Download Fat but slim: Criteria of seed attractiveness for earthworms

Pedobiologia 54S (2011) S159–S165
Contents lists available at SciVerse ScienceDirect
Pedobiologia - International Journal of Soil Biology
journal homepage: www.elsevier.de/pedobi
9th International Symposium on Earthworm Ecology
Fat but slim: Criteria of seed attractiveness for earthworms
Julia Clause, Pierre Margerie, Estelle Langlois, Thibaud Decaëns, Estelle Forey ∗
Laboratoire d’Ecologie – EA 1293 ECODIV, FED SCALE, Bâtiment IRESE A, Place E. Blondel, UFR Sciences et Techniques, Université de Rouen, F-76821 Mont Saint Aignan Cedex, France
a r t i c l e
i n f o
Article history:
Received 30 November 2010
Received in revised form 3 August 2011
Accepted 12 August 2011
Keywords:
Above- and belowground interactions
Adaptation strategy
Ingestion/digestion
Oil content
Palatability
Seed trait
a b s t r a c t
Earthworms were shown to significantly affect seeds and seedlings survival via their ingestion and digestion for nutritive purposes. Such selective feeding of earthworms on plant seeds is likely to favour certain
plant species and to affect seed bank composition, plant recruitment and plant community structure.
Relationships between earthworms and seeds, particularly seed traits that determine attractiveness of
seeds for earthworms, are yet to be determined. In this study, the influence of six seed traits was tested
on the ingestion, digestion and germination of seeds by two earthworm species (Lumbricus terrestris,
anecic and Satchellius mammalis, epigeic). The seed traits tested were their length, width, weight, shape,
oil content and the presence of trichomes on their surface. Each earthworm species was introduced into
a microcosm with eleven seed species from a chalk grassland that represented those different traits.
Ingested, digested and germinated seeds were counted after voiding the guts of the earthworms. Univariate and multivariate analyses showed that seed length, width, weight and seed oil content could
significantly affect the ingestion of seeds for both earthworm species. Seed width and seed oil content
were the two traits that influenced the digestion of seeds the most, but only for L. terrestris. We also
found that seed ingestion was earthworm species-specific but we found no correlation between earthworm traits and number of ingested or digested seeds. Few seeds germinated from L. terrestris casts
and no seeds germinated from S. mammalis casts. Implications in terms of plant evolution strategies are
further discussed.
© 2011 Elsevier GmbH. All rights reserved.
Introduction
Earthworms are among the most studied macroinvertebrates
and are considered as engineers of ecosystems (sensu Jones et al.
1994) that strongly affect soil processes and provide critical ecosystem services over a range of spatio-temporal scales (Lavelle 1997;
Lavelle et al. 2006). Indeed, their presence at the plant–soil interface indicates their importance in ecosystem dynamics via both
their indirect and direct impacts on plants and plant communities (Grant 1983; Willems and Huijsmans 1994; Scheu 2003;
Ehrenfeld et al. 2005). Previous studies have widely demonstrated
that earthworms influence plant performance indirectly – mainly
by changing soil nutrient availability, soil structure and microbial
community and functioning (Lee 1985; Lavelle et al. 1997; Scheu
2003; Eisenhauer et al. 2007; Laossi et al. 2010). Direct effects of
earthworm populations on plants include consumption of dead
roots (Lee 1985), but also changes in the distribution, survival
and establishment of seeds through seed predation and dispersion
(Scheu 2003; Eisenhauer and Scheu 2008; Eisenhauer et al. 2009).
Earthworms are increasingly recognised as important dispersers and predators of seeds (Eisenhauer et al. 2010; Forey et al.
∗ Corresponding author. Tel.: +33 232 76 94 55; fax: +33 235 14 66 55.
E-mail address: [email protected] (E. Forey).
0031-4056/$ – see front matter © 2011 Elsevier GmbH. All rights reserved.
doi:10.1016/j.pedobi.2011.08.007
in press). They can positively or negatively affect the distribution,
survival and establishment of seeds. First, displacement of seeds
by ingestion and burial might protect seeds from above-ground
seed predators, such as birds or insects (Azcarate and Peco 2003)
and reduce exposure to stressful environmental conditions such
as fire, drought or frost (Shumway and Koide 1994; Willems and
Huijsmans 1994). Additionally, it has been shown that seed ingestion and earthworm mucus secretion on egested seeds potentially
change germination rates (Ayanlaja et al. 2001; Eisenhauer et al.
