Functional diversity in fruit-frugivore interactions: a field experiment
with Mediterranean mammals
José M. Fedriani and Miguel Delibes
J. M. Fedriani (fedriani@ebd.csic.es) and M. Delibes, Estacio´n Biológica de Doñana (CSIC), Avda. Américo Vespucio s/n, Isla de la Cartuja,
ES-41092 Sevilla, Spain.
Using field seed sowings, we assessed how four mammal species (Meles meles, Vulpes vulpes, Sus scrofa, and Oryctolagus
cuniculus) influenced seed germination in three fleshy-fruited Mediterranean shrubs (Corema album, Pyrus bourgaeana,
and Rubus ulmifolius). We predicted that gut passage and removal away from mother plants would enhance the quantity,
speed, and asynchrony of seed germination. Results showed that percent germination was altered by gut passage, but that
the magnitude and even the direction of such effects varied according to plant and disperser species. Likewise, dispersal
away from mother plants affected the percentage and germination speed in some species but not others. Gut passage
increased asynchrony of germination in Rubus and Pyrus, and removal from the mother plant increased asynchrony in
Rubus, which likely enhances plant fitness in unpredictable environments. Gut passage generally had a stronger effect on
germination than removal away from mother plants, but for some species both factors were similarly influential.
Therefore, the combined effects of both seed dispersal services varied individually among fruit and frugivore species,
leading to unusually high functional diversity in this seed dispersal mutualism.
Dispersers provide a central service to plants by moving
their seeds away from the maternal plant to a new
environment, which often results in higher survival,
colonization of vacant areas, and/or genetic flux enhancement (Levin et al. 2003, Howe and Miriti 2004, Spiegel
and Nathan 2007). In addition, plants dispersed in animal
interiors (i.e. endozoochorous) may benefit from other
services, such as changes in the probability and speed of
germination due to scarification, deinhibition, or other
effects that take place during seed processing and delivery
(see reviews in Robertson et al. 2006, Traveset et al. 2007).
However, the magnitude and even the direction of such
effects often depend on the testing environment (laboratory,
greenhouse, field; Robertson et al. 2006, Traveset et al.
2007). For example, because germination depends on
the conditions present in the immediate environment
(resources, pathogens, etc.; Fenner and Thompson 2005),
it is possible that key microhabitat conditions interact with
seed treatment, leading to unexpected patterns of germination. Though we understand the separate outcomes of gut
passage on seed germination, the paucity of field studies
(but see Rodrı́guez-Pérez et al. 2005) has limited our
knowledge. We still know very little about the consequences
of gut passage when acting in concert with other components of the dispersal process, such as the removal from
the mother plant. For instance, what is the relative
importance of gut passage versus removal from mother
plant on different aspects (percent, speed) of endozoochore
germination?
Fleshy-fruited plants are typically dispersed by dozens of
vertebrate species (mostly non-flying mammals and birds,
but also reptiles and bats; Herrera 2002). The origin, preservation, and potential adaptive value of such unspecialized
mutualisms are subjects for current debate (Bolmgren and
Eriksson 2005, Thompson 2005). In particular, Izhaki and
Safriel (1990) proposed that, while passing through
vertebrate guts, seeds experience a variable scarification
process that might diversify their timing of germination (i.e.
asynchrony), which may be advantageous in unpredictable
environments (Cohen 1966, Simons 2007, Venable et al.
2008). Nevertheless, given that vertebrate dispersers deliver
ingested seeds to different microhabitats (Jordano and
Schupps 2000), dispersal from the maternal environment
might also be important in the diversification of germination timing. Even though Izhaki and Safriel’s hypothesis has
received ample support (Izhaki et al. 1995, Traveset et al.
2001, Santamarı́a et al. 2002, Nogales et al. 2006), the joint
roles of gut passage and removal from the mother plant on
the diversification of germination timing in unknown. How
these processes vary among species is unknown and an
investigation could shed light not only on the role of
endozoochory on plant population dynamics, but also on
the generalized nature of most plant-vertebrate disperser
mutualisms.
983
In this study, we experimentally assessed the combined
effects of gut passage and removal away from the mother
plant on the percentage, the speed, and synchrony of
germination of three mammal-dispersed species in a
Mediterranean shrubland. The chosen plant species (Iberian
pear Pyrus bourgaeana, blackberry Rubus ulmifolius, and
Portuguese crowberry Corema album) are all fleshy-fruited,
though they vary in relevant traits such as seed size and coat
thickness. Several herbivorous and carnivorous mammals
with differing morphological and physiological traits consume the fruit of these species in our study area. While we
expected that the effect of passage through mammal guts
generally would enhance the quantity and the speed of
germination, as well as diversify the germination timing (i.e.
asynchrony), we predicted that patterns would vary among
plant and disperser species (Traveset and Verdú 2002,
Traveset et al. 2008). Because mammals transport seeds
away from mother plants, and the resulting fungal pathogens, chemical allelopathy, and mechanical inhibition often
are associated with mother plants (Janzen 1970, Augspurger
1984, Eriksson 1995), we hypothesized that the removal
from the maternal environment would enhance germination. The initiation of germination can be affected by a
myriad of factors such as the temperature (Thompson and
Grime 1983), light environment (Silvertown 1980), and
moisture (Dubrovsky 1996), which often vary at small
spatial scales. Therefore, we predicted that the idiosyncratic
effects of removal from the mother plant on germination
timing would increase the diversification of germination.
