 inflation
parameter [39]). Assembled genomes had a median length of
7.50 Mbp, a median scaffold number of 45, and a median N50
of 465 604 bp (electronic supplementary material, Dataset S2).
We defined the ‘core genome’ as orthologous, single-copy
ITEP gene clusters found within all strains and the reference
R. leguminosarum bv. trifolii strain WSM1325 [15]. The remaining (non-core) genes comprise the ‘flexible genome’ (genes
with variable copy number and/or shared among subsets of
strains). Analysis of the flexible genome revealed high variation in gene content among strains, but no candidates strongly
associated with partner quality decline (i.e. no genes were completely absent in control strains and present in all N-fertilized
strains or vice versa). See electronic supplementary material,
Dataset S3 and text S1, for full discussion of presence-absence
variation (PAV) results. Using an ITEP pipeline, core gene
sequences were aligned and reverse-translated with the auto
flag in MAFFT v. 7.123b [40] and PAL2NAL v. 14 [41], respectively.
The ITEP GBlocks wrapper script was used for automated alignment curation (-c 24 -f 24 -n 10 -m 5 -g a) for each core gene [42].
There were a total of 2 433 core (i.e. single copy, present in all
strains) genes among all 62 strains. For analyses using the 56
strains harbouring symbiosis genes on the symbiotic plasmid
( pSym) (see below), there were a total of 3 173 core genes.
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2. Material and methods
12.3 g N m22 per year granular ammonium nitrate) and control
(unfertilized) plots located at the Kellogg Biological Station
Long Term Ecological Research Site (KBS LTER; http://lter.kbs.
msu.edu/). Weese et al. [5] found that rhizobium populations
from the N-fertilization treatment had evolved to be lower
quality partners for their hosts (i.e. resulted in lower plant
above-ground biomass and chlorophyll content), compared
with rhizobium populations from control plots (see electronic
supplementary material, Dataset S1).
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all make N-fixing rhizobia ideal systems for addressing both
mechanistic questions of mutualism evolution and more
general questions about the mode of bacterial adaptation.
In a recent quantitative genetic study of Rhizobium
leguminosarum, rhizobium symbionts of clover, we demonstrated that rhizobia from field soil populations fertilized with
N for 22 years were, on average, less beneficial for their host
plants, compared to those from control populations [5]. In particular, rhizobium strains isolated from the N-fertilized plots
resulted in host plants with 17–30% less biomass, 10–28%
fewer leaves, and 6–17% lower leaf chlorophyll (depending
on the clover host species), supporting a major prediction of
mutualism theory—that changes in the resource environment
can lead to the evolutionary decline of mutualism benefits
[21–23]. The observed phenotypic differentiation in response
to long-term experimental N-fertilization, however, does not
address the rhizobium loci targeted by selection or the evolutionary mechanisms driving reduced rhizobium quality. For
example, although numerous genes required for symbiosis
with legumes have been characterized [24], whether those
same loci underlie naturally occurring partner quality variation
remains largely unknown [25]. Similarly, partner quality could
decline in high N environments if (i) positive selection has
favoured lower quality rhizobium strains (i.e. rhizobia have
adapted to sustained high N), or (ii) selection for high-quality
strains has been relaxed (i.e. strains have simply become
defective mutualists in response to sustained high N). These
alternative hypotheses point out a critical distinction between
defective mutualists, which are simply less beneficial for their
partners, versus true ‘cheaters’, which gain a fitness benefit
from being less beneficial [26,27]. The evolutionary importance
of low-quality partners to the dynamics of contemporary mutualism dynamics remains controversial [14,26,28–31]. Thus,
identifying whether selection favours cheating can shed light
on this important issue in mutualism evolution.
Using natural populations of mutualist bacteria that
have become phenotypically differentiated in response to a
manipulated variable (N fertilization) provides the rare opportunity to address the genetic underpinnings of mutualism
evolution in response to long-term changes in the resource
environment in a well-replicated manner. Here, we compare
the genome sequences of N-fertilized and control strains of
R. leguminosarum to investigate patterns of genomic variation
between N-fertilization treatments to identify the genomic
basis of mutualism decline in rhizobia. Next, we ask whether
patterns of nucleotide variation at putatively selected genes
suggest adaptation of rhizobium mutualists, versus relaxed
selection for mutualism, in high-resource environments. Our
study merges long-term ecological field experiments and
bacterial population genomics to answer key questions about
evolutionary mechanisms in nature.