2009). Surface casts and middens might also represent important
regeneration niches for plant seedlings; seeds deposited in these
structures at the soil surface may thus have an increased probability of germinating and contributing to the indigenous vegetation
(Grant 1983; Zaller and Arnone 1999; Decaëns et al. 2003). On the
other hand, the passage of seeds through the gut of earthworms
can also alter seed germination and survival as a result of digestion and scarification of ingested seeds (McRill and Sagar 1973;
Decaëns et al. 2003; Milcu 2006; Laossi et al. 2009). For example, Decaëns et al. (2003) found that seeds lost between 70% and
97% of their germination capacity after their transit throughout
the gut of the anecic species Martiodrilus sp. Finally, seed displacement on the soil surface and along the soil profile might
modify germination success for dark and light germinating seeds
although seeds buried below a critical depth can fail to emerge
(Thompson et al. 1994).
S160
J. Clause et al. / Pedobiologia 54S (2011) S159–S165
Since Darwin (1881) shed light on the diet preferences of Lumbricids, recent studies have shown that earthworms are actually seed
predators themselves, using seeds and seedlings as part of their
food sources (Shumway and Koide 1994; Eisenhauer et al. 2009,
2010). These findings imply a deeper impact of earthworm activity
on ecosystems and plant communities than previously recognised.
Additionally, they suggest that earthworms actively select seeds
according to their nutritional needs. This selection of seeds can
be species-specific both for seeds and earthworms. Studies have
shown that earthworms may prefer small seeds over large ones
(Shumway and Koide 1994; Eisenhauer et al. 2009), that they favour
forbs over grasses species (Zaller and Saxler 2007; Eisenhauer et al.
2009, 2010), and that they respond to seed smell or taste (Willems
and Huijsmans 1994) and coat texture (Shumway and Koide 1994).
A variation of those traits could potentially induce changes in seed
palatability. More traits characterizing seeds and palatability are
still to be tested such as their nutritive value, their shape or the
presence of trichomes on their surface. Satchell (1967) showed that
earthworms preferred protein- and carbohydrate-rich litters over
litters with lower protein content. Furthermore, Eisenhauer et al.
(2009) showed that seed preferences differed among earthworm
species, depending on their body size, but also on species-specific
feeding habits that need to be explored.
Little research has been done on the selective ingestion and
digestion of seeds. Studies on seed–earthworm interactions may
have potential applications in plant restoration and conservation
because such above- and belowground interactions might induce
a modification of seed bank composition, structure and dynamics, thus significantly impacting plant communities and ecosystem
properties (Lee 1985; Bakker et al. 1996; Bekker et al. 1998;
Eisenhauer et al. 2009).
In this study, we investigated the selectivity of seed ingestion
by earthworms and the way ingested seeds are digested during
gut transit. An experiment was set up with seeds of eleven plant
species and two earthworm species from two different ecological
groups (Lumbricus terrestris L., anecic; Satchellius mammalis Savigny, epigeic) in order to test the relationships between seed traits
(size, shape, presence of trichomes and oil content) and their probability for ingestion, digestion and subsequent germination. We
expect food selection to vary between both earthworm species due
to their different food requirements. Some of the traits studied here
were already shown to influence seed selection by earthworms,
while other traits related to nutritive value and seed palatability need further investigation. The specific objectives of this study
were to (1) investigate whether the ingestion of seeds is earthworm
species-specific, (2) detect which traits earthworms select for preferential seed ingestion and digestion, and (3) discuss implications
of such selective ingestion, if any, for plant species.
Earthworms were washed immediately in clean water and placed
for 48 h on a moistened filter paper before being introduced to a
mixture of soil and sterilized horse dung for one week. After 48 h of
fasting (Eisenhauer et al. 2009) they were weighed (Table 1). At the
end of the experiment (see below) their size (length and width) was
further measured with a Leica MZ6 binocular, DFC 255 camera and
the Leica LAS AF software (http://www.leica-microsystems.com)
after fixing them into pure alcohol (Table 1).
