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Plant Ecol (2007) 193:211–222
DOI 10.1007/s11258-006-9259-4
O R I G I N A L A RT I C L E
Seed characteristics and susceptibility to pathogen attack in
tree seeds of the Peruvian Amazon
Elizabeth G. Pringle Æ Patricia Álvarez-Loayza Æ
John Terborgh
Received: 30 July 2006 / Accepted: 11 December 2006 / Published online: 23 January 2007
Springer Science+Business Media B.V. 2007
Abstract Many studies now suggest that pathogens can cause high levels of mortality in seeds
and seedlings. Recruitment from seed to sapling is
an important bottleneck for many tree species,
and if specialist or generalist pathogens have
differential negative effects among species of
juvenile trees, then they may have a significant
impact on forest community structure. To explore
the effects of differential pathogen attack among
tropical tree species, we quantified pathogen
attack on the seeds of 16 tree species from the
southeastern Peruvian Amazon and asked which
seed characteristics, including size, hardness, germination time and mode, shade tolerance, and
fruit type, were most closely correlated with
susceptibility to pathogens. Shade tolerance and
seed weight were positively and significantly
E. G. Pringle P. Álvarez-Loayza J. Terborgh
Center for Tropical Conservation, Duke University,
PO Box 90381, Durham, NC 27708, USA
Present Address:
E. G. Pringle (&)
Department of Biological Sciences, Stanford
University, Stanford, CA 94305, USA
e-mail: [email protected]
Present Address:
P. Álvarez-Loayza
Department of Plant Biology and Pathology, Rutgers
University, 59 Dudley Road, New Brunswick, NJ
08901, USA
correlated with susceptibility to pathogen attack
by ecological trait regressions (ETRs), and correspondence analysis indicated that there might
be increased attack rates in species with brightly
colored, pulpy fruits (often dispersed by primates). Only shade tolerance was significantly
correlated with pathogen attack when the analyses accounted for phylogenetic relatedness between species. Thus, contrary to standard
predictions of size-defense ratios, our results
suggest that larger, shade-tolerant seeds tend to
be more susceptible to pathogen attack than
smaller, light-dependent seeds. Moreover, differential pathogen attack may shape seed community composition, which may affect the successful
recruitment of adults.
Keywords Light dependence Fungi Plant
pathogens Recruitment limitation Seed
dispersal Seed weight
Introduction
The role that plant pathogens play in natural
forest communities is the subject of a growing
body of research but remains poorly understood
(Coley and Barone 1996; Gilbert 2002). Janzen
(1970) and Connell (1971) proposed that hostspecific seed and seedling predators could have a
major effect on forest community composition
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and structure. Indeed, recruitment limitation as a
result of high mortality among seeds and seedlings appears be an important phenomenon for
many tree species (Clark et al. 1999), and these
bottlenecks may play a particularly important
role in tropical forests, contributing to their
unique floral diversity (Hubbell et al. 1999).
Although both animal and microbial predators
could be important mortality agents for juvenile
plants, knowledge of the specific effects of
pathogens has lagged behind that of animals,
partly because of ecologists’ limited understanding of pathogen dispersal and host-specificity in
natural systems (Burdon 1987; Gilbert 2002).
Previous studies that have looked at soil-borne
pathogens in juvenile plants have generally shown
significantly negative effects of both true fungi
and oomycetes in a variety of ecosystems (Augspurger 1984; Crist and Friese 1993; Lonsdale
1993; Dalling et al. 1998; Packer and Clay 2000;
Hood et al. 2004), and the few reports that have
examined soil-borne pathogen effects on several
species in parallel suggest that the severity of
these effects also varies among species (Augspurger 1984; Augspurger and Kelly 1984; Burdon
1987; Dalling et al. 1998). Thus, host-specific
pathogens could potentially play an important
role in recruitment limitation and therefore in
shaping forest communities. Moreover, if susceptibility to generalist pathogens, which may include
Aspergillus spp., Penicillum spp., and Cephaleuros
virescens (P. Álvarez-Loayza unpublished data),
varies among species, then non-host-specific
pathogens could also affect community composition.
