Pervasive interactions between foliar microbes and soil nutrients
mediate leaf production and herbivore damage in a tropical
forest
Eric A. Griffin1,2, S. Joseph Wright3, Peter J. Morin4 and Walter P. Carson2
1Smithsonian Environmental Research Center, 647 Contees Wharf Rd, Edgewater, MD 21037, USA; 2Department of Biological Sciences, University of Pittsburgh, A234 Langley Hall, 4249
Fifth Avenue, Pittsburgh, PA 15260, USA; 3Smithsonian Tropical Research Institute, Apartado 0843-03092, Balboa, Panam
a; 4Department of Ecology, Evolution, and Natural Resources,
Rutgers University, 14 College Farm Rd, New Brunswick, NJ 08901, USA
Author for correspondence:
Eric A. Griffin
Tel: +1 770 561 5731
Email: eag46@pitt.edu
Received: 8 April 2017
Accepted: 21 June 2017
New Phytologist (2017)
doi: 10.1111/nph.14716
Key words: leaf production, niche
differentiation, plant microbiome, plant–
microbe interactions, plant–microbe–
herbivore interactions, soil resource
availability, tropical forests.
Summary

 Producing and retaining leaves underlie the performance and survivorship of seedlings in
deeply shaded tropical forests. These habitats are characterized by conditions ideal for foliar
bacteria, which can be potent plant pathogens. Leaf production, retention and susceptibility
to enemies may ultimately depend upon interactions among soil nutrients and foliar microbes,
yet this has never been tested.

 We experimentally evaluated the degree that foliar bacteria and soil resource supply medi-
ate leaf dynamics for five common tree species (five different families) in a Panamanian forest.
We reduced foliar bacteria with antibiotics for 29 months and measured leaf production,
retention and damage for seedlings nested within a replicated 15-yr factorial nutrient enrich-
ment experiment (nitrogen, N; phosphorus, P; potassium, K).

 Our results demonstrate that when we applied antibiotics, soil nutrients – particularly N –
always regulated seedling leaf production (and to a lesser extent herbivore damage) for all
five tree species. In addition, it was common for two macronutrients together to negate or
completely reverse the impact of applying either one alone.

 Our findings of frequent plant–microbe–nutrient interactions are novel and suggest that
these interactions may reinforce plant species–environment associations, thereby creating a
fairly cryptic and fine-scale dimension of niche differentiation for coexisting tree species.
Introduction
The production, retention and defense of leaves underlie the per-
formance of seedlings and saplings that live for years in deeply
shaded forest understories where soil resources are scarce and ene-
mies cause significant mortality. In tropical forests, the amount
of damage on seedlings from both pathogens and herbivores is
much higher than in temperate forests (Coley & Aide, 1991;
reviewed by Coley & Barone, 1996; Gilbert, 2002; but cf. Moles,
2013). Consequently, leaf production is costly for shade-tolerant
species that inhabit tropical forest understories because their
seedlings invest heavily in structural and chemical defenses
(Coley, 1983; Coley & Aide, 1991; Reich et al., 1992; Wright &
Cannon, 2001; Wright et al., 2004; Gilbert, 2005). Moreover,
tropical leaves in resource-poor habitats persist up to nine times
longer and take up to three orders of magnitude longer (in days)
to payback returns on carbon investments vs leaves in high-
resource habitats (Chabot & Hicks, 1982; Chazdon & Fetcher,
1984; Coley, 1988; Williams et al., 1989; Kikuzawa, 1991;
Sobrado, 1991; Coley & Barone, 1996; Westoby et al., 2000;
Wright et al., 2004). Thus, because leaf production is costly and
retention is essential, understanding the mechanisms that under-
lie leaf dynamics is critical to understanding seedling perfor-
mance.
In addition to contending with nutrient-poor soils and deep
shade, seedlings in tropical forests also occur in a habitat rich in
foliar microbes (Chazdon & Fetcher, 1984; Vitousek & Sanford,
1986; Wright & Van Schaik, 1994; Wright et al., 2004; Poorter
& Bongers, 2006; Poorter et al., 2009; Griffin et al., 2016;
reviewed by Griffin & Carson, 2015). Because UV radiation is
low, and temperature and humidity are high, foliar microbes,
particularly bacteria, are likely to be diverse and abundant, and
thus potentially key regulators of leaf dynamics (Griffin & Car-
son, 2015; Griffin et al., 2016). Indeed, leaves are one of the
world’s largest microbial habitats occupying an area twice the size
of the Earth’s land area (Vorholt, 2012). On average, foliar bacte-
ria occur in densities of 1–10 million cells cm2 and, moreover,
an average of over 500 taxa occurred on single trees in a tropical
forest in Panama (Lindow & Brandl, 2003; Delmotte et al.,
2009; Vorholt, 2012; Kembel et al., 2014). Leaves of seedlings in
the understory are almost certainly teaming with bacteria because
of the numerous ways bacteria are able to colonize leaf surfaces

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and subsequently access leaf interiors (reviewed by Griffin & Car-
son, 2015). In a recent study, Griffin et al. (2016) demonstrated
that foliar bacteria in a tropical forest caused up to a 49% reduc-
tion in growth rates for seedlings of three common tree species.
These results suggest that foliar bacteria are commonly
pathogenic and may typically regulate leaf dynamics, although
data are nearly nonexistent (but cf. Griffin et al., 2016).
Leaf dynamics may be strongly dependent on soil nutrient
availability, because the low availability of macronutrients com-
monly reduces plant performance even in shaded habitats
(Wright et al., 2011; Pasquini & Santiago, 2012; Santiago et al.,
2012; Pasquini et al., 2015). For example, Santiago et al. (2012)
demonstrated that 2 years of soil nutrient additions increased
seedling height growth by up to 24% (see also Pasquini et al.,
2015). Furthermore, Griffin et al. (2016) found that the degree
to which foliar bacteria were harmful to seedlings varied among
tree species and was often ameliorated by the greater availability
of soil nutrients, particularly potassium. This suggests that the
impact of foliar bacteria may typically depend on the availability
of macronutrients (Griffin et al., 2016). Oddly, the degree to
which soil nutrients interact with plant-associated microbes to
regulate plant performance in situ, however, remains little stud-
ied. This is surprising, because plant-associated microbes are criti-
cal mediators of plant functional traits and trophic interactions
(Friesen et al., 2011; van der Putten et al., 2013; Turner et al.,
2013; Averill et al., 2014; van der Heijden et al., 2015).