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pR132501 (pSym)
pR132502
pR132503
pR132504
pR132505
0.20
0.15
FST
0.10
0.05
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0
−0.05
(b)
0.25
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FST
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(a)
chromosome
0.10
0.05
0
−0.05
0
1 000 000 2 000 000 3 000 000 4 000 000 5 000 000 6 000 000 7 000 000
genome position (bp)
Figure 1. Patterns of genetic structure (FST) and gene presence and absence across the genome for N-fertilized and control R. leguminosarum. (a) Gene-by-gene FST
between N-fertilized and control rhizobia for all (3 173) core genes using the subset of 56 strains possessing key symbiosis genes. The region of the symbiotic
plasmid ( pSym) (in pink) that houses symbiosis genes of interest (e.g. nif and nod genes) is highlighted in light blue. (b) Gene-by-gene FST between highand low-quality R. leguminosarum, independent of N treatment of origin, for all core genes in the 56-strain subset. Red circles indicate significant FST values
( p , 0.05); red triangles indicate six genes with significant FST in the top 1% of values in both FST analyses; the dark blue line denotes the 99th percentile;
the solid black line represents a 13 gene sliding window analysis of mean FST values, and the dashed lines represent average FST in the chromosome and plasmids.
(c) Genome-wide patterns of variation
To determine evolutionary relationships among the experimental
strains, we concatenated aligned core gene sequences on the chromosome and five plasmids, including the pSym. We used the GTR-G
model with 100 bootstrap replicates in RAxML v. 8.0.19 [43] to generate a core gene phylogeny and visualized the distribution of
partner quality on the tree by mapping mean trait values using
ITOL v. 2 [44]. To test whether nucleotide variation in the rhizobium
genome was structured geographically and/or between N fertilization treatments, we used Arlequin v. 3.5.1.2 [45] to carry out separate
hierarchical analyses of molecular variance (AMOVA) for core gene
single nucleotide polymorphisms (SNPs) on the chromosome (2 521
single-copy genes present in all strains) and the core plasmid
genomes (238, 195, 8, 113, and 98 genes in plasmids pR132501–
pR132505, respectively). Strain, plot within treatment (six plots per
N treatment), and N treatment were included as factors. The first
100 000 SNP sites were used for AMOVA on the chromosome
due to data input limitations of Arlequin. AMOVA on remaining
chromosomal SNP sites (data not shown) did not alter the results.
(d) Patterns of gene loss at a key symbiosis gene region
of the pSym
Many genes known to be critical for the formation and maintenance of symbiosis with legumes [46] exist within a symbiosis
island roughly centred at 0.303 Mega base pair (Mbp) on the
pSym of the reference strain, R. leguminosarum bv. trifolii
WSM1325 [15], and we were interested in testing whether
genes at this region were differentiated between N-fertilized
and control rhizobia. We found that six strains (four N-fertilized
strains, 40, 538, 643, and 773 and two control strains, 160 and 3)
lacked many of the genes in this region and that the average partner quality for these six strains was below the average for all
other strains (significantly so for plant biomass; t ¼ 22.048,
p ¼ 0.045; electronic supplementary material, Dataset S2). This
left a large gap in the core gene alignment at this critical region
of interest (‘Results’, and gap on the pSym in electronic supplementary material, figure S1 and figure 1 of text S1); therefore,
we used the 56 strains that possessed the region of the pSym in
population genetic analyses of the core genome to test for patterns
of differentiation at the symbiosis gene region of the pSym.