Theory predicts that there will be trade-offs
among offspring traits when a given amount of
parental energy is expended in reproduction, and
further, that these trade-offs will result in the
correlation of certain traits (Moles and Westoby
2004). For example, small seeds generally have
less physical protection and are shade intolerant,
lacking the nutrient reserves needed to germinate
successfully from deep in the soil or emerge
above existing ground vegetation (Foster and
Janson 1985; Mazer 1989; Pons 1992). In contrast,
larger seeds usually have both thicker seed coats
and greater reserves (Howe and Richter 1982;
Westoby et al. 1996; Pearson et al. 2002; Moles
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Plant Ecol (2007) 193:211–222
and Westoby 2004). However, the production of
larger seeds generally results in a smaller number
of seeds per unit of parental investment (Smith
and Fretwell 1974; Westoby et al. 1996; Henery
and Westoby 2001), and host-specific predators
may be more prevalent in larger-seeded species
(Janzen 1969; Hubbell 1979).
Among the few studies that have addressed the
effects of disease in natural plant communities,
fewer still have looked at the effects of pathogens
specifically on seeds (but see Crist and Friese
1993; Lonsdale 1993; Dalling et al. 1998; Schafer
and Kotanen 2004). In addition, little is known
about the relationships between seed traits and
susceptibility to disease because these previous
investigations have typically involved only one or
two species. In one of the few exceptions, Augspurger and Kelly (1984) found no significant
relationship between seed size and pathogen
attack on new seedlings of 18 tree species in a
wet forest in Panama. However, that study was
confined to wind-dispersed species, all of which
have relatively small seeds for tropical trees
(Westoby et al. 1996).
In this study, we tested various predictions
about the relationships between seed traits and
susceptibility to disease, including: (i) seeds with
longer germination times should be less susceptible because they must withstand longer exposure to pathogens; (ii) seeds with harder and/or
thicker coats should be less susceptible; (iii) seeds
whose germination modes result in retention of
greater energy reserves should be less susceptible
because of their greater ability to recover from
attack; (iv) dispersal mode, which presumably
selects for a variety of seed traits (Gautier-Hion
et al. 1985; Hammond and Brown 1995), should
be correlated with susceptibility; (v) shade-tolerant species should be less susceptible than lightdependent species because pathogens tend to be
more prevalent in shaded, moister soil (Augspurger 1984); (vi) large-seeded species should be less
susceptible than small-seeded species because
large seeds typically have both greater energy
reserves and harder and/or thicker coats; and (vii)
as suggested by previous studies of the effects of
light variation and fungal pathogens on the
success of seedlings (Augspurger 1984; Augspurger and Kelly 1984; Hood et al. 2004), ungermi-
Plant Ecol (2007) 193:211–222
nated seeds should also be more susceptible to
pathogens under greater shade and be attacked
less frequently in the presence of a broad-acting
fungicide.
We designed a series of experiments to evaluate these relationships and potential trade-offs
using seeds of 16 tree species from the southeastern Peruvian Amazon, analyzing the data with
inter-specific ecological trait regressions (ETRs)
and phylogenetically independent contrasts
(PICs) (Felsenstein 1985). Our results represent
a first step towards making predictions about the
effect of pathogens on seed communities and how
these effects may influence long-term forest
composition.
Methods
Study site and species
The experiments were performed over a period of
7 months (September 2004–March 2005) in a
greenhouse in a clearing at Cocha Cashu Biological Station, located in Manu National Park, Perú
(1151¢ S, 7119¢ W; see Terborgh 1990 for
description). Seeds of 16 species were collected
Fig. 1 Tree species and characteristics, with diagram of
tentative phylogenetic relationships. Seed weight represents a species average ± SD; shade tolerance is measured
as number of saplings per adult per hectare; fruit type is a
list of characteristics with the order: fruit color, pulp
attachment score (0–3, None–High), dehiscence (I or D);
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and planted while the parent tree was still
fruiting, and thus within about 2 weeks of landing
on the forest floor. Species were chosen to
represent a variety of taxonomic groups, dispersal
modes, life histories, fruiting phenology, and
abundance (Fig. 1). We included only species
represented by at least five fruiting adults within
the station’s trail system, with the exception of
Swietenia macrophylla (mahogany), which was
included with only two fruiting adults due to its
distinction as a valuable, highly endangered
timber species.