Foliar bacteria and soil nutrients may commonly interact to
regulate leaf dynamics. These interactions could alter host plant
nutrient status and plant defenses, thereby altering the rate of leaf
loss or gain, and changing the amount and type of damage on
small seedlings and saplings (Coley, 1983; Coley et al., 1985;
Bazzaz et al., 1987; reviewed by Coley & Barone, 1996). In addi-
tion, pathogenic bacteria may, like fungal pathogens, cause pre-
mature leaf abscission when plants shed leaves in response to
infection (Ostry, 1987; Eyal et al., 1993; Patterson, 2001; David-
son et al., 2011). Thus, interactions between foliar bacteria and
soil nutrient availability may commonly regulate patterns of
enemy damage. For example, studies among agricultural crop
species have shown that nutrient availability may mediate the
negative impacts of foliar pathogens (Dordas, 2008; Johnson
et al., 2010). Potassium in particular tends to mitigate the severity
of pathogen damage, likely because potassium fortifies plant cell
walls to confer protection from pathogen entry (Dordas, 2008).
Ultimately, variation in nutrient availability may interact with
bacterial communities to favor some plant species over others as
resources vary patchily across the landscape (Griffin et al., 2016).
If so, then interactions among foliar bacteria and soil nutrient
availability could reinforce plant species–environment associa-
tions, thereby creating a fairly cryptic and fine-scale dimension of
niche differentiation (Griffin et al., 2016).
In the present study we assess whether foliar bacteria interact
with soil nutrients to govern leaf dynamics for seedlings of co-
occurring tree species in the shaded understory. We tested the fol-
lowing hypotheses within a tropical forest in Panama: the degree
to which soil nutrients mediate leaf dynamics (production, reten-
tion, and enemy damage) varies substantially among co-occurring
tree species; the degree to which foliar bacteria mediate leaf
dynamics varies substantially among tree species; and interactions
among soil nutrients and foliar bacteria will be frequent, thereby
mediating leaf dynamics among co-occurring host plant species.
To address these hypotheses, we experimentally reduced foliar
bacteria for 29 months for seedlings of five common tree species.
These seedlings were nested within a fully factorial, well-
replicated nutrient enrichment experiment (nitrogen (N), phos-
phorus (P), and potassium (K) and all combinations) that com-
menced in 1998 (Yavitt & Wright, 2008; Yavitt et al., 2009,
2011; Wright et al., 2011; Pasquini & Santiago, 2012; Santiago
et al., 2012; Pasquini et al., 2015; Griffin et al., 2016).
Materials and Methods
Study site and fertilization experiment
We conducted this study in a mature, seasonally moist, semi-
deciduous tropical secondary forest (c. 200 years old) on the
Gigante Peninsula in Panama, which is part of the Barro
Colorado Nature Monument. The soils are oxisols, alfisols,
inceptisols and acric nitisols (Turner et al., 2016). The site
receives 2600 mm of rainfall annually, though < 10% of this falls
during the four month dry season (January–April).
Beginning in 1998, we applied nitrogen (N), phosphorus (P)
and potassium (K) by hand four times a year between June and
November in a 29 29 2 factorial design (Supporting
Information Fig. S1). We replicated each treatment four times
along a mild elevational and soil gradient, using 32 plots,
measuring 409 40 m separated by at least 40 m (Yavitt et al.,
2009; Turner et al., 2016). We applied 125 kg N ha1 yr1 as
urea, 50 kg P ha1 yr1 as triple super-phosphate, and 50 kg K
ha1 yr1 as KCl.
Study species
We selected five common and relatively shade tolerant woody
species from five different families; these species vary in life his-
tory traits and spanned a wide range of maximum adult heights.
Alseis blackiana Hems. (Rubiaceae) is a mid-canopy tree,
Desmopsis panamensis Saff. (Annonaceae) and Heisteria concinna
Standl. (Olacaceae) are understory treelets, Sorocea affinis Hemsl.
(Moraceae) is a small tree, and Tetragastris panamensis Kunze.
(Burseraceae) is a canopy tree (Dalling et al., 2001; Wright et al.,
2003, 2010; Gilbert et al., 2006; nomenclature follows Garwood,
2009). Hereafter, we refer to each species by genus or by four-
letter abbreviations in figures (ALBL, Alseis; DEPA, Desmopsis;
HECO, Heisteria; SOAF, Sorocea; TEPA, Tetragastris).
Antibiotic applications
Within the inner 309 30 m of each fertilization plot, we ran-
domly assigned three c. 20–30 cm tall seedlings of each species
for antibiotic treatment and another three for control treatment
(sterile water; n = 941 seedlings). Beginning in January 2010, we
carefully sprayed antibiotics or sterile water to all seedlings to
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saturation every 10–15 d for 29 months. We placed a plastic
sheet around the base of each seedling to prevent exposing soil
microbes to either treatment; soil samples verified that neither
the antibiotic, nor water, altered soil bacterial abundance or rich-
ness (see Griffin et al., 2016).
We alternated the antibiotic treatments between streptomycin
(up to 100 ppm of Agri-mycin 17; Hummert International #02-
0150; Earth City, MO, USA) or a joint treatment of oxytetracy-
cline and gentamicin (up to 1752 ppm of Agry-Gent Plus 800;
Qu
ımica Agron
omica de M
exico, Chihuahua, M
exico). These
are the three most commonly used broad-spectrum antibiotics in
temperate and tropical agricultural crops and they can reduce
bacterial abundance on the leaf surface as well as inside leaves by
up to 85% (McManus et al., 2002; Vidaver, 2002; Traw et al.,
2007; Griffin et al., 2016). Streptomycin (Agri-mycin) and gen-
tamycin (Agry-gent) inhibit protein synthesis for Gram-negative
bacteria, and oxytetracycline (Agry-gent) inhibits both Gram-
positive and Gram-negative bacteria (Chopra & Roberts, 2001;
McManus et al., 2002; Ding & He, 2010; Nelson & Levy,
2011). All products have limited nontarget effects, including
those on fungi (Ingham & Coleman, 1984; Colinas et al., 1994;
Chopra & Roberts, 2001; Thiele-Bruhn & Beck, 2005).
Leaf production and retention
We recorded the total number of leaves on each seedling at
the beginning of the experiment and after 29 months of antibi-
otic or control treatments. Thus, we define the rate of leaf
change for each seedling after 29 months as ‘leaf production’
(see Statistical analyses). In addition, we randomly selected and
marked four leaves from each seedling and estimated enemy
damage (see Enemy damage) at the outset as well as enemy
damage and how many of the original leaves remained after
14 months of applications (retention). We recorded leaf reten-
tion and enemy damage after only 14 months because almost
all leaves (c. 98%) had fallen before the end of the experiment
(29 months). We excluded Alseis from the herbivore, pathogen
and retention analyses at month 14 because all leaves turnover
each dry season (Dalling et al., 2001). Finally, all data were
collected blindly so the observer was unaware of antibiotic
treatment and soil nutrient additions.