To evaluate our predictions, we sowed seeds of the three
plant species that had passed through the gut of four
mammalian dispersers, as well as control seeds, both
beneath and away from reproductive conspecifics (simulating non-dispersed and dispersed seeds, respectively). We
monitored seedling emergence for twenty-one months and
evaluated the combined effects of mammal ingestion and
dispersal away from mother plants on the quantity, speed,
and asynchrony of seed germination.
Methods
Study sites, plants, and dispersers
The study was carried out from November 2005 to
September 2007 at Doñana National Park (510 km2;
3789?N, 6826?W; elevation 0—80 m), located on the right
bank of the Guadalquivir delta in southwestern Spain. The
climate is Mediterranean sub-humid and characterized by
dry, hot summers (June—September) and mild, wet winters
(October—January). Annual rainfall varies widely, ranging
during the last twenty-five years from 170—1028 mm
(mean9SD=583.09221.1 mm). Though most rain (‘80%)
falls between October and March, there is a marked
interannual seasonal variability in rainfall.
The three species (hereafter referred as Rubus, Pyrus and
Corema) are relatively common in Doñ ana, though typically
do not co-occur in the same sites; thus, three sites where
each of the three species is abundant were selected for our
study. Our study sites are also characterized by a variable
understory of Halimium halimifolium, Pistacea lenticus,
Ulex spp., and Juniperus phoenicea, and scattered Quercus
984
suber and Pinus pinea trees. Pyrus is a monoecious small tree
(‘3—6 m high), Rubus is a monoecious shrub (1—2 m
high), and Corema is a dioecious shrub (‘0.5—1 m high).
The three species grow singly or in small clumps, often
separated by open spaces with some forbs and grasses. They
all flower during spring (March—May) and produce fleshy
fruits that ripen during the fall and winter (September—
December; Jordano 1984, Fedriani and Delibes unpubl.).
Their seeds differ in many traits, including size, shape, and
coat thickness (Table 1), which are presumably relevant for
germination.
In Doñ ana, several abundant herbivores (e.g. wild boar
Sus scrofa [40—50 kg], European rabbit Oryctolagus cuniculus
[‘1 kg]) and less common mammalian carnivores (e.g.
Eurasian badger Meles meles [6—8 kg], red fox Vulpes vulpes
[5—7 kg]) are the main dispersers of Pyrus and Corema
(Fedriani and Delibes 2009, unpubl.). Rubus, however, also
includes birds among its major dispersers (Jordano 1984).
As a consequence of their movement behaviors, mammal
species in our study area frequently deliver feces (with
ingested seeds) both beneath and within a few meters from
reproductive plants in open interspaces (Fedriani and
Delibes 2009). Conditions beneath reproductive plants
(e.g. shaded, relatively humid, copious litter) visibly
contrast with conditions present in the open spaces
surrounding them (sunny, dry, less litter).
Mammal-ingested and control seeds
Because we focus on the possible scarification effect of gut
passage on germination, we compared the germination
behavior of control (non-ingested) seeds with that of
mammal-ingested seeds. To avoid pulp inhibitory effects
(Samuels and Levey 2005, Robertson et al. 2006), the pulp
attached to control seeds was removed by hand. For the
purpose of this study, ‘‘removal from the mother plant’’
refers to either ripe fruit being picked by frugivores directly
from fruiting plants (typical for Corema) or fallen fruit
being picked up beneath them (typical for Pyrus). Finally,
germination was defined as the emergence of any seedling
part from the seed (Izhaki and Safriel 1990); thus, we use
seed germination and seedling emergence interchangeably.
To obtain control seeds, ripe fruits were collected in
November 2005 from 15 to 20 individuals of each species
well-distributed throughout their respective study sites.
Viable seeds were extracted from collected fruits and their
pulp was removed. Seeds were pooled into three samples
(one per species) and stored in paper bags in the dark at
room temperature (abortions and seeds showing signs of
pathogens or insect damage were discarded). Mammalingested seeds were removed from fresh feces of wild
animals (‘20 feces per mammal species at each site)
collected in the field (Traveset et al. 2001, Nogales et al.