Seed preparation and experimental procedure
Immediately after collection, seeds were scrubbed
clean of pulp and allowed to air-dry for 1–3 days
before weighing or planting, a procedure that
yields germination success similar to that of
naturally dispersed seeds (Leiberman and Leiberman 1986; Palmeirim et al. 1989). In order to
begin with the highest possible percentage of
viable seeds, all seeds were examined visually for
damage, and non-floating species were checked
for pre-experiment insect infestation by placing
them in water; only seeds appearing healthy and
un-infested were planted.
and pathogen susceptibility is measured as the number of
seeds observed with pathogens divided by the total
number of seeds (n = 150 for each species). Note that in
the case of S. mombin, seeds that suffered from pathogen
attack frequently survived. For details see ‘‘Methods’’
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Soil was collected from within tree plots
surrounding the station (Terborgh et al. 2002).
Seeds were placed individually in the soil so that
approximately half of each seed remained exposed to the surface for easier observation, and 10
seeds from a single adult were planted per
9 · 9 · 9-cm open-topped bag of soil; a total of
150 seeds was planted for each species. Soil bags
had holes to allow drainage and were watered
every other day or with enough frequency to keep
the soil consistently moist. Each species was
maintained for 3 months, and observations of all
seeds were made every 6 d, recording germination, seedling establishment, and presence or
absence of pathogens as assessed by visual
inspection.
After 3 months, seedlings were measured, and
all seeds that had not germinated and could be
found (>90% of the ungerminated seeds for each
species) were inspected, scored as alive or dead
according to a visual examination of embryonic
tissue, and, if dead, assigned an apparent cause of
death: externally visible pathogen attack; internal
decomposition; failed germination; or insect damage that had occurred since planting. Seeds that
were scored as ‘‘with pathogens’’ included seeds
that had visible fatal or non-fatal attack before
germination during the 3-month observation
period and those that never germinated and were
scored as decomposed at the end of the experiment. No distinction was made between true
fungi, oomycetes, and bacterial pathogens for
these experiments. The few seeds that could not
be found at the end of the experiment were not
included in the analyses. We considered the
proportion of seeds attacked by pathogens to be
a measure of susceptibility to pathogens.
Seed characteristics
We determined several seed characteristics for
each species, including average fresh seed weight
of ~30 seeds, median germination time, germination mode, seed hardness, fruit type, and shade
tolerance (Fig. 1, Appendix 1, and see below).
The median number of days to germination was
calculated for the seeds that germinated in each
soil bag replicate and averaged for each species.
Germination mode is described as either
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hypogeal, when the germinating seed retains its
cotyledons below ground as stored energy, or
epigeal, when the germinating seed is elevated
above ground and sheds its seed coat to expose
photosynthetic cotyledons. Seed hardness was
used to estimate seed-coat thickness and evaluated by biting a subset of seeds of each species
and ranking them from 1–4: 1 = falls apart easily,
disintegrates; 2 = possible to break but pieces
stay firm; 3 = possible to leave marks or crack
with a lot of force; 4 = nearly impossible to crack.
Gautier-Hion et al. (1985) found that fruit
color, dehiscence, and pulp type were significantly
related to disperser choice in an Old World
tropical forest, and these findings are supported
by observations of dispersers in Manu (P.
Álvarez-Loayza unpublished data). Thus, due to
the difficulty of objectively identifying seed dispersers in a fauna-rich tropical forest, we used
these three fruit traits to approximate mode of
dispersal. According to Gautier-Hion et al.
(1985), orange and yellow indehiscent fruits with
juicy, sticky pulp tend to be dispersed by primates; red and purple dehiscent fruits with more
easily detached aril pulp tend to be dispersed by
birds; and brown indehiscent fruits with fibrous
pulp tend to be dispersed abiotically or by
terrestrial mammals. Each type of fruit was given
a binary presence/absence score for each nominal
category of color, dehiscence, and ranked score
corresponding to the strength of attachment of
fruit pulp to seeds during the efforts to completely clean them (0–3, None–High), and species
were then treated as replicates in a design matrix
that was analyzed by ordination (see ‘‘Data
analysis’’).