Enemy damage
We estimated percentage of leaf area removed by leaf-chewing
herbivores and percentage damage by pathogens (chlorosis or
lesions) for four randomly selected leaves from each seedling fol-
lowing the protocols of Schnitzer et al. (2002) and Mangan et al.
(2010). As stated above, we estimated damage at the beginning
of the experiment and then 14 months after either antibiotic or
control applications. We based percentage loss estimates on a
template of artificial (paper) leaves with 24 levels of damage: 0%,
1%, 2.5%, 5%, 7.5%, 10% and in 5% increments up to 100%
area removed (Carson & Root, 2000; Schnitzer et al., 2002).
Although some insect damage may cause lesions and chlorosis
(e.g. Miller & Davidson, 2005), the primary causes of this type
of damage are fungi, bacteria and viruses (e.g. Garcia-Guzman &
Dirzo, 2001; Myster, 2002; Mangan et al., 2010; E. Griffin, pers.
obs.). Hereafter, we will refer to percentage area loss by leaf-
chewing insects as ‘herbivore damage’ and chlorosis and lesions
as ‘pathogen damage’.
It is important to note that we also measured the mean
canopy openness (0–100%) above each seedling with a concave
densitometer at beast height at each time point (0 months,
14 months, 29 months). Canopy openness had no effect on
any of the models.
Statistical analyses
We performed MANOVAs to evaluate whether nutrient addi-
tions caused changes in plant performance metrics. We chose to
use MANOVAs because all five species were nested within the N,
P and K treatments, and MANOVAs allowed us to avoid pseudo-
factorialism by adjusting for correlated response variables among
species (Morrison, 1976; Morin, 1983; Winer et al., 1991; Hurl-
bert, 2013). We assessed the significance of nutrient additions
using Wilks’ Criterion, one of four standard test statistics com-
monly used to evaluate the MANOVA (Morrison, 1976),
defined by (determinant (E))(determinant (E+ H))1, where H is
the matrix of sums-of-squares and cross products calculated
among treatment means (e.g. +N and N), and E is the ele-
ment-wise squared difference between each observation and the
mean vectors for that group (Morin, 1983).
We used the method of linear discriminant functions, some-
times called canonical analysis of discriminance (CAD), to iden-
tify which species contributed to significant differences among
our treatments for each response (Fisher, 1936; Legendre &
Legendre, 1998). CAD follows directly from the calculations
used to determine test statistics in the multivariate analysis of
variance (MANOVA). Species that are significantly correlated
with the values of the discriminant function scores are those that
contribute to significant differences among the treatments exam-
ined in the MANOVA. Although single-species ANOVAs often
yield similar results to those detected by CAD, the ANOVAs can
miss correlated responses among the five variables (species
responses; see Methods S1), and their significance levels are not
adjusted for multiple tests on potentially correlated variables
(Morin, 1983). For a full explanation of the discriminant func-
tion analysis, see Methods S1.
MANOVA models for control individuals We performed a first
set of MANOVAs to evaluate seedling leaf production rate (L),
percentage leaf area attacked by herbivores and pathogens (H and
P), and leaf retention (R). We did this to test differences among
nutrient addition treatments (N, P, K and all combinations) for
control seedlings only. We calculated leaf production rates
(leaves/leaf month1) for each seedling as:
L ¼ ðloge L1  loge L0Þ=ðt1  t0Þ
(L0 and L1, initial and final leaf number; t1 t0, time period
(29 months; Santiago et al., 2012)).

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We estimated herbivore and pathogen damage among all
seedlings before treatments began (Time = 0 months). Estimating
damage at the beginning of the experiment allowed us to evaluate
how over 15 years of soil nutrient additions impacted leaf damage
among all seedlings. We calculated leaf retention R by determin-
ing the proportion of four randomly selected leaves from each
seedling that remained after 14 months.
The average for each species in a plot (e.g. L for leaf produc-
tion) for species i was
LðiÞ ¼ Lði; controlÞ
Because all five species were nested within treatment plots (and
nonindependent), our response vectors for each metric (e.g. L) in
plot j was:
Lj ¼ ðLð1Þ; Lð2Þ; Lð3Þ; Lð4Þ; Lð5ÞÞj
where numbers 1 through 5 represent each plant species. Alseis
was not included in the retention analysis because their leaves
had flushed within two months of the beginning of the
experiment.
MANOVA models for differences among antibiotic and control
individuals We performed a second set of MANOVAs to evalu-
ate the differences in leaf production rates (dL) after 29 months
of applications and herbivore damage (dH), pathogen damage
(dP) and leaf retentions (dR) after 14 months of applications.
We calculated the difference in plot mean values (e.g. dL, d H ,
dP and dR) for replicated individuals of each species with or
without antibiotic applications in each plot. Thus, for leaf
production,
dLðiÞ ¼ Lði; antibioticÞ  Lði; controlÞ
For herbivore or pathogen damage rates, we only included sur-
viving leaves after 14 months for species (i) and evaluated as:
H ðiÞ ¼ ðH14; i H0; iÞ=ð1H0; iÞ or PðiÞ
¼ ðP14; i  P0; iÞ=ð1 P0; iÞ
Therefore, our response vector for each performance metric
(e.g. dLj ) in plot j was:
dLj ¼ ðdLð1Þ; dLð2Þ; dLð3Þ; dLð4Þ; dLð5ÞÞj
where the numbers 1 through 5 refer to the five plant species. A
MANOVA of this response vector tests whether dLj differed
across nutrient treatments. Alseis was not included in the herbi-
vore, pathogen, or retention MANOVA analyses because its
leaves flushed two months after the experiment began.
We logit transformed all proportional data (Warton & Hui,
2011). We used SAS 9.4 (SAS Institute, Cary, NC, USA) to run
MANOVAs.