2005) during the same time period and near the fruiting
plants from which control seeds were obtained. Mammal
feces were identified at the species level on the basis of
shape, odor, and color (Fedriani et al. 1999). Fecal samples
were air dried and, as above, stored individually in paper
bags in the dark at room temperature. Each fecal sample
was later washed using a sieve under running water. Seeds
were immediately and carefully removed and dried. Then,
0.02390.001 (40)
0.01690.001 (40)
0.04090.004 (40)
0.07190.004 (40)
0.09090.005 (40)
0.07590.006 (40)
Corema album
Pyrus bourgaeana
Rubus ulmifolius
0.2690.01 (45)
9.5490.43 (60)
1.1390.05 (45)
3.0490.31 (45)
3.0091.60 (22)
37.7391.61 (45)
9.7290.21 (137)
73.53922.22 (66)
2.4890.03 (450)
4.5990.11 (20)
8.5490.31 (20)
2.8990.08 (20)
3.3990.08 (20)
5.4890.21 (20)
1.8890.04 (20)
Seed coat thickness/seed
diameter
Seed coat thickness
(mm)
Seed diameter (mm)
Seed length (mm)
Seed weight (mg)
No. viable seeds per fruit
Fruit weight (g)
Table 1. Fruit and seed characteristics of the three plant species studied (mean91SE). From 10 to 15 reproductive individuals of each species we estimated seed diameter and coat thickness of twenty
randomly selected seeds. A transversal slice per seed (16—18 mm wide) was created using a freezing microtome and immediately examined under a microscope (10—40 x). Coat thickness was measured
using a calibrated scale (i.e. reticule); we multiplied the number of ocular divisions spanned by the coat width by a conversion factor according to the magnification used. Numbers in parentheses indicate
sample sizes. Because seed coat thickness was variable within each sample, two measurements were recorded per seed; thus, sample size for coat thickness was double that for seed diameter.
they were examined with 20—40x magnification glasses.
Only unharmed seeds (i.e. not crushed or fractured) were
selected for the sowings.
Despite our intensive sampling effort, we could not
gather an acceptable number of mammal-ingested seeds for
some plant-disperser pairs due to different contingencies of
our study system. For example, rabbits destroyed and
ground up all ingested Pyrus seeds, and the few badger
feces found in the Corema study site seldom contained
seeds. Consequently, some interacting pairs of species were
not accounted for. Succesful plant-disperser pairs and the
number of mammal-ingested seeds sown were: Corema
seeds ingested by fox, rabbit, and boar (360 seeds per
treatment); Rubus seeds ingested by badger and fox (360
seeds per treatment), and Pyrus seeds ingested by badger
(340 seeds). The corresponding numbers of control seeds
were sown for each plant species.
Field seed sowing and statistical analysis of the
effects of endozoochory on the percent and speed
of germination
To evaluate the combined effect of passage through
mammal guts (i.e. ‘‘gut passage’’) and the removal from
the mother plant (i.e. ‘‘removal’’) on seed germination, we
sowed experimental seeds in the field late in November
2005. In each of the three study localities, we haphazardly
chose 17—18 reproductive individuals (or random blocks)
separated by at least 15 m, and used an experimental design
whose factors were ‘‘gut passage’’ (mammal-ingested and
control seeds) and ‘‘removal’’ (beneath and away [ ]5 m in
open microhabitat] from any reproductive conspecific). The
open microhabitat was chosen as the target deposition site
because is clearly the most pervasive arrival site for
mammal-dispersed seeds in our study area. For example,
systematic fecal surveys carried out in our three study sites,
in which all microsites were searched evenly, showed that
most mammal feces (67.5%, n =1540) were deposited in
the open microhabitat; remaining samples were found
beneath seventeen different shrub/tree species (Fedriani
and Delibes unpubl.). Depending on the number of plantdispersers pairs (from 1 to 3, see above) associated to each
random block, we set, at least, the following four treatment
combinations: 1) mammal-ingested seeds beneath a reproductive conspecific, 2) control seeds beneath a reproductive
conspecific, 3) mammal-ingested seeds away from any
reproductive conspecific, and 4) control seeds away from
any reproductive conspecific. In each experimental block,
we placed one seed depot (see below) per disperser species,
plus one for control seeds, both beneath and away from a
reproductive conspecific (e.g. for Pyrus four seed depots
were set per block). Each seed depot consisted of an openbottomed plastic beaker (7 cm diameter) pushed partly into
the ground (cf. Robertson et al. 2006). Ten viable seeds of a
particular treatment were sown in each depot (e.g. for Pyrus
40 seeds were sown per block), and then buried (‘0.5 cm
depth) with in situ soil previously sieved to remove nonexperimental seeds. To evaluate potential contamination by
non-experimental seeds (e.g. seeds within bird droppings),
we placed in each block two extra depots, following the
procedure as above except with no sown seeds. To keep out
985
vertebrate seed predators (Fedriani and Delibes 2009), all and Grambsch 2000). The inverse-logarithm transformed
regression coefficients give the relative likelihood of
depots were covered with a 1-cm mesh cage (28 x18 x
13 cm). Sowings were checked monthly from January germination during the overall study period related to
2006 to September 2007. Ball-headed needles of variable changes in each explanatory variable (after adjustment for
the effects of the other variables in the model). Since we
colors were placed next to each emerged seedling upon
each check, allowing us to distinguish between monthly only considered data for seeds that had germinated by the
end of our field experiment, the relative likelihood of
seedling cohorts.