Finally, a continuous scale of juvenile shade
tolerance was approximated by the number of
tagged and identified saplings per adult in a
1 hectare plot of mature forest free of large gaps
and adjacent to the station (Appendix 1); the tree
and sapling plots were established in 1974–1975
and 1993, respectively (Terborgh et al. 2002). The
number of saplings of each species growing under
the forest canopy serves as an indication of the
ability of juveniles to establish in the shade, and
that number was divided by the number of tagged
adults in the plot to correct for species abundance. The total number of saplings should be
Plant Ecol (2007) 193:211–222
affected little, if at all, by total seed input because
of the severity of recruitment limitation in tropical forests (Hubbell 1979; Hubbell et al. 1999).
Shade and fungicide experiments
Additional experiments to test the effects of light
levels and fungicide on pathogen success were
conducted with two of the study species, Spondias
mombin and Sapium marmieri. Shade consisted
of two layers of 2-mm mesh hung 10 cm above
germinating seeds. Fungicide treatments were
conducted with Captan (N-trichloromethylthio4-cyclahexene-1,2-dicarboximide), which is active
against both true fungi and oomycetes (G.S.
Gilbert personal communication). Seeds were
soaked in 1 g/l Captan solution for 3 min before
planting, and seeds and soil were sprayed with the
same solution every 2 weeks thereafter. Shaded
and unshaded control seeds were mock-treated
with water.
Data analysis
Statistical analyses were performed using the
statistical software JMP IN 5.1.2 (SAS Institute
2004), except as described below. Seed weights
are reported ± SD, and all other experimental
means are reported ± SE. Nonparametric tests
were used when soil bags were considered separately instead of being averaged by species in
order to handle the large numbers of zeros
without violating the assumptions of ANOVA;
ANOVA was used for all other tests of significance except as noted. In all tests that assume
normality and homogeneity of variance, seed
weights, and the fractions of seeds attacked by
pathogens were log-transformed, and the data
from the additional experiments on S. mombin
and S. marmieri were arcsine-transformed.
Explained variance of multi-variate species
comparisons was calculated using step regression
in a general linear model, but because both seed
weight and light dependence, which we treat as
variables in the x dimension, are measured with
error, the assumptions of model I-type linear
regression are not fully satisfied, and the slopes of
the predictive equations are inaccurate. Thus, to
minimize the residual variance in both x and y
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and calculate the proportional relationship between them (Sokal and Rohlf 1995), we also
performed model II-type Standardized Major
Axis (SMA) regressions using the program
(S)MATR (Falster et al. 2003).
Multiple correspondence analysis (MCA) ordination was performed on a binary design matrix
of fruit traits using CANOCO 4.5 (Ter Braak and
Smilauer 2002), fitting a unimodal model and
biplot scaling with a focus on inter-species
distances. Fruit traits were designated ‘‘species’’
in the CANOCO matrix, and species’ fruits were
designated as ‘‘samples’’, i.e., replicates. Frequency of pathogen attack was then described
as one of three unique categorical variables: low
(<mean – SD/2), medium (mean ± SD/2), or high
(>mean + SD/2). Pathogen attack was then added
as a supplementary variable in the design matrix
and analyzed as part of the MCA in order to
obtain an estimate of the relationship between
pathogen attack and fruit traits, a technique
known as predictive mapping (StatSoft 2006).
The ordination diagram of the first two axes
produced from the MCA output illustrates both
species values, which approximate inter-species
covariances, and the v2 distances between these
values, which approximate the similarity of each
value to the next. Apeiba membranacea was
removed as a color replicate because it was the
only black fruit.
Finally, in addition to ETRs, in which pairs of
traits were assigned coordinates corresponding to
each species, we also analyzed PICs of traits of
interest to correct for phylogenetic relatedness. A
phylogeny of the species was assembled in the
program Phylomatic (Webb and Donoghue 2005)
using data from the Angiosperm Phylogeny
Group tree (Fig. 1). Species that were not recognized by the program were added as polytomies
(unresolved relationships) by hand according to
Gentry (1996); three of these species, which are
members of the family Bombacaceae (Matisia
spp. and Quararibea witti), were identified as
congenerics by Gentry and are treated as such in
our analysis (Fig. 1). PICs of log-transformed
data were calculated using the software Phylocom
(Webb et al. 2004; see Moles et al. 2005 for
description of methods), which can analyze trees
with polytomies; branch lengths were designated
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Plant Ecol (2007) 193:211–222
equal. Contrasts were then analyzed as linear
regressions, except that lines of fit were forced
through the origin (Garland et al. 1992).