Results
Antibiotic efficacy
We previously demonstrated that Agry-gent and Agri-mycin sig-
nificantly decreased mean abundance of epiphytic and
Table 1 MANOVA results for the effects of nitrogen (N), phosphorus (P)
and potassium (K) on plant performance metrics for control seedlings
treated with sterile water of five species (Alseis blackiana, Desmopsis
panamensis, Heisteria concinna, Sorocea affinis and Tetragastris
panamensis)
Effect
Leaf
production
Leaf
retention
Herbivore
damage
Pathogen
damage
N 1.64 8.71*** 1.79 0.87
P 1.47 1.80 4.31** 1.63
K 6.90*** 2.28 2.67 1.22
N9 P 2.36 5.71** 3.69* 0.62
N9 K 3.38* 2.58 1.54 1.03
P9 K 2.62 2.80 1.10 0.48
N9 P9 K 1.76 2.26 1.80 0.17
We did not include Alseis in the leaf retention analysis because their leaves
turnover annually. Entries are F-values determined fromWilks’ Criterion.
Degrees of freedom are 5,20 for the leaf production, herbivore damage
and pathogen damage analyses, and 4,20 for leaf retention. Detection of a
significant treatment effect indicates that the mean values of the leaf met-
ric differ for the corresponding nutrient addition.
Significance: *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001.
Table 2 Pearson correlation coefficient values (r) for correlations between the discriminant function scores of significant nutrient effects compared to the
original variables (species) in MANOVAs for control seedlings treated with sterile water
Metric Nutrient Alseis Desmopsis Heisteria Sorocea Tetragastris
Production K 0.84*** 0.61*** 0.052*** 0.28 0.08
Production N9 K 0.73*** 0.62*** 0.50*** 0.23 0.33
Retention N NA 0.39* 0.34 0.77*** 0.55***
Retention N9 P NA 0.48*** 0.48*** 0.84*** 0.15
Herbivory P 0.02 0.12 0.32 0.56*** 0.56***
Herbivory N9 P 0.17 0.18 0.26 0.81*** 0.44*
Significant r correlations indicate a nutrient9 species interaction for the corresponding leaf metric and corresponding nutrient addition (N, nitrogen; P,
phosphorus; K, potassium).
*, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001.
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endophytic bacteria by over 50% on our focal species and also
decreased microbial morphotype richness by over 20% (Griffin
et al., 2016). Each of these antibiotic treatments were equally
effective and remained so over time; also, their impact was very
similar among nutrient treatments and plant species (Griffin
et al., 2016). Thus, our antibiotics worked in the sense that they
reduced foliar bacterial loads, although our results are likely con-
servative because we only reduced bacterial abundance and rich-
ness by c. 50%.
At the beginning of the experiment, mean leaf numbers were
7.53 for Alseis (n = 181), 10.59 for Desmopsis (n = 188), 5.81 for
Heisteria (n = 191), 5.32 for Sorocea (n = 187) and 6.70 for
Tetragastris (n = 193). Below, if we do not report the impact of
the antibiotic or specific nutrient or nutrient combination, it is
because it did not have a significant impact on leaf dynamics. It is
important to note that c. 10% seedlings died during the entirety
of the experiment, and mortality did not differ among tree
species or nutrient or antibiotic applications.
The impacts of macronutrients on leaf dynamics separate
from the effects of antibiotics
Leaf production For three species, N and K interacted to cause
increases or decreases in leaf production by 55–88% (Tables 1, 2;
Fig. 1). K addition caused Alseis to produce far fewer leaves, but
adding K and N together significantly reduced the negative
effects of K alone (Tables 1, 2; Fig. 1b). For two other species, K
addition alone caused the production of significantly more leaves
0.04
*
* *
0.03
0.02
0.01
0.00
–0.01
ALBL DEPA HECO SOAF TEPA –N +N
–N +N–N +N
Species
Alseis
Desmopsis Heisteria
Treatment Treatment
0.04
–K
+K
–K
+K
–K
+K–K
+K
0.03
0.02
0.01
0.00
–0.01
0.04
0.03
0.02
0.01
0.00
–0.01
0.04
0.03
0.02
0.01
Le
af
 p
ro
du
ct
io
n 
ra
te
(le
av
es
 p
er
 le
af
 m
on
th
)
Le
af
 p
ro
du
ct
io
n 
ra
te
(le
av
es
 p
er
 le
af
 m
on
th
)
0.00
–0.01
(a) (b)
(c) (d)
Fig. 1 Significant effects of potassium (K) and nitrogen (N)9 K on leaf production rates among control seedlings of Alseis blackiana (ALBL), Desmopsis
panamensis (DEPA), Heisteria concinna (HECO), Sorocea affinis (SOAF) and Tetragastris panamensis (TEPA) after 29months of nutrient enrichment. (a) A
significant K9 species interaction effects on leaf production for Alseis, Desmopsis and Heisteria (K effect: F5,20 = 6.90, P = 0.001; Alseis: P < 0.0001;
Desmopsis: P = 0.0002; Heisteria: P = 0.002). (b–d) N9 K interaction effects on leaf production (F5,20 = 3.38, P = 0.023; (b) Alseis: P < 0.0001; (c)
Desmopsis: P = 0.0002; (d) Heisteria: P = 0.004). Bars represent mean values  SE.

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(Fig. 1a), as did adding N alone, but adding both K and N
together did not cause an additive increase in leaf production
(significant N9K interaction, Fig. 1c,d).
Leaf retention We found a very consistent and significant effect
of adding N alone, or adding N and P together on leaf retention.
N addition alone decreased leaf retention for all four species (sig-
nificant for three or four) but this decrease disappeared or was
even reversed (Desmopsis) when N and P were added together
(N9 P interactions, Tables 1, 2; Fig. 2a–d; Alseis was not
included). These findings demonstrate that a strong effect of
adding one soil resource could be entirely offset by the simultane-
ous addition of another.
Herbivore and pathogen damage For two species, significant
N9 P interactions mediated herbivore damage (Tables 1, 2;
Fig. 3). For Sorocea, adding P reduced damage, however, adding
N and P together increased damage (Tables 1, 2; Fig. 3b). Con-
versely, for Tetragastris, adding P more than doubled herbivore
damage but this effect disappeared when we added N and P
together (Tables 1, 2; Fig. 3c). Nutrient additions never signifi-
cantly altered pathogen damage. Overall, mean herbivore damage
across all tree species was 8.5% ( 0.35 SE) and pathogen dam-
age was 6.1% ( 0.26 SE; Fig. 4).