Seeds of eleven plant species observed in chalk grasslands (Saint
Adrien site, Normandy) were provided by The Botanic Garden of
Caen (Lower-Normandy, France) and used to test for ingestion preferences in relation to six different traits: length, width, weight,
shape, presence of trichomes and oil content. The eleven species
were Achillea millefolium L., Arrhenatherum elatius (L.) p. Beauv.
ex J. Presl & C. Presl, Brachypodium pinnatum (L.) p. de Beauvois,
Daucus carota L., Lotus corniculatus L., Medicago lupulina L., Origanum vulgare L., Sanguisorba minor Scop., Teucrium chamaedrys
L., Teucrium scorodonia L. and Urtica dioica L. These species were
selected because the combination of the six seed traits was different for all seed species. Average weight (calculated per 1000
seed weights) and oil content data were obtained through the Kew
Garden seed databank (http://data.kew.org/sid/). Length and width
traits were measured on twenty seeds of each species with a Leica
MZ6 binocular, DFC 255 camera and the Leica LAS AF software
(http://www.leica-microsystems.com). Trichomes were characterized by their presence or absence on the seeds; they were not
enumerated. Data traits for seeds are reported in Table 1.
Materials and methods
Digestion of plant seeds and germination after gut passage
Species choice and sampling
Earthworms were removed and placed on moistened filter
paper for 48 h in Petri dishes (darkness, 15 ◦ C) to void their guts
(Eisenhauer et al. 2009). All casts from the box and Petri dishes were
then broken up and all remaining seeds were counted under binocular observation. The difference between the number of ingested
seeds and the number of seeds found in casts represented digested
seeds. Finally, casts were left in a temperature-controlled phytotron
during 3 weeks (day/night, 12/12 h and 18/15 ◦ C) and seedlings
were counted to test for germination of all egested seeds. Additional germination assays were run as control tests. Twenty seeds
from each of the eleven plant species were placed on a moist filter
paper in a Petri dish. Each Petri dish was replicated ten times and
left in a temperature-controlled phytotron (day/night, 12/12 h and
18/15 ◦ C) for 3 weeks after which seedlings were counted. Filter
Ten mature individuals of both S. mammalis and L. terrestris were
collected from a grassland soil in April 2010. Epigeic species reside
mainly in the upper organic soil layers and cause limited mixing of mineral and organic layers. Anecic species are known to be
large earthworms living in deep vertical burrows up to 2 m depth,
but predominantly feed on litter on the soil surface. They move
vertically, forming burrows and aggregates that can potentially
contain seeds (Willems and Huijsmans 1994; Regnier et al. 2008). In
sampled grasslands, S. mammalis and L. terrestris were found in densities of 10.03 and 11.44 individuals per square meter respectively
(Decaëns et al. 2008). After watering one square meter with a 0.4%
formalin solution, earthworms were hand sorted for up to 15 min.
Ingestion of plant seeds
The experiment was set up following Eisenhauer et al. (2009).
Ten earthworms of each species were placed on moist filter paper
for 48 h to void their guts (15 ◦ C, darkness). Each individual was
then placed in a 14.7 cm × 10.3 cm plastic box with 200 g of airdried, seed-free 2 mm-sieved soil from the grassland site, which
had been sprayed with 40 ml of water, and had twenty seeds of
each of the eleven plant species randomly placed at the soil surface. The purpose of presenting a large number of seeds from each
seed species in a large amount of soil over a short period of time
was to increase food choices and avoid seed re-ingestion by earthworms. During the experiment, twenty boxes (ten replicates × two
earthworm species) were incubated in the dark for 24 h at 15 ◦ C.
Thereafter, earthworms and identified casts were removed and
the number of remaining seeds per box was counted for 40 min
under binocular observation. As a precautionary measure, boxes
were incubated for 3 weeks (day/night, 12/12 h and 18/15 ◦ C) to
allow for germination and to avoid missing any seeds. The soil was
turned regularly to increase chances of seed germination. Missing
seeds were considered ingested.
J. Clause et al. / Pedobiologia 54S (2011) S159–S165
S161
Table 1
Traits values of the 11 seed species and 2 earthworm species used for the experimentation. For length, width and weight, mean and standard deviation are given, except
for seed weight calculated from the weight of 1000 seeds units (see “Materials and methods section” for details about data acquisition). Seed shape, length and width were
measured and determined on a panel of 20 seeds. Germination assays were run on a panel of 10 seeds and were replicated 10 times.