Results
Germination success averaged 43% over all
species. The proportion of seeds attacked by
pathogens ranged from 0 to 0.75 (Fig. 1) and
varied significantly among the species analyzed
(Wilcoxon Rank Test, P < 0.0001). Among soil
bags in which at least one seed was attacked by
pathogens, the total number of seeds attacked per
bag exhibited a downward-sloping distribution
(data not shown), suggesting that the probability
of attack for each seed was independent of
whether a pathogen was attacking another seed
in the same bag. Visible pathogen attack led to
mortality in ~90% of all cases, with the exception
of seeds of S. mombin, which often suffered a
brief period of attack by a locally common
saprophyte but later produced healthy seedlings.
tributed to a total inertia, or Pearson v2 for the
design matrix divided by the total number of
observations, of 2.5. Eigenvalues for the first two
axes that are closer to 1 than to 0 indicate a
relatively high degree of correspondence between
traits (Leps and Smilauer 2003); these values
especially reflect traits that are distant from the
origin and cluster together. A plot of the first two
axes (Fig. 2) showed that high indices of pathogen
attack clustered around the negative end of the
first axis with fruit traits that are associated with
dispersal by primates: orange/yellow fruits; indehiscence; and juicy pulp, as approximated by high
adherence of pulp to the seed (categories 2 and
3). In addition, red/purple fruits, dehiscent fruits,
and intermediate pulp adhesion (fruit traits associated with dispersal by birds) clustered around
the positive end of the first axis, and, although
Relationships between seed characteristics
and pathogen susceptibility
Several of the seed traits that we tested were not
significantly correlated by ETRs with pathogen
susceptibility. These included median germination time (P = 0.13), seed hardness (P = 0.3), and
germination mode (P = 0.08), although it should
be noted that the mean proportion of seeds
attacked was lower for seeds with hypogeal
germination (0.09 ± 0.04) than for those with
epigeal germination (0.33 ± 0.14).
However, fruit traits were related to susceptibility to pathogens by MCA ordination. The first
four axes of the MCA explained 77% of the
variance in our trait data (Table 1), which conTable 1 Summarized results of the MCA of fruit traits
and pathogen susceptibility
Axis 1 Axis 2 Axis 3 Axis 4
Eigenvalues
0.657
Cumulative
26.3
percentage
variance of trait data
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0.522
47.1
0.416
63.8
0.34
77.4
Fig. 2 Trait values and inter-trait distances for the first
two axes obtained in an MCA. The first axis is horizontal;
the second axis is vertical. Gray circles indicate groups of
traits discussed in the text. 0–3 pulp = strength of
attachment of fruit pulp to seeds, see ‘‘Methods’’; colors
(brown, green, orange/yellow, red/purple) = fruit color;
D = dehiscent; HPA = high pathogen attack; I = indehiscent; LPA = low pathogen attack; MPA = medium pathogen attack
Plant Ecol (2007) 193:211–222
slightly less clustered, brown fruits and zero pulp
adhesion (fruit traits associated with dispersal by
wind or terrestrial mammals) fell along the
second axis near low pathogen attack. The first
two axes for fruit traits explained 26% of the
variation in high pathogen attack, 58% of the
variation in medium pathogen attack, and 55% of
the variation in low pathogen attack; these
percentages reflect the fractions of the weighted
regression sums of squares of each variable over
the total sums of squares for all the traits.
Interestingly, both shade tolerance and seed
weight were positively and significantly correlated
with the frequency of detectable pathogen infection over the 16 species (Fig. 3). SMA regression
217
led to steeper slopes for the ETRs between
pathogen susceptibility and shade tolerance or
seed weight (0.20 ± 0.14 and 0.38 ± 0.18, respectively) than were calculated through linear regression (0.11 and 0.23, respectively). Stepwise
regression (Forward direction, P to enter = 0.15)
showed that these two variables, along with a
non-significant interaction effect (P = 0.11), accounted for 66% of the variation in the frequency
of pathogen attack (Table 2). In order to investigate whether these relationships were conflated
by evolutionary relationships between species, we
also calculated PICs for susceptibility to pathogens, seed weight, and shade tolerance, and
regressed contrasts for susceptibility against those
for seed weight and shade tolerance. There was a
significant, positive correlation between PICs for
shade tolerance and pathogen attack (P < 0.02),
but the positive correlation between PICs for seed
weight and pathogen attack was not significant
(P = 0.08).