To analyze the data on seedling emergence, we used germination can be interpreted in terms of germination
generalized linear mixed models using the SAS macro speed (i.e. the higher the likelihood, the faster the
GLIMMIX (Littell et al. 1996). One model per plant germination).
species was fitted with gut passage, removal, and their
interaction as fixed effects. Experimental block was included
as a random factor. Because of the binomial nature of the Effects of endozoochory on germination asynchrony
response variable in all models (number of emerged Because sampling variance approaches population variance
seedlings/number of seeds sown [n =10]), we used binowith sample size (Zar 1999), an evaluation of the role of
mial error and logit link function. Adjusted means and endozoochory on germination asynchrony (Izhaki and
standard errors were calculated using the LSMEANS
Safriel 1990) requires that sample size effect (i.e. number
statement and back-transformed using the appropriate of emerged seedlings) be controlled (Simons 2007). This
Taylor’s series approach (Littell et al. 1996). To compare could be particularly crucial when the number of emerged
the effects of different levels of any significant main factor, seedlings significantly varies across different treatment
we calculated the differences between their least-square
combinations, which was the case of our study (e.g. for
means. When the interaction between any two factors was Pyrus, the number of seedlings emerged from control seeds
significant, we performed tests for the effect of a given [n =103] almost doubled that emerged from badgerfactor at the different levels of the other factor (‘‘tests of ingested seeds [n =53]). Therefore, we used random
simple main effects’’), using the SLICE option in the samples of each plant species, which were generated from
LSMEANS statement of the MIXED procedure (Littell our empirical emergence data (overall, 504 emergence
et al. 1996). Thus, GLIMMIX allowed: the modeling of
records), to calculate a metric of the diversity in germinaour response variable according to the specific distribution
tion timing by means of the Shannon’s index (H ?):
of its residuals, the achievement of a mixed factorial design,
n
X
and a full evaluation of main effects and their interactions.
Pi (ln Pi );
However, generalized linear models do not make allowances H ? =—
i =1
for censored data; thus, because some of our experimental
seeds could have germinated after our study period (twenty- where Pi is the proportion of seeds germinated during
one months), we reanalyzed our data on percentage of the ith month of the study period (e.g. Labouriau and
Pacheco 1978, Izhaki and Safriel 1990, Ranal and Santana
germination by means of failure-time analyses, by fitting
Cox proportional hazard regression models that account for 2006).
In our simulations, the diversity of germination timing
censored data (Therneau and Grambsch 2000). Results
from this second set of analyses were clearly consistent with was monitored for each treatment combination as a
previous ones; therefore, we show only the results from function of sample size (number of emergence times
GLIMMIX analyses, which most comprehensively disen- recorded). For the sake of simplicity, we distinguished
between two gut passage levels (i.e. mammal-ingested
tangle the effect of main factors and their interactions on
[irrespective of the species] and control seeds), and two
percent germination.
The effects of gut passage and removal on seed removal levels (beneath and away from mother plant). An
germination speed were tested using failure-time analyses, iterating procedure was devised in Visual FoxPro (1998)
by fitting Cox proportional hazard regression models to and, for any given sample size, we ran 20 iterations to
data consisting of the number of months between sowing generate the plausible ranges of diversity in germination
timing given our empirical data. To analyze the response
and seedling emergence for each seed. To separate the
effects on germination speed from those on percentage of variable (H ?) by controlling for sample size, we fitted a
germination, we only considered seeds that had germinated generalized linear mixed model for each plant species using
by the end of our field experiment (Figuerola et al. 2002, the GLIMMIX macro. In all models, both gut passage and
Santamarı́a et al. 2002). As above, one model was fitted per removal from mother plant were included as fixed factors;
plant species. Block was included in each model as a sample size was included as a covariate, and the iteration
Simulated diversity indexes
‘‘frailty’’ (i.e. random) term, and the significance of each (n =20) as a random factor.
1/2
were
transformed
[(x+1)
]
to achieve homogeneity of
factor and interaction was evaluated by backwards-stepwise
variances
and,
thus,
normal
error
and identity link function
elimination from the full model (Therneau and Grambsch
2000). In comparing successive models, we calculated the were specified in all models (Littell et al. 1996).
double absolute difference of their respective EM-likelihood
algorithms, and compared that value against a chi-square
Results
with k-1 degrees of freedom, k being the number of levels
(or combination of levels) of the factor (or interaction)
being tested. For the frailty factor we also assumed a chi- Seedling emergence in our field sowing was low for all
square distribution with one degree of freedom (Therneau species, with Rubus and Corema having the highest and
986
lowest percentage of germination, respectively (Fig. 1).