Seed species
Family
Length (mm)
Daucus carota
Achillea millefolium
Lotus corniculatus
Medicago lupulina
Origanum vulgare
Teucrium chamaedrys
Teucrium scorodonia
Arrhenaterum elatius
Brachypodium pinnatum
Sanguisorba minor
Urtica dioica
Apiaceae
Asteraceae
Fabaceae
Fabaceae
Lamiaceae
Lamiaceae
Lamiaceae
Poaceae
Poaceae
Rosaceae
Urticaceae
2.8
1.8
1.4
2.6
0.8
1.8
1.3
7.9
9.1
3.2
3.2
±
±
±
±
±
±
±
±
±
±
±
0.4
0.1
0.1
0.2
0.1
0.2
0.1
0.8
0.8
0.6
0.7
Width (mm)
1.5
0.6
1.2
2.2
0.5
1.4
1.1
1.5
1.6
1.7
0.8
±
±
±
±
±
±
±
±
±
±
±
0.2
0.1
0.1
0.2
0.1
0.1
0.1
0.3
0.2
0.4
0.1
Weight (mg)
Shape
Trichomes
Oil content (%)
Germination (%)
1.0
0.2
1.0
1.6
0.1
1.3
0.8
2.8
2.8
7.3
0.1
Oval
Oval
Round
Round
Round
Round
Round
Linear
Linear
Oval
Oval
Yes
No
No
No
No
No
No
Yes
No
No
No
19.3
27.0
6.5
5.5
35.0
10.0
13.0
7.2
2.3
9.4
27.9
40
89
18
12
56
59
52
68
55
72
89
Earthworm species
Length (mm)
Width (mm)
Weight (g)
Satchellius mammalis
Lumbricus terrestris
31 ± 5.2
109 ± 14.3
2.3 ± 0.3
7.1 ± 0.7
0.13 ± 0.04
3.6 ± 1.0
papers of all germination trials were wetted regularly in order to
keep filter papers moist.
Data analysis
The effect of earthworm species on the number of ingested,
digested and germinated seeds was analysed using non-parametric
tests (Kruskal–Wallis tests) for individual seed species, and also for
all species altogether. To determine whether the variations of continuous seed or earthworm variables (weight, length, width and oil
content) were related to ingestion, digestion or germination percentage, data were analysed by non-parametric regression for each
earthworm species using the Ecosim software (Acquired Intelligence Inc. & Kesey-Bear, http://garyentsminger.com/ecosim.htm).
For discrete seed traits (shape and trichomes), Chi-Square tests
were performed. All these analyses were done using JMP software 8
(SAS 2008). Multiple testing corrections using the Truncated Product Method (TPM) of Zaykin et al. (2002) were done on processes
showing statistical significance (i.e. ingestion, digestion or germination). Data before and after TPM adjustment are presented in
Table 2.
The variability of seed morphological characteristics was
described using a Multiple Correspondence Analysis (MCA)
(Tenenhaus and Young 1985). The MCA is a multivariate data
analysis technique for nominal categorical data that provides a
typology of a series of objects described with qualitative variables. To run the analyses on the complete set of seed traits
listed in Table 1, we previously categorised each continuous variable in four classes. For each trait, four classes were created in
order to keep the same statistical weight for all traits. Classes
were defined to cover the totality of variations for each trait and
the limits of classes were adapted so that species distribution
across classes was as close to a normal distribution as possible.
For seed length, A ≤ 1 mm, B = 1–2 mm, C = 2–3 mm, D ≥ 3 mm; for
seed width, A ≤ 1 mm, B = 1–1.5 mm, C = 1.5–2 mm, D ≥ 2 mm; for
seed weight, A ≤ 0.5 mg, B = 0.5–1 mg, C = 1–2 mg, D ≥ 2 mg; for seed
oil content, A ≤ 5%, B = 5–10%, C = 10–15%, D ≥ 15%. In addition, we
calculated the palatability of seeds – their tendency to be readily ingested by the worms – by averaging, for each plant species,
the % of seeds ingested by the two earthworm species. Palatability
categories were defined as follows: low ≤ 20%, medium = 20–40%,
high ≥ 40%. These categories were projected on the MCA axes to
see how they were associated with the different seed traits. All
multivariate analyses were done using the ade4 module (Dray and
Dufour 2007) within the R environment (R Development Core Team
2004).
Results
Importance of earthworm species on the ingestion, digestion and
germination of all seed species
The total number of seeds ingested and digested by L. terrestris (respectively 28.4 ± 4.5% and 24.1 ± 5.9%) and S. mammalis
(24.0 ± 5.9% and 23.6 ± 5.1%) was found to be similar (P = 0.111 and
P = 0.820). Earthworm traits (Table 2) did not have any significant
influence on the total percentage of ingested and digested seeds. No
seeds germinated in S. mammalis casts out of the 528 ingested and
only 34 seeds germinated in L. terrestris casts out of 624 ingested.