(e) Genetic differentiation between N-fertilized
and control strains
We expected that genes underlying phenotypic differentiation
between N-fertilized and control rhizobia should be genetically
differentiated (have elevated FST) between N-fertilized and control
groups, and also be associated with symbiotic partner quality
(i.e. differentiate low- and high-quality strains). Thus, we
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Forms of selection acting differently in N-fertilized (versus control) plots should generate distinct patterns of nucleotide
variation at candidate genes. If positive directional selection
has favoured low-quality strains in the N plots, for example,
N-fertilized strains should have reduced nucleotide diversity
( p) at candidate genes compared to control strains, relative to
the rest of the genome [48– 51]. Alternatively, relaxed selection
in the N plots should lead to increased p at candidate genes
compared to control strains. We calculated p within N-fertilized
and control populations for each core gene on the chromosome
or pSym using PROSEQ v. 3.5 [52] and then performed a sliding
window analysis of p as described above (394 windows
total). The other four plasmids were excluded due to a relative
deficiency of core genes with significantly elevated FST.
R v. 3.0.3 was used to plot the regression of sliding window
results for N-fertilized and control populations, and to generate
the 95% confidence and prediction intervals to identify regions
of the genome (sliding windows) with anomalous p.
3. Results and discussion
We found that the vast majority of genetic variation occurred
among individual strains (AMOVA; electronic supplementary
material, table S1), consistent with other bacterial populations that are quite variable even at fine spatial scales [53,54].
We also detected spatial genetic structure (significant amongplot variance; electronic supplementary material, table S1),
suggesting some limits to gene flow at a rather fine geographical scale in these rhizobia [55,56]. We found little evidence of
genome-wide differentiation between N-fertilized and control
rhizobia, as evidenced first by an intermixed core genome
phylogeny (electronic supplementary material, figure S2),
and second by the lack of between-treatment variance in
the AMOVAs (electronic supplementary material, table S1A).
This finding is consistent with a previous analysis of the
internal transcribed spacer (ITS) locus [5] and facilitates the
identification of genomic outliers that are particularly structured due to differential selection in N-fertilized versus control
(a) Genetic differentiation between N-fertilized versus
control and high- versus low-quality rhizobia
The top 1% of FST values in our comparison of N-fertilized
and control rhizobium populations (FST 0.074) included
20 chromosomal genes, nine pSym genes, and a single gene
on both plasmid pR132502 and pR132505 (electronic supplementary material, Dataset S4). Twenty-five of these
genes were significantly differentiated (17 chromosomal
genes: mean FST ¼ 0.090, s.d. ¼ 0.016; eight pSym genes:
mean FST ¼ 0.125, s.d. ¼ 0.037). These outliers stand out
against a backdrop of low overall genomic differentiation
(mean FST ¼ 0.022, s.d. ¼ 0.019, 99th percentile ¼ 0.074), and
the sliding window analysis revealed a particularly strong
FST peak at the symbiosis gene region of the pSym (blue vertical
line in figure 1a). Genes among the top 1% of FST values were
significantly enriched for pSym genes (32% of genes in the
top 1% were on the pSym, versus the null expectation
of 8.8% from the core genome, two-tailed Fisher’s exact test,
p ¼ 0.001). Evidence from flexible genome analyses (i.e. genes
with variable copy number and/or presence among strains)
also revealed that six relatively low-quality strains were
missing several symbiosis genes on the pSym (see pSym gap
in electronic supplementary material, figure S1), further
implicating the symbiosis island as an area of interest.
Consistent with the above findings, genes in the top 1%
of FST values comparing low versus high partner quality
rhizobia were also enriched for pSym genes (48% on the
pSym, p , 0.0001), and sliding window analysis revealed an
island of high differentiation at the symbiosis region on the
pSym (figure 1b), compared to a low genomic background
(mean FST ¼ 0.027, s.d. ¼ 0.022, 99th percentile ¼ 0.086).
Specifically, the top 1% of FST values (FST 0.086; 34 genes) in
this high- versus low-quality comparison included 19 on the
chromosome (14 significant; mean FST ¼ 0.100, s.d. ¼ 0.017)
and 14 on the pSym (13 significant; mean FST ¼ 0.189, s.d. ¼
0.053), and a single gene on plasmid pR132502 ( p . 0.05;
electronic supplementary material, Dataset S4).