Overall, 15.8% of seeds germinated (504 of 3200 seeds
sown). Species-specific germination percentages were
23.6% (255 of 1080), 22.5% (153 of 680), and 6.7%
(96 of 1440) for Rubus, Pyrus, and Corema, respectively.
Extra depots set without seeds showed that contamination
by non-experimental seeds was negligible for the three
species; specifically, only one seedling of one fleshy-fruited
species (Rubus) emerged in a total of 106 control pots.
Seedlings emerged (%)
(A) Rubus ulmifolius
p < 0.020
50
ns
ns
40
ns
30
20
Effects of endozoochory on the percent of
germination
10
Badger
Fox
Control
Our linear model for Pyrus revealed that there was a
significantly higher percentage of germination for control
seeds as compared with badger-ingested seeds (Table 2,
Fig. 1C). Also, Pyrus seeds germinated in higher proportion
beneath reproductive conspecifics than away. No significant
interaction between removal and gut passage was found
(Table 2). For Rubus, we found a significant gut passage
effect on the percentage of seed germination (Table 2).
Differences among least square means indicated that seeds
ingested by badgers germinated in higher proportions than
those ingested by fox and control seeds (t >2.38, DF =
1057, p B0.020; Fig. 1A); however, no significant differences were found between fox-ingested and control seeds
(p =0.867). No significant effect of removal or its interaction with gut passage was found for Rubus (Table 2).
For Corema, we found a significant effect of gut passage
(Table 2), indicating that seeds ingested by all three
mammals germinated in a higher proportion than control
seeds (t >3.91, DF =1415, p B0.0001); however, no
differences were found among seeds ingested by individual
mammal species (rabbit, boar, and fox; t B1.22, DF =
1415, p >0.224; Fig. 1B). Also, removal showed a marginally significant interaction with gut passage (Table 2); thus,
seeds ingested by boars germinated more often away from
than beneath reproductive conspecifics (F1, 1415 =7.11, p =
0.008; Fig. 1B), whereas for fox- and rabbit-ingested seeds,
as well as for control seeds, there was no significant removal
effect (F1, 1415 B0.27, p >0.208).
Seedlings emerged (%)
(B) Corema album
p < 0.0001
30
ns
**
ns
ns
Rabbit
Boar
Fox
Control
20
10
0
(C) Pyrus bourgaeana
Seedlings emerged (%)
70
p < 0.0001
60
**
50
40
30
**
20
10
0
Badger
Control
Figure 1. Corrected mean percentages (91 SE) of seedling
emergence for Rubus (A), Corema (B), and Pyrus (C) seeds ingested
by different mammalian dispersers (and for control seeds) beneath
(open circles) and away (black circles) from reproductive conspecifics (ns, not significant; **, p B0.01).
Effects of endozoochory on germination speed
Seedling emergence took place during the first few months
after sowing (January—April 2006) for Pyrus and Rubus,
whereas for Corema germination spanned a long period
Table 2. Main results of our generalized linear mixed models testing the effects of passage through mammal guts and removal from the
mother plant on the final percentage of seed germination.
Rubus
Fixed effects
Gut passage (GP)
Removal (R)
GP xR
Random effect
Block
Corema
Pyrus
DF
F
p
DF
F
p
DF
F
p
2, 1057
1, 1057
1, 1057
4.21
1.13
0.5
Wald-z
2.33
0.015
0.288
0.609
p
0.010
3, 1415
1, 1415
3, 1415
7.52
2.07
2.54
Wald-z
2.04
B0.0001
0.151
0.055
p
0.021
1, 660
1, 660
1, 660
27.82
16.46
0.36
Wald-z
2.13
B0.0001
B0.0001
0.551
p
0.017
987
(showing two germination peaks, during the first and
second rainy seasons; Fig. 2). For Pyrus, the Cox model
showed no significant effect of any factor on the speed of
germination given our temporal unit of resolution (Table 3),
likely because most (>90%) seeds germinated during their
second month after sowing (Fig. 2E, F). For Rubus,
however, there were significant main effects of both gut
passage and removal on the speed of seed germination
(Table 3). Even though most Rubus seeds germinated
between the second and fourth month after sowing (Fig.
2A, B), seeds ingested by badgers and foxes germinated 1.5
(B) Rubus ulmifolius – away
100
Accumulated emergence (%)
Accumulated emergence (%)
(A) Rubus ulmifolius – beneath
and 1.4 times faster than control seeds, respectively. Also,
Rubus seeds away from reproductive conspecifics germinated 1.4 times faster than those sown beneath them. For
Corema, we also found a significant effect of gut passage
(Table 3), indicating that, overall, seeds ingested by rabbits
and foxes germinated faster than control seeds; however, gut
passage and removal showed a marginally significant
interaction (Table 3), indicating that fox-ingested seeds
germinated faster beneath conspecifics, whereas for the
remaining treatments (rabbit, boar, and control) there were
no marked differences related to removal (Fig. 2C, D).