The impact of antibiotics and macronutrients on leaf
dynamics
Leaf production The regular application of antibiotics and soil
nutrients over 29 months caused increases or decreases in leaf
production for all five species ranging from 38% to 140%
(Tables 3, 4; Figs 5, 6). However, the effects of antibiotics were
1.0
0.8
0.6
0.4
0.2
0.0
DEPA
*
*
*
Le
af
 re
te
nt
io
n 
ra
te
 (r
at
e 
of
 re
m
ai
ni
ng
le
av
es
 a
fte
r 1
4 
m
on
th
s)
Le
af
 re
te
nt
io
n 
ra
te
 (r
at
e 
of
 re
m
ai
ni
ng
le
av
es
 a
fte
r 1
4 
m
on
th
s)
HECO SOAF TEPA
Species Treatment
Desmopsis
Heisteria Sorocea
–N +N
–N
+N
–P
+P
–P
+P
–P
+P
Treatment
–N +N
Treatment
–N +N
1.0
0.8
0.6
0.4
0.2
0.0
1.0
0.8
0.6
0.4
0.2
0.0
1.0
0.8
0.6
0.4
0.2
0.0
(a) (b)
(c) (d)
Fig. 2 Significant effects of nitrogen (N) and N9 phosphorus (P) on leaf retention rates among control seedlings of Desmopsis panamensis (DEPA),
Heisteria concinna (HECO), Sorocea affinis (SOAF) and Tetragastris panamensis (TEPA) after 14months of nutrient enrichment. (a) A significant
N9 species interaction effect on leaf retention for Desmopsis, Sorocea and Tetragastris (N effect: F4,20 = 8.71, P = 0.0003; Desmopsis: P = 0.027; Sorocea:
P < 0.0001; Tetragastris: P = 0.001). (b–d) N9 P interaction effects on leaf retention (F4,20 = 5.71, P = 0.003; (b) Desmopsis: P = 0.005; (c) Heisteria:
P = 0.005; (d) Sorocea: P = 0.001). Bars represent mean values  SE.
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always governed by N9 K interactions, and to a lesser extent
N9 P interactions (Table 3). For example, for Desmopsis, antibi-
otics increased leaf production in plots fertilized with P but this
was reversed in plots fertilized with both P and N (Table 4;
Fig. 5b). For Sorocea, antibiotics increased leaf production but
only in plots fertilized with both N and P (Fig. 5c).
For four of five species, the impact of antibiotics was strongly
governed by N9K interactions (Table 4; Fig. 6). For three of
five species, the response was consistent: adding antibiotics and K
together increased leaf production; however, antibiotics had no
effect when K and N were added together (Figs 6a,c,d, 3a). For a
fourth species, Heisteria, applying antibiotics and K together
decreased leaf production unless N and K were applied together
14
12
10
8
6
4
2
0
ALBL
M
ea
n 
le
af
 h
er
bi
vo
re
 d
am
ag
e
(%
 le
af
 re
m
ov
ed
)
M
ea
n 
le
af
 h
er
bi
vo
re
 d
am
ag
e
(%
 le
af
 re
m
ov
ed
)
M
ea
n 
le
af
 h
er
bi
vo
re
 d
am
ag
e
(%
 le
af
 re
m
ov
ed
)
DEPA HECO
Species
–N +N
–P
+P
–P
+P
–P
+P
*
*
–N +N
Treatment
Sorocea
Tetragastris
SOAF TEPA
5
4
3
2
1
0
5
4
3
2
1
0
(a)
(b)
(c)
Fig. 3 Significant effects of phosphorus (P) and nitrogen (N)9 P on leaf
herbivore damage rates among control seedlings of Alseis blackiana
(ALBL), Desmopsis panamensis (DEPA), Heisteria concinna (HECO),
Sorocea affinis (SOAF) and Tetragastris panamensis (TEPA) at the
beginning of the experiment (Time = 0months). (a) A significant
P9 species interaction effect on herbivore damage for Sorocea and
Tetragastris (P effect: F5,20 = 4.31, P = 0.01; Sorocea: *P = 0.001;
Tetragastris: *P = 0.001). (b, c) N9 P interaction effects on herbivore
damage (F5,20 = 3.69, P = 0.016; (b) Sorocea: P < 0.0001; (c) Tetragastris:
P = 0.013). Bars represent mean values  SE.
25
20
15
10
5
0
ALBL DEPA HECO SOAF TEPA
Species
Herbivore damage
Pathogen damage
M
ea
n 
le
af
 d
am
ag
e 
(%
 le
af
 re
m
ov
ed
 o
r d
is
ea
se
d)
Fig. 4 Mean insect herbivore and pathogen damage (chlorosis or lesions)
among leaves of Alseis blackiana (ALBL), Desmopsis panamensis (DEPA),
Heisteria concinna (HECO), Sorocea affinis (SOAF) and Tetragastris
panamensis (TEPA) at the beginning of the experiment. We estimated
percentage leaf area removed by leaf-chewing herbivores and percentage
damage by pathogens for four randomly selected leaves from each
seedling before we began applying antibiotics (n = 3572 leaves). Bars
represent mean values  SE.
Table 3 MANOVA results for the effects of nitrogen (N), phosphorus (P)
and potassium (K) on plant performance metric differences between
antibiotic and control seedlings of five species (Alseis blackiana,
Desmopsis panamensis, Heisteria concinna, Sorocea affinis and
Tetragastris panamensis)
Effect
Leaf
production
Leaf
retention
Herbivore
damage
Pathogen
damage
N 1.97 1.76 1.51 2.48
P 1.49 0.90 0.19 1.13
K 3.52* 0.25 0.20 0.97
N9 P 3.39* 0.37 0.93 2.12
N9 K 4.45** 2.41 3.19* 1.20
P9 K 0.56 1.13 1.94 0.92
N9 P9 K 1.16 1.18 2.58 1.05
Detection of a significant treatment effect indicates that the mean values
of the leaf metric differ among antibiotic and control individuals for the
corresponding nutrient addition. We did not include Alseis in the leaf
retention analysis because their leaves turnover annually. Entries are F-
values determined fromWilks’ Criterion. Degrees of freedom are 5,20 for
leaf production, and 4,20 for the leaf retention, herbivore and pathogen
damage analyses.
Significance: *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001.

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(Fig. 6b). Overall, the effects of the antibiotics were often
reversed when two macronutrients were added together.
Leaf retention We never detected any impact of antibiotics on
leaf retention under any combination of our nutrient treatments
(Table 3). Note that in the second set of analyses we evaluated
the difference in means between the antibiotic and control indi-
viduals. This simply means that there were no differences in how
antibiotic individuals responded to nutrient enrichment vs con-
trol individuals, not that there were no nutrient effects on reten-
tion (e.g. Tables 1, 2; Fig. 2a–d).