Putting these two analyses together, only six genes were
among the most differentiated (top 1%) in both FST comparisons: nifH, nifA, nifD, fixC, nodB, and an electron transfer
flavoprotein subunit (Rleg_4928)—all on the pSym (blue
vertical line in figure 1). Together, these results implicate
genes in this region as players in the evolution of reduced
cooperation in response to N fertilization. A strong history of
molecular genetic work using rhizobial mutants and changes
in gene expression during various stages of symbiosis has
revealed the necessity of many rhizobium genes (particularly
those in this region) for the establishment and maintenance
of symbiosis with legumes (e.g. [15,59–64]). Ecological genetic
studies such as ours, which focus on natural variation and the
loci underlying ecologically relevant traits, can complement
mutant analyses by helping to resolve the loci targeted by
contemporary natural selection [6,7,25,65].
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(f ) Patterns of nucleotide variation at candidate
selected genes
plots [57,58]. We posited that, compared to the rest of the
genome, rhizobium genes underlying mutualism decline
should: (i) be genetically differentiated between N-fertilized
and control rhizobia, (ii) be associated with partner quality phenotypes (rhizobium effects on plant growth and chlorophyll
content), and (iii) show anomalous patterns of nucleotide
diversity consistent with recent selection.
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calculated FST for core genes in two ways. First, we identified
genes showing elevated FST between N-fertilized and control
strains (i.e. between the groups of rhizobia originally collected
from N-fertilized versus control plots). Second, we ordered
strains according to rank partner quality (rather than N treatment) by summing the ranks of all plant growth traits (see
ranks in electronic supplementary material, Dataset S2) and
split the strains into two groups: ‘high-quality’ (eight N-fertilized
and 20 control strains) versus ‘low-quality’ (16 N-fertilized and
12 control strains). We then identified genes showing elevated
FST between these two quality groups. In conjunction with the
ITEP toolkit, we used a collection of ‘pop_genome’ scripts [47]
(https://github.com/nyoungb2/pop_genome) to carry out
population genetic analyses with core gene alignments. Population pairwise FST was calculated with default parameters for
each core gene using ARLECORE v. 3.5.1.3 [45]. The 99th percentile
of FST values was calculated with the ‘quantile’ function in R
v. 3.0.3 (R Core Team, 2014; http://www.R-project.org/). We
performed a sliding window analysis in R to calculate the
mean of pairwise FST values using a window size of 13 genes
(to roughly match the size of the region spanning the betweentreatment differentiated symbiosis genes; see ‘Results’) and a
step-size of seven genes (452 windows total).
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N-strain p
(a)
control p
chromosome
5
pR132501 (pSym)
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genome position (bp)
mean N-strain p
(b)
4 000 000
5 000 000
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0.03
mean control p
(c)
0.06
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0.02
0.02
0.01
0.01
0
0
0
0.01
0.02
0.03
mean control p
0.04
0
0.04
Figure 2. Patterns of within-treatment nucleotide diversity (p) across the chromosome and symbiotic plasmid ( pSym) for 56 R. leguminosarum strains. (a) Points
(N ¼ 394) represent sliding window means (window size ¼ 13 genes), and dashed lines represent genome-wide averages. (b) Chromosome nucleotide diversity
(p) in N-fertilized versus control populations; black points represent the 360 chromosomal sliding windows from (a). Correlation coefficient (R) ¼ 0.81, p , 0.0001.
(c) pSym nucleotide diversity (p) in N-fertilized versus control groups; black points represent the 34 pSym sliding windows from (a). Correlation coefficient (R) ¼
0.56, p , 0.001. Blue triangles denote two sliding windows (which overlap by seven genes) that house the significant symbiosis gene region displaying high FST in
both FST analyses. The 95% confidence and prediction intervals are shown in purple and grey, respectively.
(b) Patterns of nucleotide diversity in N-fertilized versus
control strains
The genome-wide average p was higher among N-fertilized
strains compared with control strains, resulting from a few
N-fertilized strains (209, 231, and 717) that dominated a
clade more distantly related to the majority of other strains
(electronic supplementary material, figure S2). Nevertheless,
p covaried between the two groups for both the chromosomal and pSym windows (chromosome R ¼ 0.81, p , 0.0001;
pSym R ¼ 0.56, p , 0.001; figure 2b,c), suggesting that the
relative selective constraints among genes are similar across
most of the genome for N-fertilized and control groups.