80
60
40
20
0
1
2
3
4
5 19
20
60
40
20
21
0
(C) Corema album – beneath
100
80
60
40
20
0
0
3
6
9
12
15
18
4
5 19
20
21
60
40
20
0
3 19
80
60
40
20
0
0
Accumulated emergence (%)
80
2
3
3
6
9
12
15
18
21
(F) Pyrus bourgaeana – away
100
1
2
100
21
(E) Pyrus bourgaeana – beneath
0
1
(D) Corema album – away
Accumulated emergence (%)
Accumulated emergence (%)
80
0
0
Accumulated emergence (%)
100
20
Months after sowing
Rabbit – beneath
Boar – beneath
Fox – beneath
Badger – beneath
Control – beneath
21
100
80
60
40
20
0
0
1
2
3 19
20
21
Months after sowing
Rabbit – away
Boar – away
Fox – away
Badger – away
Control – away
Figure 2. Cumulative percentages of seedling emergence for the three fleshy-fruited species (A—B, Rubus; C—D, Corema; and E—F, Pyrus)
ingested by different mammalian dispersers, both beneath and away from reproductive conspecifics. Note break in x-axis for Rubus and
Pyrus.
988
Table 3. Main results of our Cox regression models testing the effects of passage through mammal guts and removal from the mother plant on
the speed of seed germination.
Rubus
Corema
DF
2
x
p
DF
Fixed effects
Gut passage (GP)
Removal (R)
GP xR
2
1
5
6.6
7.2
6.2
0.05
0.01
0.287
Frailty term
Block
1
3.0
0.083
Effects of endozoochory on germination asynchrony
Simulated indices of temporal diversity of germination (H ?)
showed a strong pervasive sample size effect for all three
species (p B0.0001; Fig. 3A—C), supporting the convenience of our approach. Conversely, no iteration effect (p :1)
was found for any species. For Pyrus, passage through
mammal guts clearly diversified the germination timing
(F1, 1516 =194.88, p B0.0001; Fig. 3B). Also, removal had
a strong effect (F1, 1516 =125.0, p B0.0001), with germination timing of seeds sown beneath mother plants more
diverse. The significant interaction between both factors
(F1, 1516 =26.75, p B0.0001) indicated that, for a given
sample size, the effect of gut passage was higher away from
mother plants (Fig. 3C). For Rubus, removal had a strong
main effect (F1, 2533 =144.9, p B0.0001), being germination timing of seeds sown away from mother plants more
diverse. Gut passage did not have any significant effect as
main factor (F1, 2536 =0.65, p =0.420), though its significant interaction with removal (F1, 2536 =28.91, p B0.0001)
indicated that the diversification in germination timing
related to passage through mammal guts was stronger away
from mother plants (Fig. 3A). For Corema, only five
seedlings emerged from control seeds (all of them in the
twelfth month after sowing); thus, we only tested differences in germination diversification for seedlings emerged
from mammal-ingested seeds (n =91), and found that
germination was much more diverse beneath reproductive
conspecifics (F1, 898 =852.2, p B0.0001; Fig. 3B).
Discussion
Effects of endozoochory on germination under field
conditions
Recent studies of the role of endozoochory on seed
germination highlight different results linked to the
particular conditions used in the experiments (laboratory,
glasshouse, field) and call for further evaluations under field
conditions (Robertson et al. 2006, Traveset et al. 2007).
The percentages of germination found in our field sowings
were similar to those reported for the same species in assays
carried out in the field (Rodrı́guez-Pérez et al. 2005 for
Rubus; Calviñ o-Cancela 2004 for Corema), but clearly lower
than those found under controlled conditions (Traveset
et al. 2001, Rodrı́guez-Pérez et al. 2005 for Rubus). Thus,
our study supports the idea that sowing conditions are of
great importance in determining germination outcomes.
Pyrus
2
p
DF
x
p
3
1
7
8.2
1.4
13.8
0.05
0.237
0.055
1
1
3
1.2
2.4
0.0
0.273
0.121
1
1
13.2
0.001
1
0.06
0.807
x
2
The effect of gut passage on the proportion of germination was not related to either our absolute nor relative
measurements of seed coat thickness (i.e. coat thickness and
coat thickness/seed diameter ratio, respectively; Table 1).
For instance, Rubus and Corema have similar coat or
endocarp thickness; however, enhancement of germination
by gut passage was most evident for Corema. Such
interspecific differences in response to gut passage could
be related to seed coat hardness, rather than thickness
(Traveset et al. 2008). For Pyrus (the species with the lowest
coat thickness/seed diameter ratio) gut passage decreased
germination, as found in previous studies (Nogales et al.