Herbivore damage For three species, the impact of antibiotics
was consistent and regulated entirely by N9K interactions
(Tables 3, 4; Fig. 7). Specifically, antibiotics decreased damage
when neither N nor K was applied, or when both were applied
together; conversely, antibiotics increased damage when just N
was applied (Table 4; Fig. 7). This was most pronounced for
Heisteria and Sorocea (Fig. 7b,c). These results mirror the strong
N9K interactions that regulated leaf production (Fig. 6). To
our surprise, we never detected any impact of the antibiotic on
pathogen damage under any combination of nutrient treatments
(Table 3).
Discussion
Our results demonstrate that soil nutrients and broad-spectrum
antibiotics regulate leaf dynamics among five species in a deeply
shaded forest understory. Specifically, potassium (K) was a key
resource in regulating leaf production (K and N9 K interac-
tions), nitrogen (N) was key for leaf retention (N and N9 P
interactions), and phosphorus (P) was key for regulating herbi-
vore damage (P and N9 P interactions; Tables 1, 2; Figs 1–3).
Notably, antibiotics caused increases in leaf production for four
of five species (Fig. S2). More importantly, the impact of
broad-spectrum antibiotics and soil nutrients interact to regulate
leaf dynamics and, to a lesser extent, herbivore damage. This
was true for all five of our focal tree species, which came from
five different plant families, and vary in adult stature and other
life history traits. The direction and magnitude of the impact of
antibiotics were entirely dependent upon interactions with soil
nutrients for all five species (Table 4). N was a key resource
involved in each of these interactions for both leaf production
and herbivore damage (i.e. significant N9 K and N9 P inter-
actions). For example, it was striking that applying antibiotics
caused a dramatic increase (up to 140%) in leaf production in
plots enriched with K, but this increase was entirely reversed if
N was applied together with K (e.g. Alseis (Figs 5a, 6a) showed
highly significant N9 K interactions). These types of reversals
occurred for two other species (N9 K interactions for leaf pro-
duction; Fig. 5c,d) and also for three species for the effects of
antibiotics on herbivore damage (N9 K interactions; Fig. 7a–
c). Our results provide compelling evidence that the impact of
foliar microbial communities depends upon soil resource avail-
ability and, in particular on two macronutrients, N and K. Fur-
thermore, our results strongly suggest that the application of
antibiotics in combination with a single macronutrient (typi-
cally K) decreases the abundance of pathogenic bacteria, often
increasing leaf production. However, the benefits of reducing
bacteria, or the indirect effects of reducing bacteria (see
Potential mechanisms underlying plant–microbe–nutrient
interactions), were often negated when N and another macronu-
trient were applied together. Overall, our findings support
recent studies that challenge the conventional wisdom that light
is only a limiting resource for tropical seedlings and moreover
demonstrate that soil nutrient availability and foliar bacteria are
major drivers of plant performance (e.g. Pasquini & Santiago,
2012; Santiago et al., 2012; Pasquini et al., 2015; Griffin et al.,
2016).
Our results support all three hypotheses and demonstrate that
antibiotics alter foliar bacteria to such a degree that it can substan-
tially mediate leaf production and herbivore damage. Moreover,
the magnitude of the impacts of nutrients on leaf production was
host specific, and N and K availability regulated these responses.
Thus, we suggest that without considering microbial communi-
ties, it is difficult to understand the mechanisms that regulate
seedling performance and dynamics among co-occurring species
and thus key aspects of forest regeneration (e.g. Mangan et al.,
2010). Moreover, these interactions appear strong enough to fre-
quently change the rank-order performance of seedlings of differ-
ent species across forest understories because soil resources and
most likely bacteria as well, are patchily distributed at both large
and small spatial scales (e.g. John et al., 2007; Baldeck et al.,
2013; Condit et al., 2013; Kembel et al., 2014). For example, for
our five species, antibiotic applications changed the rank-order
performance for two of five species for leaf-production and
Table 4 Pearson correlation coefficient (r) for correlations between the discriminant function scores of significant nutrient effects compared to the original
variables (species) in MANOVAs of antibiotic and control differences
Metric Nutrient Alseis Desmopsis Heisteria Sorocea Tetragastris
Production K 0.93*** 0.01 0.32 0.31 0.36*
Production N9 P 0.15 0.86*** 0.04 0.46* 0.02
Production N9 K 0.47*** 0.26 0.52* 0.57** 0.78***
Herbivory N9 K NA 0.50** 0.84* 0.59*** 0.25
Significant r correlations indicate nutrient9 species9 antibiotic interactions for the corresponding leaf metric and nutrient addition (N, nitrogen; P, phos-
phorus; K, potassium). Alseiswas not included in the herbivore, pathogen or retention analyses because its leaves turnover annually.
*, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001.
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herbivore damage, and four of five species for seedling growth
(Fig. S2; growth data from Griffin et al., 2016). Notably, how-
ever, Griffin et al. (2016) only detailed the interplay between soil
nutrients and foliar bacteria and their impacts on seedling growth
among tree species. Here, we went one step further to measure
the impacts of these interactions on leaf production and retention,
two key underlying mechanisms for seedling growth. Addition-
ally, we demonstrate that interactions between antibiotics and soil
nutrients impacted plant growth and leaf production differently
under different experimental conditions and sometimes in oppos-
ing directions. For example, Griffin et al. (2016) demonstrated
that antibiotics mediated seedling growth when P, K and N9 P
were added; however, here we demonstrated that antibiotics
mediated leaf production when K, N9 P and N9K were added.
In another example, Griffin et al. (2016) demonstrated that
antibiotics increased Desmopsis growth rates when N and P were
added; however, in this study antibiotics decreased Desmopsis leaf
production rates when N and P were added. Thus, species-specific
impacts of foliar bacteria vary among nutrient additions for differ-
ent performance metrics, even if those metrics correlate with one
another to a certain degree (e.g. growth and leaf production). For
these reasons, we argue that foliar bacteria should be considered
an entirely independent yet cryptic plant functional trait that reg-
ulates plant phenotypes and performance metrics among species.
Ultimately, we hypothesize that these interactions may well repre-
sent a novel dimension of fine-scale niche differentiation, thus
promoting the long-term coexistence of plant species (Griffin
et al., 2016).
Although speculative, we argue that combinations of two
nutrients together (particularly N) make leaves more vulnerable
to enemies, thus negating or reversing the effects of enrichment
on performance. These results are consistent with findings by
both Andersen et al. (2010) and Santiago et al. (2012), who sug-
gested that enhanced herbivory caused by nutrient enrichment
could mask growth responses to fertilization. This conclusion,
however, runs counter to our result where the effects of nutrients,
the application of antibiotics, or their interaction never signifi-
cantly altered pathogen damage, and had significant but variable
effects on herbivore damage for three species. It is possible that
we did not pick up many significant herbivore or pathogen
effects because the majority of leaves we selected were fully
expanded and potentially already accrued most of their damage.