Two chromosomal sliding window regions fell below the
95% prediction interval in the p correlation (figure 2b),
suggesting positive selection in N-fertilized environments;
however, these regions did not possess genes with FST in
the top 1% of either FST analysis. Two pSym sliding
window regions also fell below the 95% prediction interval
(blue triangles in figure 2c), and these regions contained the
six pSym loci from the top 1% of the FST outlier analyses
above. Reduced nucleotide diversity at the six pSym genes
revealed in the FST outlier analyses suggests positive selection
on these genes in N-fertilized environments. In other words,
these results are consistent with natural selection favouring
less beneficial rhizobia in high N conditions. Manipulative
experiments are required to test definitively whether lowquality N strains have higher fitness than control strains in
high N environments; these experiments are underway.
One selective agent favouring lower partner quality in high
N plots might be a shift towards the saprophytic lifestyle at the
expense of symbiosis. Among other ecological changes
in the Long Term Ecological Research (LTER) study since N
fertilization began, the availability of clover hosts has declined
in N-fertilized plots [5,66], likely leading to more frequent/
prolonged saprobic phases for R. leguminosarum. Genetic
trade-offs between symbiotic and free-living lifestyles could
drive positive selection for less beneficial rhizobia in high N
environments. For example, selection could favour mutualists
that invest more in storage compounds such as poly-3-hydroxybutyurate (PHB), which can increase rhizobium fitness in the soil
but likely divert carbon away from N fixation and thus from host
benefits [67]. Both molecular and experimental evolution studies
suggest the potential for such a trade-off between saprotrophy
and mutualism. Rhizobium etli (PHB synthase) and R. tropici
(glycogen synthase) mutants have higher rates of nitrogen
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(c) Potential functions of key candidate genes
(d) Bacterial adaptation in nature
Bacterial symbionts are key players in many plant and animal
mutualisms [83 –85], yet the historical dearth of ecological
genetic studies in bacteria has meant that the rules governing
adaptation in natural populations of bacteria are still being
elucidated [86]. The balance between natural selection and
recombination within natural bacterial populations is one
important unresolved aspect. With little recombination, selection acting at one or more loci should result in genome-wide
sweeps that purge previously existing genetic variation
within the population [19,87]. Many recent microbial studies,
however, have discovered adaptive regions of reduced
genetic diversity amidst high background polymorphism
[18,88–91]—implying abundant recombination. Using a
population genomic approach, we found a genomic island of
differentiation between rhizobium populations that have
experienced different fertilization histories and that this region
stands out against a genome-wide background of recombination/admixture between the two groups. These findings are
congruent with a recent study of a natural R. leguminosarum
population from the UK, in which high rates of recombination
sufficient to prevent divergence by drift were found [20].
Thus R. leguminosarum in nature appear to form dynamic,
diverse populations that are unified by gene flow despite selection acting at one or more loci. In our case, where genomic
islands of differentiation (this study) and large phenotypic
differences [5] were detected, selection due to N addition must
be exceptionally strong.
Because the rhizobium populations were not assayed when
the LTER experiment began, we cannot know with certainty
whether particular nucleotide-level differences underlying
our phenotypic differentiation have arisen through mutation
since that time (22 years ago), or instead pre-dated the establishment of the N plots. These alternatives represent two
distinct mechanisms of evolution [92]. On the one hand, new
mutations could have arisen and increased in frequency atop
a diverse phylogeny of rhizobium strains (selection on
new mutations); on the other hand, pre-existing alleles could
have increased in frequency (selection on standing genetic
variation). Given the levels of nucleotide diversity found
throughout the genome, in addition to our observations of
substantial standing variation in partner quality variation
within the control plots [5], we suspect that the alleles at the
key outlier region on the pSym pre-dated the establishment of the N plots—i.e. that selection has acted on standing
genetic variation. Evolution from standing variation should
proceed more quickly than evolution from new mutations
[93,94], potentially allowing populations to adapt more
quickly. Indeed, how quickly N-fertilized populations of rhizobia might return to control levels of partner quality after a
return to low N conditions is an interesting open question.