Effects of light levels and fungicide
Consistent with previous experiments on seedlings
(Augspurger 1984; Augspurger and Kelly 1984;
Hood et al. 2004), we found a higher frequency of
pathogen infection among ungerminated seeds in
the shade than in the higher-light control and
fungicide treatments for both species tested
(Fig. 4). Approximately 6.0 ± 2% of S. marmieri
seeds were attacked in the shade treatment, which
was significantly higher than the zero seeds
attacked by pathogens in both the control and
fungicide treatments (Student’s t-test, P < 0.001).
Table 2 Full model statistics for the effect of seed weight
and saplings per adult as a proxy for shade tolerance on the
proportion of seeds attacked by pathogens (data grouped
by species)
Fig. 3 SMA regressions of the positive ETR relationships
between susceptibility to fungal attack and (a) seed weight
(P < 0.005) and (b) shade tolerance, estimated by the
number of saplings per adult (P < 0.01). Arrows indicate
outlier species, which were included in the regression
analysis, but which may have special properties (see
‘‘Discussion’’)
Fixed effect
df
F
P
Explained
variation (R2)a
Seed weight
Saplings/adult
Seed weight ·
saplings/adult
1
1
1
12.37
10.69
3.06
0.0042
0.0067
0.1059
0.35
0.22
0.09
a
R2 values indicate the contribution of each effect to the
model’s total explained variation (R2 = 0.66)
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Plant Ecol (2007) 193:211–222
Germination time, seed hardness, and
germination mode
Fig. 4 Proportion + SE of Spondias mombin (black) and
Sapium marmieri (hatched) seeds with pathogens under
shade cloth, control greenhouse conditions, or fungicide
treatment (for each treatment, n = 15 soil bags, each with
10 seeds). Letters indicate statistically significant differences among means of arcsine-transformed data by
Student’s t-test comparisons (P < 0.05 for S. mombin;
P < 0.001 for S. marmieri). Note that in the case of
S. mombin, seeds that suffered from pathogen attack
frequently survived (see ‘‘Results’’)
Similarly, in S. mombin, 26 ± 6% of seeds were
attacked by pathogens in the shade as opposed to
21 ± 4% in the higher-light control, and an even
smaller proportion of seeds was attacked in the
fungicide treatment (11 ± 3%). However, the only
statistically significant difference for S. mombin
was between the mean frequency of attack in the
shade and fungicide treatments (Student’s t-test,
P < 0.05).
Discussion
In this study, we examined the relationships
between seed traits and susceptibility to pathogens. Contrary to prediction, we found that there
was no significant relationship between either
germination time or seed hardness and susceptibility to pathogens, that shade-tolerant seeds
were generally more susceptible than light-dependent seeds, and that larger seeds were generally
more susceptible than smaller seeds. However, in
accord with prediction, we found that hypogeal
germinators had a lower mean susceptibility than
epigeal germinators, that certain fruit traits were
related to pathogen susceptibility, and that seeds
of both S. mombin and S. marmieri were more
susceptible to pathogens in the shade.
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Overall germination success was relatively high,
perhaps because of the favorable growth conditions (relatively high-light levels, regular watering) provided by the greenhouse. We expected to
find that seeds that germinate more slowly and
have harder endocarps were better defended
against pathogen attack, but we found no correlation between these traits. Although the correlation between germination mode and pathogen
attack was not statistically significant, the mean
susceptibility of hypogeal germinators was less
than that of epigeal germinators. Epigeal species
tend to germinate quickly and may thus invest
little in immediate pre- and post-germination
defense (Coley et al. 1985), whereas hypogeal
germinators have substantial ability to recover
from early seedling damage due to their withheld
energetic reserves (Green and Juniper 2004). Our
data suggest that hypogeal seeds themselves may
also be more resistant to attack. Additional
experiments with higher sample sizes will be
necessary to determine whether this relationship
is indeed significant.