For example, almost 70% of lifetime enemy damage to tropical
leaves occur within the first few weeks of leaf expansion (Coley
& Aide, 1989; Kursar & Coley, 2003; reviewed by Coley &
Barone, 1996). Moreover, it is important to point out that plants
infected with foliar pathogens may suffer reduced performance
yet often show no visible symptoms (reviewed by Griffin & Car-
son, 2015). For example, Pseudomonas tomato, a plant pathogen,
can decrease tomato leaf production by as much as 30% without
showing any sign of infection (Bashan & Okon, 1981). Indeed,
we found that antibiotics or their interactions with nutrients
caused no differences in foliar pathogen damage yet caused
increases in leaf production by up to 140%. Thus, our results
lead us to suggest that high nutrient tissue concentrations, partic-
ularly for N and K together and N and P together, may com-
monly make host plants more vulnerable to plant enemies.
Indeed, some authors have argued that increased vulnerability to
enemies may select against luxury consumption under low light
and nutrients, particularly for N (Ostertag, 2010; Sayer & Banin,
2016). Nevertheless, our study demonstrates there were pervasive
interactions between antibiotic and macronutrient treatments,
0.03
*
*
0.02
0.01
0.00
–0.01
–0.02
0.03
0.02
0.01
0.00
–0.01
–0.02
0.03
–N +N
–N +N
Treatment
–P
+P
–P
+P
–K
+K
Sorocea
Desmopsis
ALBL DEPA HECO
Species
L 
(a
nt
ib
io
tic
) –
 L
 (c
on
tro
l)
L 
(a
nt
ib
io
tic
) –
 L
 (c
on
tro
l)
L 
(a
nt
ib
io
tic
) –
 L
 (c
on
tro
l)
SOAF TEPA
0.02
0.01
0.00
–0.01
–0.02
(a)
(b)
(c)
Fig. 5 Significant effects of antibiotic applications and potassium (K) and
nitrogen (N)9 phosphorus (P) on leaf production rates (L) among
seedlings of Alseis blackiana (ALBL), Desmopsis panamensis (DEPA),
Heisteria concinna (HECO), Sorocea affinis (SOAF) and Tetragastris
panamensis (TEPA) after 29months of applications and nutrient
enrichment. When bars are above the line, antibiotic applications increased
leaf production and when below the line, antibiotic applications decreased
production. (a) For Alseis and Tetragastris, applying antibiotics increased
leaf production when K was added (K effect: F5,20 = 3.52, P = 0.0192;
Alseis: *P < 0.0001; Tetragastris: P = 0.0427). (b, c) For Desmopsis and
Sorocea, antibiotics and N9 P regulated leaf production (significant
antibiotics9N9 P interaction, F5,20 = 3.35, P = 0.0233; Desmopsis:
P < 0.0001; Sorocea: P = 0.0141). Bars represent mean values  SE.

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particularly N treatments, on leaf production for all five tree
species. Ultimately, foliar bacteria appear to be a highly cryptic
component of plant performance, and future studies should con-
sider the impacts of pathogens even when there are no visible
symptoms on plant hosts.
Potential mechanisms underlying plant–microbe–nutrient
interactions
Although our antibiotic treatments reduced bacterial abundance
and richness (Griffin et al., 2016), it remains unclear whether
the impacts of antibiotics are due to bacterial reduction, changes
in the species composition of bacterial communities, indirect
effects (e.g. changes in fungal pathogens due to reductions in
bacteria), or a combination of these. Indeed, whereas many
species of bacteria are plant pathogens, others are mutualists,
and thus like some fungal endophytes, produce compounds
such as growth hormones that increase plant resistance to
pathogens and herbivores (see Arnold et al., 2003; Herre et al.,
2007; Mejia et al., 2008, 2014 for leaf fungal endophytes;
mutualist bacteria reviewed by Griffin & Carson, 2015). Griffin
et al. (2016) recently demonstrated that antibiotics in general
increased tropical seedling growth and here, antibiotics
increased leaf production for four of five species (Fig. S2).
Moreover, we found that in four cases, antibiotics, in combina-
tion with the addition of a single macronutrient, increased leaf
production (Figs 5, 6). Thus, our findings suggest that bacteria
typically function as plant pathogens. In one case, however,
antibiotics in combination with a single macronutrient
decreased performance (Figs 5, 6). Because the mechanisms are
0.04
Alseis
L
L 
(a
nt
ib
io
tic
) –
 L
 (c
on
tro
l)
L 
(a
nt
ib
io
tic
) –
 L
 (c
on
tro
l)
Heisteria
Sorocea Tetragastris
0.03
0.02
0.01
0.00
–0.01
–0.02
0.04
0.03
0.02
0.01
0.00
–0.01
–0.02
0.04
–K
+K
–N +N –N +N
Treatment Treatment
–K
+K
–K
+K–K
+K
0.03
0.02
0.01
0.00
–0.01
–0.02
0.04
0.03
0.02
0.01
0.00
–0.01
–0.02
(a) (b)
(c) (d)
Fig. 6 Significant effects of antibiotic applications and nitrogen (N)9 potassium (K) on leaf production rates (L) among seedlings of Alseis blackiana,
Heisteria concinna, Sorocea affinis and Tetragastris panamensis after 29months of applications and nutrient enrichment. When bars are above the line,
antibiotic applications increased leaf production and when below the line, antibiotic applications decreased production. All panels illustrate significant effects
of antibiotics and N9 K on leaf production rates (significant antibiotics9N9 K interaction; F5,20 = 4.45, P = 0.0069). (a, c, d) For Alseis, Sorocea and
Tetragastris, applying antibiotics increased leaf production when K was added; however, antibiotics had no effect on leaf production when K and N were
added together (Alseis: P = 0.0064; Sorocea: P = 0.0006; Tetragastris: P < 0.0001). (b) For Heisteria, applying antibiotics decreased leaf production when K
was added, however, antibiotics had no effect on leaf production when K and N were added together (P = 0.0022). Bars represent mean values  SE.
New Phytologist (2017) 
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still unclear, future experiments are needed using genomic tech-
niques to critically evaluate how antibiotics alter bacterial
communities to allow for a deeper understanding of how particu-
lar bacterial taxa drive leaf dynamics. Moreover, it is possible
that antibiotic applications cause direct effects (e.g. toxic) on
nontarget organisms (e.g. fungi); however, empirical studies
have looked for this and found little evidence (Ingham & Cole-
man, 1984; Colinas et al., 1994; Chopra & Roberts, 2001;
Thiele-Bruhn & Beck, 2005). Nevertheless, more reductionist
approaches are imperative to link bacterial communities to plant
physiology and plant performance.