Significant effects of earthworm species were observed on seed
ingestion depending on seed species (Fig. 1). L. terrestris ingested
significantly more seeds of A. millefolium, M. lupulina, S. minor, T.
chamaedrys and T. scorodonia than S. mammalis (Fig. 1). Conversely,
S. mammalis ingested more U. dioica seeds than L. terrestris (Fig. 1).
No earthworm effect on seed digestion was observed. The influence
of earthworms on germination was difficult to detect due to the low
number of seeds egested by both species (Table 2). As no germination occurred in S. mammalis Petri dishes, these data were omitted
in Table 2. No additional seeds that could have originated from the
primary horse dung – soil mixture or from the sieved grassland soil
were found in mesocosms or casts. Results from the germination
assays showed that smaller seeds such as U. dioica or A. millefolium
had the highest germination rate of all seeds (89%, Table 1). Most
other seeds had germination rates above 50% except for D. carota, L.
corniculatus and M. lupulina that showed germination rates of 40%,
18% and 12% respectively.
Relationships between seed traits and seed ingestion or digestion
The shape and the presence of trichomes on seeds did not significantly affect the feeding habits of either earthworm species
(Table 2). However, the seed width, length, weight and oil content
significantly affected overall seed ingestion (Table 2). Both earthworm species fed preferentially on small, narrow or light seeds
rather than on long, large or heavy seeds (Fig. 2a). At the earthworm species level, significant results for length were found only
for L. terrestris whereas the impact of the seed weight on seed ingestion was only significant for S. mammalis. Moreover, seeds with high
oil content were also eaten preferentially (Fig. 2b). This trend was
similar for both earthworm species with a slightly higher significant effect for S. mammalis. Only seed width and oil content were
correlated with seed digestion and only for L. terrestris (Table 2).
S162
J. Clause et al. / Pedobiologia 54S (2011) S159–S165
Table 2
Relationships between earthworm traits or seed traits and the ingestion, digestion or germination of seeds after ingestion by two different earthworm species. No germination
occurred for S. mammalis. Correlation coefficients (r) are given for continuous traits (weight, length, width and oil content) and X2 are given for discrete traits (shape and
trichomes). Significant P-values (P < 0.05) are in bold characters. P1: P-value before TPM adjustment, P2: P-value after TPM adjustment. Only data for the ingestion by both
earthworms and digestion by L. terrestris were adjusted with the TPM method as other processes did not show primary significant results.
L. terrestris
S. mammalis
Ingestion
Earthworm traits
Weight
Length
Width
Seed traits
Length
Width
Weight
Shape
Trichomes
Oil content
Digestion
r or X2
P1
P2
0.17
0.35
0.41
0.317
0.156
0.119
0.726
0.335
0.212
−0.58
−0.76
−0.28
4.87
1.39
0.74
0.005
0.004
0.207
0.090
0.240
0.008
0.009
0.004
0.472
0.105
0.600
0.017
r or X2
Germination
Ingestion
Digestion
P1
P2
r or X2
P
r or X2
P1
P2
r or X2
P
0.0014
0.11
−0.05
0.491
0.356
0.462
0.928
0.735
0.858
−0.22
−0.06
0.32
0.307
0.381
0.153
−0.53
−0.50
0.24
0.062
0.079
0.261
0.132
0.200
0.409
−0.49
−0.52
0.21
0.077
0.073
0.286
−0.29
−0.71
−0.13
2.75
0.06
0.57
0.185
0.008
0.320
0.250
0.810
0.003
0.194
0.011
0.589
0.395
0.986
0.003
0.49
0.29
0.07
0.16
0.50
−0.43
0.130
0.172
0.270
0.920
0.480
0.071
−0.53
−0.75
−0.48
2.16
0.35
0.81
0.035
0.008
0.032
0.340
0.550
0.001
0.074
0.009
0.041
0.610
0.824
0.001
0.05
−0.53
−0.04
0.33
0.40
0.39
0.592
0.067
0.329
0.850
0.710
0.891
The results of the MCA are given in Fig. 3. The two first axes
(CA1 and CA2) explained 51% of the total variance of the trait table
(29.6% and 21.4%, respectively). The eigenvalue diagram is given in
Fig. 3c. Fig. 3d shows how the different trait categories were associated with the first two axes, while Fig. 3a represents the projection
of the seed species on this first factorial plane. Three main groups
of species clearly emerge from the analysis. The first group (positive coordinates on CA1) was composed by species (B. pinnatum, S.