2005). This antagonistic effect is likely related to the strong
gut treatment by badgers on Pyrus seeds (Fedriani and
Delibes unpubl.). As predicted, the effect of gut passage was
dependent on the mammal species (Barnea et al. 1991,
Nogales et al. 2005, Rodrigez-Pérez et al. 2005). For
example, Rubus passage through badger guts increased
germination percent, while passage through fox gut did
not have a significant effect. That badgers had a negative
effect on Pyrus, but a positive effect on Rubus is likely
related to the different seed traits of these two species
(Table 1). Rubus has a relatively thick coat (to its seed
diameter), which likely resists abrasion in the badger’s gut;
in contrast, it appears that the relatively thin coat of Pyrus
is unable to withstand badger gut treatment. The differences among dispersers might be due to interspecific
differences in morphology and physiology of their guts, as
well as to differences in retention times and/or to the types
of food ingested along with the seeds (Levey and Karasov
1992, Charalambidou et al. 2005). Gut passage also altered
germination speed across species, similar to that observed
for ability to germinate, i.e. Corema experienced the greatest
acceleration of germination, followed by Rubus, and then
Pyrus. Finally, our simulations indicated that gut passage
increased the asynchrony of germination for Rubus and
Pyrus, corroborating results from previous studies (Izhaki
and Safriel 1990).
Our experimental results also showed a marked effect of
removal from the mother plant on germination behavior.
Pyrus seeds unexpectedly germinated in higher proportion
beneath than away from reproductive individuals. This
result is consistent with the regular presence of recently
germinated seedlings beneath conspecifics (Fedriani and
Delibes unpubl.) and might be related to a more favorable
microenvironment (e.g. light [Silvertown 1980, Pons
2000], moisture [Dubrovsky 1996]) beneath reproductive
trees. For Corema the effect of removal on the proportion of
germination seemed contingent not only on whether seeds
989
(A) Rubus ulmifolius
Temporal diversity of
germination (H' )
1.2
1.0
0.8
0.6
0.4
0.2
0
10
20
30
Number of seedlings
40
50
10
20
30
Number of seedlings
40
50
40
50
(B) Corema album
Temporal diversity of
germination (H' )
1.2
1.0
0.8
0.6
0.4
0.2
0
(C) Pyrus bourgaeana
Temporal diversity of
germination (H' )
0.8
0.6
0.4
0.2
0.0
0
10
20
30
Number of seedlings
Control – away
Control – beneath
Mammal – away
Mammal – beneath
Figure 3. Shannon index of germination asynchrony of randomly
generated samples plotted against number of seedlings of Rubus,
Corema, and Pyrus. Asynchrony was simulated using our empirical
data accounting for the effect of gut passage (mammal ingested or
control seeds) and removal from the mother plant. When sample
size allowed it, simulations were run up to n =50. Each simulation
was run for 20 iterations to generate the plausible ranges of
variation in germination timing given our empirical data. Note
that, for Corema, only five seedlings emerged from control seeds
(all of them in the twelfth month after sowing); thus, we only
simulated germination diversification of mammal-ingested seeds
(n =91) in relation to removal from the mother plant.
990
had been ingested by mammals, but also on the particular
disperser species that had consumed the seeds. A comparable interaction between gut passage and the environment
of seed sowing was found for the duck-dispersed helophyte
Scirpus litoralis under controlled conditions (Espinar et al.
2004), suggesting that the interaction between the effects of
gut passage and the microenvironment of seed arrival could
be a widespread phenomenon. In addition, the speed of
germination was also dependent on removal from the
mother plant (Fig. 3). Corema seeds ingested by foxes
germinated faster beneath reproductive conspecifics,
whereas Rubus seeds tended to germinate earlier away
from conspecifics. Finally, our simulations for the three
species indicated that removal from the mother plant
altered germination asynchrony suggesting this also is an
important determinant of seed germination behavior.
In general, our study indicates that gut passage had a
stronger effect on germination than removal from mother
plant; however, for some species both factors were similarly
influential in some seed germination aspects (e.g. both the
amount and synchrony of germination in Pyrus were
strongly altered by both gut passage and the removal
from the mother plant). Thus, the joint effect of these two
components of the dispersal process on different aspects of
germination should be considered in future studies. It is
possible other processes not accounted for in this study
could also affect the germination patterns of our target
species. For instance, by moving seeds from the maternal
surrounding to a new environment, frugivores often assist
escape from post-dispersal seed predators (e.g. granivorous
rodents; Janzen 1970, Wenny 2000), a fitness advantage
that appears to occur at least for Pyrus (Fedriani and Delibes
2009). Frugivores also provide fruiting plants with services
such as deinhibition or fertilization (Samuels and Levey
2005, Robertson et al. 2006), which could interact with the
microenvironment of seed delivery, affecting amount and
timing of seed germination. In Doñ ana, Pyrus seeds within
uneaten fruits are most often subject to depredation by
invertebrates or decay late in the dispersal season (Fedriani
and Delibes unpubl.). Thus, frugivorous mammals likely
provide an important disinhibition service to Pyrus by
removing the pulp attached to seeds and allowing their
germination. Nonetheless, the possibility that the physicochemical and biological processing of intact Pyrus fruits in
the field partly reproduce processes happening in animal
guts cannot be ruled out (Traveset et al. 2007) and deserves
further research. Finally, some of our experimental seeds
could have entered dormancy and germinated after the 21month monitoring period (Calviñ o-Cancela 2004), which
would likely result in an even higher variability in
germination timing. Although some of these processes
may play a part in germination, we do not expect that
they would significantly alter the overall patterns reported
given the length and sample sizes of our study.