Fruit type
The relationship between fruit type (and thereby
presumed primary disperser) and susceptibility to
pathogen attack may be caused by relationships
between unidentified common qualities of these
seeds. For example, dispersers may select for seed
shape (Howe and Vandekerckhove 1981), and
differences in surface:volume ratios or in surface
texture may affect pathogen success. However,
Lambert (2001) found that a primate-dispersed
tree species benefited from dispersal in part
because seeds that fell directly from the parent
tree with sticky pulp, typical of primate-dispersed
species, had much higher rates of fungal attack
than seeds that had been spat out by dispersers.
Thus, our result that primate-dispersed seeds
tended to be more susceptible to pathogen attack
may simply indicate differences between natural
dispersal and even the most careful experimentally imposed dispersal, in which some pulp may
have remained.
Plant Ecol (2007) 193:211–222
Light dependence and seed weight
Although we predicted that shade-tolerant seeds
would need to be better defended because they
germinate in shaded soil where pathogens may be
more prevalent, the positive ecological and evolutionary correlation between high-light dependence and low susceptibility to pathogen attack is
probably advantageous to gap-colonizing species,
as their seeds may need to persist for long periods
in shaded soil before they encounter high-light
conditions (Pons 1992). In addition, the positively
correlated ETRs between seed weight and pathogen attack suggests either that seed weight itself
plays a role in pathogen susceptibility or, perhaps,
that there are selective pressures for gap colonizers to produce seeds that are small as well as
pathogen resistant. The relationship between seed
weight and susceptibility remained positive but
lost statistical significance when the relationships
were analyzed using PICs. However, the low
baseline rates of pathogen attack may mean that
this correlation would be difficult to detect
reliably with PICs without larger sample sizes
and a better resolved phylogenetic tree.
Previous observations of soil seed-bank composition have shown that light-dependent, pioneer
species and, to a lesser extent, smaller-seeded
species are over-represented in comparison to
shade-tolerant, primary-forest, larger-seeded
species (Hopkins and Graham 1987; Garwood
1989; Thompson 1992; Dalling et al. 1997; Bekker
et al. 1998; Hulme 1998; Moles et al. 2000; Pearson
et al. 2002). Although this phenomenon can be
explained in part by the greater numbers of
smaller, light-dependent seeds produced, it also
seems possible that the greater resistance of many
light-dependent seeds to pathogen attack and
decomposition contributes to this effect.
Pearson et al. (2002) proposed that larger seeds
are better defended than smaller seeds against
both seed predators and pathogens because they
have thicker seed coats. However, this proposal
was based on a study exclusively of pioneer
species. Among groups of species that span a
wider range of light dependence, there is equivocal evidence on the relationship between seed
size and predation (Moles et al. 2003), and we
have shown here that seed size may be positively
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correlated with susceptibility to pathogen attack.
Also in contrast to our results, Dalling et al.
(1998) found a >90% mortality rate in two species
of pioneers, which was attributable in large part
to attack by fungal pathogens. However, these
experiments were carried out with seeds that were
80- and 500-fold smaller, respectively, than the
smallest seeds in our experiments, and were also
completely buried in soil. There is preliminary
evidence that there may be different frequencies
of pathogen attack on buried seeds than on seeds
exposed to the surface among the species tested
in our study (P. Álvarez-Loayza unpublished
data). Further experiments that include an even
larger range of seed sizes and comparison of
attack on seeds at different depths in the soil are
required to begin to understand the full complexity of seed–pathogen interactions.
Effects of light levels and fungicide
As expected, we found higher frequencies of
pathogen attack in the shade, suggesting the
interesting possibility that there may be spatial
heterogeneity in the importance of seed pathogens in the forest. In addition, the marked
seasonality in Manu, with ~95% of the yearly
2000 mm of rain falling between November and
May (Terborgh 1990), and a concomitant increase
in cloudy days during these months, may also
contribute to temporal variation in pathogen
effects. The reduction of pathogen attack on
S. mombin seeds through the application of
fungicide suggests that a significant proportion
of the pathogens that attacked our seeds were
either fungi or oomycetes.