minor and A. elatius), which produce large and heavy seeds, of linear to oval shape and with low oil contents (Fig. 3a). Another group
(negative scores on both CA1 and CA2) corresponded to species (A.
millefolium, U. dioica, O. vulgare) with small seeds of oval shape,
Fig. 1. Percentage (mean ± se) of (A) the ingestion and (B) digestion of plant
species seeds by two earthworm species (Lumbricus terrestris in grey, and Satchellius
mammalis in black). Ael: Arrhenaterum elatius, Ami: Achillea millefolium, Bpi: Brachypodium pinnatum, Dca: Daucus carota, Lco: Lotus corniculatus, Mlu: Medicago lupulina,
Ovu: Origanum vulgare, Smi: Sanguisorba minor, Tch: Teucrium chamaedrys, Tsc:
Teucrium scorodonia, Udi: Urtica dioica. For ingestion data, significance of non parametric test within seed species is indicated: ns, not significant; *P < 0.05; **P < 0.01;
***P < 0.001. There was no effect of earthworm species on digestion data.
with no trichomes and high oil content. Finally, the remaining
species (negative scores on CA1 and positive scores on CA2) were
characterized by seeds of medium size, round to oval shapes and
intermediate oil content. The projection of the three palatability
categories on the first factorial plan of the MCA clearly highlights
some relationship between seed traits and their ingestion by earthworms (Fig. 3b). The less palatable category corresponded to seeds
that have low oil contents (oil content categories A, B and C, Fig. 3d)
and that are of large to medium size (length, width and weight categories B, C and D). Conversely, the more ingested seeds tended
to be smaller (category A for length, width and weight variables)
and richer in oil (category D). The only exception was L. corniculatus seeds, which were highly ingested despite their relatively large
size and their medium oil content. Although not so clearly related
to palatability, the absence of trichomes on the seed coat is likely to
be important, as all highly palatable seeds did not have trichomes.
Fig. 2. Relationships between seed traits and seed ingestion by the 2 earthworm
species for (A) seed width and (B) seed oil content. Correlation coefficients (r) and P
values are given for each species.
J. Clause et al. / Pedobiologia 54S (2011) S159–S165
S163
Fig. 3. Multiple Correspondence Analysis (MCA) of the palatability of seed species by earthworm according to seed traits. (C) eigenvalues diagram, and projection on axis 1–2
of (D) the different trait categories (A) seed species and (B) palatability categories by the two earthworm species. Palatability categories were defined as follow: low (L) ≤ 20%,
medium (M) = 20–40%, high (H) ≥ 40%. Categorised continuous variables were encoded (see “Materials and methods section” for details) from A class (smallest values) to D
class (highest values).
Discussion
Are seed ingestion and digestion dependent on earthworm
characteristics?
Studies on species specific effects of earthworms on seed ingestion and digestion are scarce. Earthworm characteristics, such as
size, age, habits and species identity potentially have an impact
on seed choice. For example, Eisenhauer et al. (2009) showed an
influence of earthworm size on ingestion patterns. These authors
found that small endogeic earthworms (Aporrectodea rosea and
Allolobophora chlorotica) ingested small seeds (<1.4 mm width) in
low numbers, and that larger endogeic species (e.g. Octolasion
tyrtaeum and Aporrectodea caliginosa) ingested seeds of all investigated plant species, independently from their body weight. This
suggested that seed ingestion was not related to earthworm size
alone. In the present study, earthworm traits such as length, width
and weight were not correlated with the total number and percentage of seed ingested or digested, and the biggest species (L.
terrestris) did not ingest more seeds than the smallest (S. mammalis). This is surprising, as we would not expect small earthworms
to eat as many seeds as bigger earthworms since they would only
select small seeds that fit their mouth size. Our findings might
be due to the methodology used for counting or to the nature of
the mesocosm that was more suitable for S. mammalis than for
L. terrestris. Indeed, it was quite shallow and did not allow for deep
burial. These aspects are further discussed. Similar to other studies,
we found that the rate of seed ingestion was earthworm speciesspecific (McRill and Sagar 1973; Grant 1983; Shumway and Koide
1994; Eisenhauer et al. 2009) and that the pattern of seed ingestion was different between the two species. L. terrestris ingested
twice the amount of seeds of S. minor compared to that ingested by
S. mammalis, while the latter ingested twice the number or seeds
of U. dioica than L. terrestris. In this study, although we compared
an anecic with an epigeic species, we cannot directly attribute this
contrasted seed selectivity to a group difference (no replicates), but
only to a difference of species identity. To our knowledge, only one
experiment has compared selective ingestion of seeds or seedlings
by different earthworm ecological groups (Asshoff et al. 2010).