Benefits of endozoochory in unpredictable
environments
Passage through mammal guts increased the proportion of
seed germination for two of the three plant species tested.
These species are likely benefiting from their interactions
with dispersers, though a higher germinability does not
always translate into higher seedling survival (Schupp
2007). In our study, we sowed 3200 seeds of the three
species, from which 504 seedlings emerged. Of them, only
five Rubus seedlings (two from badger, two from fox and
one control) survived after their first summer, preventing
comparisons of the effects of different treatments on seed
survival. Most seedlings died due to desiccation and, thus,
the extreme dry summers preceding and following our field
sowing (only 0.5 and 0.0 mm of rainfall during the
summers of 2005 and 2006, respectively) likely accounted
for the reported low seedling survival, a common feature in
Mediterranean ecosystems (Pugnaire and Valladares 2007).
Conceivably, given the high variability in rainfall in our
study area (see Methods), seedling survival during benign
summers (e.g. summers of 2000 and 2007, with 103 and
134 mm of rainfall, respectively) should be higher and the
presumed benefits of endozoochory, such as a higher
proportion of germination, would result in higher recruitment. Experimental field studies disentangling the relative
importance of gut passage and removal from the mother
plant on long-term seedling survival during benign and
harsh years are clearly needed.
Passage through mammal guts changed the timing of
germination, generally accelerating it and increasing its
diversification (i.e. asynchrony). Early germination often
enhances plant fitness (Verdú and Traveset 2005, De Luis
et al. 2008) by allowing early seedlings to outcompete later
seedlings. Nonetheless, such effects can be contingent on
individual species (Seiwa 2000) and on a myriad of biotic
and abiotic factors (Gó mez 2004). Both theoretical and
empirical evidence indicates that diversification of germination timing (asynchrony) often results in fitness advantages
under environmental unpredictability in annuals (Cohen
1966, Simons 2007, Venable et al. 2008). Though
perennials do not rely on persistent seed banks (Thompson
et al. 1997), a comparable beneficial mechanism has been
proposed for fleshy-fruited species (Izhaki and Safriel
1990). The advantages of germination diversification can
arise within a reproductive season by reducing sibling
competition (Nilsson 1994) or by increasing the spatial
variance of seedling density (Geritz 1995). Besides, under
the temporal environmental unpredictability that characterizes Mediterranean habitats, germination asynchrony
among reproductive seasons (the case of Corema; Calviñ oCancela 2004) might result in an overall fitness benefit by
spreading the risk of encountering conditions particularly
unsuitable for survival (Simons 2007). Given the results
from this study, the contrasting treatments provided by
different dispersers (Traveset and Verdú 2002), and the
myriad of microhabitats where they deliver ingested seeds
(Jordano and Schupp 2000), endozoochory might represent
a virtually unlimited source of diversification of germination behavior. Consequently, we extend the proposal of
Izhaki and Safriel (1990) to suggest that both gut passage
and the removal from the mother plant provide chief
services to fleshy-fruited plants, acting as a ‘‘randomization
mechanism’’ diversifying their germination patterns, with
potential advantages under environmental unpredictability.
In conclusion, seed rains generated by vertebrate
dispersers are likely to be heterogeneous not only in
densities, distributions, and genotypes (Jordano and Godoy
2002), but also in germination patterning. Our study
indicates that the consequences of endozoochory for
germination vary with each interacting plant-animal pair
and also with the specific environment to which seeds
arrive. The ecological services provided by a particular
disperser appeared to interact to increase the singularity of
the species’ overall service, suggesting lack of redundancy
and expendability (sensu Kareiva et al. 2003) of any
disperser species, and leading to unusually high functional
diversity in this seed dispersal mutualism. The interaction
among fleshy-fruited plants and vertebrate dispersers does
not take place on a species-to-species basis, but it is often
the sum of numerous interactions involving dozens of
species (Herrera 2002, Thompson 2005); thus, fleshyfruited plants most likely benefit from such unspecialized
interactions through both germination enhancement and
diversification in germination timing.
Acknowledgements — We are indebted to Gemma Calvo, Mó nica
Váz, and innumerable volunteers for their enthusiastic field and
lab assistance. We thank Andy Green, Xavi Picó, Anna Traveset,
Kevin Burns, Ken Thompson and two anonymous reviewers for
helpful comments on earlier drafts. Kimberly Holbrook thoroughly reviewed the English, and Enrique Collado kindly assisted
us in computer programming. The Spanish Ministerio de Medio
Ambiente (15/2003 grant) and Ministerio de Educación y Ciencia
(CGL2007-63488/BOS) supported this study.
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