We suggest that future studies address the mechanisms and
pathways by which foliar bacteria and other enemies impact
plant hosts in situ. On the one hand, enemy attacks often trigger
plants to produce proteins, phenolics and alkaloids for protection
from both pathogens and insects; thus, bacterial infection may
induce plant defenses against insect enemies (Tierens et al., 2001;
Thomma et al., 2002; Wittstock & Gershenzon, 2002; Haq et al.,
2004; Kniskern et al., 2007). On the other hand, pathogen infec-
tion may upregulate the salicylic acid pathway. If so, this directly
inhibits the jasmonic acid pathway, which is a key pathway that
enhances plant defense against herbivores (e.g. Traw et al., 2003;
reviewed by Stout et al., 2006; Bari & Jones, 2009; Pieterse et al.,
2009; Thaler et al., 2012). Thus, upregulation of microbial defenses
in response to attack may leave the host plants more vulnerable to
herbivore attack (Glazebrook, 2005; Koornneef & Pieterse, 2008).
Studies addressing the mechanisms underlying enemy attack,
however, have almost exclusively focused on Arabidopsis thaliana
or agricultural crop species. Thus, future studies should address the
interactions among microbes, herbivores and plant hosts, in species-
rich ecosystems where enemy pressure is substantial.
Implications for plant diversity maintenance and niche
differentiation
Studies have demonstrated that increased soil nutrient availability
decreases realized niche space of co-occurring species, yet it
remains uncertain whether soil nutrients alone can maintain
hyper-diversity of plant species in tropical forests (e.g. Hubbell
et al., 1999; Hubbell, 2001; reviewed by Wright, 2002; Silver-
town, 2004; Kitajima & Poorter, 2008). To date, the mechanism
underlying this niche dimension is often assumed to be direct
resource competition, where different plant species are better or
worse competitors along resource gradients (Tilman, 1982).
Thus, spatial variation in soil nutrient availability favors different
species in different places. Here, we demonstrate that foliar bacte-
ria are critical mediators of interactions between plants and soil
resource availability, supporting other studies that demonstrate
the importance of plant–microbial interactions in diversity main-
tenance (e.g. Mangan et al., 2010; Schnitzer et al., 2011; Pender-
gast et al., 2013). In fact, some have proposed that plant-
associated microbes can act as stabilizing factors to increase differ-
ences in species’ performance outcomes along resource gradients
or among interactions with other trophic levels (e.g. Chesson,
2000; reviewed by Bever et al., 2010; Mordecai, 2011). In this
framework, such stabilizing processes cause intraspecific effects to
be more negative than interspecific differences (Chesson, 2000).
Thus, when any single species increases in abundance, its per cap-
ita growth rate slows relative to growth rates of other species,
which aids in species coexistence (Chesson, 2000). Indeed, we
found that foliar bacteria caused co-occurring plant species to
0.06 Desmopsis
Heisteria
Sorocea
–N +N
Treatment
–K
+K
–K
+K
–K
+K0.04
0.02
0.00
–0.02
–0.04
–0.06
0.06
0.04
0.02
0.00
–0.02
–0.04
–0.06
0.06
0.04
0.02
0.00
H
 (a
nt
ib
io
tic
) –
 H
 (c
on
tro
l)
H
 (a
nt
ib
io
tic
) –
 H
 (c
on
tro
l)
H
 (a
nt
ib
io
tic
) –
 H
 (c
on
tro
l)
–0.02
–0.04
–0.06
(a)
(b)
(c)
Fig. 7 Significant effects of antibiotic applications and nitrogen
(N)9 potassium (K) on herbivore damage rates (H) among seedlings of
Desmopsis panamensis, Heisteria concinna, and Sorocea affinis after
14months of applications and nutrient enrichment. When bars are above
the line, antibiotic applications increased herbivore damage and when
below the line, antibiotic applications decreased herbivore damage. (a–c)
For Desmopsis, Heisteria and Sorocea, applying antibiotics decreased
damage when neither N nor K was applied, or when both were applied
together; conversely, antibiotics increased damage when just N was
applied (significant antibiotics9N9 K interaction; F4,20 = 5.05,
P = 0.0353; Desmopsis: P = 0.0041; Heisteria: P < 0.0001; Sorocea:
P = 0.0005). Bars represent mean values  SE.

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perform in some cases better or in other cases worse among dif-
ferent soil resource treatments. This suggests that bacteria interact
with a familiar and long-standing key niche axis (soil nutrient
availability and its degree of patchiness). Although speculative,
we argue that plant–bacterial interactions more finely divide
niche differences among coexisting plant species and thus func-
tion to promote plant diversity.
Acknowledgements
We thank Jonathan Pruitt, Brian Traw, Sarah Pasquini, Michelle
Spicer, Nick Keiser, Nathan Brouwer and two anonymous review-
ers for helpful comments and discussions that greatly improved
the quality of the manuscript. We thank Omar Hern
andez,
Rufino Gonz
alez and Severino Fernandez for plant identification
and help with antibiotic applications in the field. We acknowledge
financial support from a National Science Foundation Graduate
Research Fellowship, a Smithsonian Tropical Research Institute
Predoctoral Fellowship, a Lewis and Clark Fund for Exploration
and Research, two Sigma Xi Grants-in-Aid of Research, and the
University of Pittsburgh, all of which supported E.A.G.’s research.
A Central Research Development Fund from the University of
Pittsburgh provided funds for W.P.C. and E.A.G.
Author contributions
E.A.G., S.J.W. and W.P.C. planned and designed the experi-
ment; E.A.G. performed the experiment and conducted field-
work; P.J.M. analyzed the data; and E.A.G., S.J.W. and W.P.C.
wrote the manuscript.
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Supporting Information
Additional Supporting Information may be found online in the
Supporting Information tab for this article:
Fig. S1 Map of study area showing the placement of nutrient
treatments within the study site.
Fig. S2 The impacts of antibiotic applications on rank-order per-
formance among plant performance metrics (leaf production,
growth rate and herbivore damage).
Methods S1 Full description of linear discriminant function
analysis.
Please note: Wiley Blackwell are not responsible for the content
or functionality of any Supporting Information supplied by the
authors. Any queries (other than missing material) should be
directed to the New Phytologist Central Office.
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