The authors showed that anecic species consistently decreased
the number of germinating seeds and the number of established
seedlings. Influence of endogeic and epigeic species was less consistent and strongly depended on the presence of earthworm species
from other ecological groups, especially anecic species, which were
shown to have a dominant effect on seedling establishment.
Although the two earthworm species showed differences in
ingestion preferences, they both actively selected seeds according
to the two same seed trait values: small width and high oil content.
Light seeds were also preferred by S. mammalis and long seeds were
preferred by L. terrestris. Few studies have tried to explain results
S164
J. Clause et al. / Pedobiologia 54S (2011) S159–S165
concerning seed traits and a comparison of different earthworm
species (Eisenhauer et al. 2009). Thus, more research will be necessary to understand if seed predation by earthworms might act
as a selection pressure for plants and their seed production. The
lack of relationship between earthworm traits and seed digestion
supports the results of Eisenhauer et al. (2009), who found no correlation between earthworm size and digestion patterns for all seed
sizes. Finally, poor germination results did not allow us to make
any conclusions on differences in seed germination here but most
studies showed a negative impact of the ingestion of seeds by earthworms (McRill and Sagar 1973; Decaëns et al. 2003; Milcu et al.
2006; Laossi et al. 2009). Seeds may be physically damaged by gut
contractions as well as chemically damaged by enzymes produced
by gut-associated microorganisms (Lattaud et al. 1998).
Which seed traits are attractive for earthworms?
Among all seed traits tested in this study, seed width and oil content were the most significantly and directly correlated to ingestion
rate by earthworms. Seed size has often been recognised as the
main seed trait selected by earthworms for ingestion (Shumway
and Koide 1994; Eisenhauer and Scheu 2008; Eisenhauer et al.
2009). Earthworms were shown to ingest small seeds rather than
larger ones (Grant 1983; Shumway and Koide 1994; Eisenhauer
et al. 2009). The strong correlation between seed width and earthworm seed palatability supports the findings by Eisenhauer et al.
(2009) who showed that the volume and surface of seeds were more
significant criteria, overall, for ingestion than seed length alone.
Physical limitations of earthworms might explain the importance
of seed width as compared to seed length. Indeed, earthworms can
only swallow seeds smaller than or as big as the size of their mouth,
which is approximately 3 mm wide for most earthworm species of
temperate regions (Shumway and Koide 1994; Zaller and Saxler
2007). Therefore, the width – being the smallest dimension – is
also the limiting criterion. Thus, it is surprising to obtain a similar number of ingested seeds for both earthworm species with no
significance related to earthworm size. Our results also show no
significant difference in digestion patterns due to seed size. Similar results were obtained by Eisenhauer et al. (2009). However, in
regards to the total number of seeds ingested, small seeds seemed
to be more easily digested by L. terrestris than larger seeds, which
also support results of Eisenhauer et al. (2010).
To our knowledge, no other study used oil content as a selective trait for earthworm ingestion. Seeds with high oil content
(O. vulgare, A. millefolium or U. dioica) were more attractive to
earthworms than seeds with low oil content (B. pinnatum or M.
lupulina), suggesting that earthworms might use seeds as a nutritive resource. We are aware that seeds traits, such as oil content,
show a degree of phenotypic plasticity in response to environmental conditions under which they develop (Fenner and Thompson
2005). Because specific seed oil content was obtained from an international data base, we might expect a slight difference between
oil content in tested seeds and those found in literature data. To
limit such important bias, we used the mean oil content calculated
on all data from the same species. Additionally, phenotypic variation is inherent in seeds, and production of seeds with different
traits was also thought to increase chances of germination in unpredictable environments (Fenner and Thompson 2005). Eisenhauer
et al. (2010) also investigated the role of earthworms as seed
and seedling predators for nutrition purposes. In order to further
investigate this aspect, other biochemical characteristics should be
studied such as seed or seedling protein, mineral or water contents.
Indeed, Satchell (1967) showed that earthworms preferred proteinand carbohydrate-rich litters to litters with lower protein content.