Generalist vs. specialist pathogens
Schafer and Kotanen (2004) found that the lethality of seed fungal pathogens was highly dependent
on the specific plant–pathogen combination because only some seeds were hosts to specialist
pathogens. In our experiments, we were unable to
isolate the pathogens, and we were thus unable to
differentiate definitively between specialist and
generalist pathogens. However, there were two
sets of unique-looking disease symptoms consisting
of pinkish, thick-stemmed hyphae, resembling
123
220
Plant Ecol (2007) 193:211–222
Rhizostilbella hibisci, and yellow, fuzzy hyphae,
resembling Aspergillus spp., that appeared solely
on Leonia glycycarpa, or Oxandra acuminata,
respectively. The fact that these symptoms were
unique to these two species could indicate that
these pathogens were more specialized, and it is
interesting to note that L. glycycarpa and O. acuminata had some of the highest overall rates of
seed mortality due to pathogens (Fig. 1) and were
in fact outliers in the regression of pathogen attack
by seed weight (Fig. 3a). It is also possible that our
experiments may have underestimated levels of
pathogen attack in the field because the greenhouse provided a refuge from specialist pathogens
that would have attacked in situ.
Conclusions
In this study, detectable pathogen attack on ungerminated seeds of 16 tree species almost always
resulted in mortality; in fact, S. mombin was the
only species whose seeds regularly managed to
escape mortality after apparent attack. Thus, we
conclude that pathogens are potentially important
agents of seed mortality in forest communities.
Unlike the effects of specialist predators and
pathogens, which may contribute to spatial separation among conspecifics by intense effects near
adult trees (Janzen 1970; Connell 1971), especially
of more common species (Leigh et al. 2004),
generalist predators and pathogens with differential
effects may increase spatial and temporal heterogeneity in forest species composition in less predictable or ordered ways. Future experiments
addressing the relationships between physical and
chemical plant defenses and pathogen attack, as
well as the specificity and distribution of pathogens
in the field, especially when phylogenetic relationships are considered in the experimental design,
should eventually shed more light on the impact of
soil pathogens on plant recruitment and survival.
Acknowledgments The Instituto Nacional de Recursos
Naturales in Perú kindly granted the permits to conduct
this research. We are grateful to N. Quinteros, D. Osorio,
and V. Swamy for helpful conversations in the field. The
manuscript benefited from comments by J. Hille Ris
Lambers, B.S. Mitchell, J.R. and R.M. Pringle, and S.
Ramı́rez. We also thank S. Ramı́rez and S. Russo for help
with the independent contrast analyses and J. Lancaster
for help with the ordination analysis. Thanks to J. White,
K. Seifert, and B. Gerald for help with fungal
identifications. This work was funded in part by the
Andrew Mellon Foundation.
Appendix 1 Tree species (ordered as in Fig. 1) and additional trait information used in the study
Species
No. of saplings/ha No. of adults/ha Germination time (days) Seed hardness Germination mode
Oxandra acuminata
110
Otoba parvifolia
51
Genipa americana
~0.1
Apeiba membranacea
~0.98
Quararibea witti
262
Matisia cordata
21
Matisia rhombifolia
1.2
Swietenia macrophylla ~0.25
Spondias mombin
2.5
Terminalia oblonga
16
Casearia fasiculata
1.0
Chrysoclamys ulei
2.5
Calophyllum
~0.25
brasiliense
Leonia glycycarpa
59
Sapium marmieri
2.5
Brosimum lactescens
~1.05
4.0
30
0.1
3.2
42
5.8
0.25
0.25
4.0
1.2
7.0
0.75
0.25
5.2
6.0
1.8
30.5
57.3
20.4
30
25
16.8
11.4
18.7
30
36.9
29.4
22.3
55.8
±
±
±
±
±
±
±
±
±
±
±
±
±
2.0
1.9
1.2
1.8
3.6
1.2
1.1
1.5
2.2
1.4
4.8
4.5
5.4
2
4
1
2
3
3
3
1
1
2
1
1
4
E
E
E
H
H
E
E
H
H
H
E
H
H
45 ± 8.0
50.8 ± 9.2
24.7 ± 2.7
1
3
1
H
H
H
For species with zero saplings in the tree plot used for the rest of the measurements, an approximate (~) number of saplings
is given based on estimates over a larger area. Germination time is the average median number of days to germination of the
soil bags (n = 15) and is reported ± SE. Seed hardness is evaluated on a scale of 1–4 (soft to very hard). Germination mode
is described as H = hypogeal or E = epigeal. For details see ‘‘Methods’’
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Plant Ecol (2007) 193:211–222
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