Data accessibility. Illumina reads and de novo genome assemblies are
available via DRYAD (http://dx.doi.org/10.5061/dryad.9gn28).
Competing interests. We declare we have no competing interests.
Funding. This work was funded by the National Science Foundation
(NSF DEB-1257938), the NSF LTER Program at the Kellogg Biological
Station (DEB-1027253), and Michigan State University AgBioResearch.
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Proc. R. Soc. B 283: 20152563
Only six symbiosis-related genes on the pSym (nifH, nifA,
nifD, fixC, nodB, and an electron transfer flavoprotein subunit) matched all three criteria for identifying the genes
underlying the response to N fertilization. Because these
genes are in close proximity, it is unclear which particular
genes have been targeted by selection; nevertheless, given
the large body of genetic work on symbiosis genes in this
region [46], several interesting candidates stand out. For
example, nifH, nifD, nifA, nodB, and fixC are among the top
1% of FST values in both FST analyses, and additionally nifK,
nifE, nifN, and nodF are in the top 1% of FST values in the
second analysis of high- and low-quality groups (a full list of
candidate loci is in the electronic supplementary material,
Dataset S4). The N-fixing (nif) genes nifH, nifD, and nifK are
responsible for the formation of the nitrogenase enzymatic
complex (Fe and FeMo proteins), which reduces atmospheric
di-nitrogen (N2) to ammonia for plant use [71]. The nifB, nifE,
and nifN genes are involved in the synthesis of the FeMo cofactor that is located at the active site of the FeMo protein, where
N2 binds and is reduced [72,73]. Additionally, nifA helps regulate the expression of various nif genes [74], while fixC is part of
the fixABCX complex involved in gene transcription regulation
in low oxygen conditions (e.g. inside the nodule) and is posited
to have a central role in electron transfer to nitrogenase [75,76].
In general, nod genes code for enzymes that generate Nod factors, rhizobium signalling molecules between rhizobia and
plant required for nodulation initiation; nodB in particular is
part of the nodABC operon, which generates the main Nod
factor structure [24].
Aside from the six symbiosis-related pSym genes discussed
above, 12 window regions on the chromosome and one
window on the pSym possessed decreased p in control, compared with N-fertilized, strains (and thus fall above the
upper bounds of the prediction interval in figure 2b,c). There
were no outliers from both FST analyses within these 13 windows; however, they did contain five chromosomal genes
that were significantly differentiated between N-fertilized
and control rhizobia (electronic supplementary material, Dataset S4), including adenylate/guanylate cyclase and FecR. It is
unclear how these genes might govern partner quality traits
per se, since they did not differentiate low- and high-quality rhizobia in the second FST comparison; nevertheless, they might
have an important role in uncharacterized phenotypic differences between N- and control strains. Adenylate cyclases
have recently been implicated in infection thread formation
[77], as well as in the negative regulation of secondary infections after initial infection thread formation [78], potentially
controlling the number of successful nodulation events.
N-fixing rhizobia inside the nodule have a high demand for
iron (e.g. nitrogenase and cytochrome synthesis), and iron
uptake could occur by means of ferric citrate [79], which
requires the cytoplasmic membrane protein FecR in Escherichia
coli [80]. A full understanding of how chromosomal and pSym
genes together determine mutualist partner quality and other
rhizobium phenotypes will require a much larger effort, ideally
implementing emerging techniques for association mapping
in bacteria [81,82].
rspb.royalsocietypublishing.org
fixation in symbiosis and hoard less carbon for life outside the
nodule [68,69], while Bradyrhizobium japonicum strains that
evolved the most significant increase in host-free fitness after
450 generations in culture showed the largest decline in partner
quality [70]. We are currently investigating whether the
N-fertilized strains in our study are better saprophytes.
Downloaded from http://rspb.royalsocietypublishing.org/ on June 18, 2017
Acknowledgements. We thank B. Gordon for laboratory assistance,
N. Youngblut and M. Friesen for sequencing and bioinformatics
advice, and R. Whitaker, Z. Cheviron, P. Tiffin, and the Heath laboratory
for comments on the manuscript. This is KBS publication no. 1903.
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