Patterns in protein content could explain the high ingestion and
digestion rates of L. corniculatus observed in this study, as it might
have been selected for its high protein content despite its low oil
content. It should be noted that an important negative correlation
exists between seed oil content and seed width (R2 = 0.68). Additional statistical analyses performed to test for the actual impact of
each of the two factors did not give significant results as both seed
traits were too strongly correlated. The data adjustment strengthens the significance of our results but we underline the importance
of conducting further experiments which would offer more small
seeds with low oil content and large seeds with high oil content.
Among other tested traits, we only found an effect of seed weight
on seed ingestion for S. mammalis and no effect on seed digestion by either earthworms. Moreover, the MCA suggests that the
weight may influence ingestion by earthworms in the same way
as seed length and width. To our knowledge, no other studies on
earthworms have tested this weight trait, which is known to be a
good predictor of plant palatability. Pirk and De Casenave (2011) for
example showed that seed weight was positively associated with
seed preferences in some ant species. Seed shape – oval, round or
linear seed – even though potentially linked with other seed size
traits, did not show direct correlation with the seed ingestion. However, the MCA indicated that linear seeds were the least ingested.
Other interesting traits should be explored like seed texture or surface, which have been shown to influence the ingestion of seeds.
Seeds with smooth seed coats were found to be more attractive to
earthworms than rough-coated seeds (Shumway and Koide 1994).
These results were suggested by the MCA in the present study, as
all seeds of the highly palatable category did not have trichomes.
Potential implications for plant adaptive strategies
Negative effects of earthworms on plant survival via a reduction
of germination rate following seed ingestion and/or digestion has
been shown by several authors (McRill and Sagar 1973; Decaëns
et al. 2003; Laossi et al. 2009). This effect was also observed in our
study, as most of the seeds that were ingested were also digested
and could thus not germinate. The physiology of seeds must also
be considered in studies, as some seeds that are egested by earthworms may not germinate due to dormancy. Also, the burial of the
seeds selected by earthworms may reduce germination opportunities as buried seeds do not have access to light, and casts may
limit the access of seeds to gas and water (Blanchart et al. 1993;
Decaëns et al. 2003). On the other hand, other studies showed beneficial impacts of the ingestion of certain seeds by earthworms
on germination and plant survival through a chemical stimulation of the germinating process (Ayanlaja et al. 2001; Eisenhauer
et al. 2009), and through the protective and nutrient-rich environment that casts provide (Grant 1983; Shumway and Koide 1994;
Willems and Huijsmans 1994). Seed transportation to the surface
(Willems and Huijsmans 1994; Zaller and Saxler 2007) makes them
more likely to access light and break dormancy after the fragmentation of casts (Decaëns 2000; Decaëns et al. 2003). Due to the
species-specific interactions and earthworm preferences demonstrated here, these positive or negative effects of earthworms on
the probability of seed germination are likely to induce contrasted
effects on seed dynamics, plant fitness and plant communities.
Consequences can be particularly significant in ecosystems with
high earthworm density or in areas invaded by exotic earthworm
populations. In those cases we can expect the seed bank to be
significantly affected by direct earthworm–seed interactions as
well as indirect interactions, depending on the earthworm density and species assemblage (Eisenhauer et al. 2007). Consequently,
the more earthworms, the more likely the seed bank is expected
to be affected (Decaëns et al. 2003). To our knowledge no study
has been done on direct earthworm–seed interactions under large
earthworm populations and this aspect must be further investigated. Consequences of earthworms–seed interactions and seed
J. Clause et al. / Pedobiologia 54S (2011) S159–S165
palatability on the selection of seed traits, as well as the possible
appearance of adaptive strategies in plants such as avoidance of
predation versus attraction for earthworms are challenging topics
that should be further explored, as they illustrate complex soil to
surface interactions and might explain plant evolutionary patterns.
Acknowledgements
Financial support for this work was provided by a grant from the
“Conseil Régional de Haute-Normandie” to Julia Clause (through
the “Grand Réseau de Recherche SER”, ESTER project, and the “FED
4116 SCALE” network). The authors are grateful to the Botanic Garden of Caen for providing seeds. Thanks also to Benoit Richard,
Matthieu Chauvat and Aurélie Husté for their technical support on
earthworm sampling advice as well as Mrs. Tina Schaefer Hayes and
Cindy Kilkenny for their language corrections of the manuscript.
The authors also thank the three anonymous reviewers for their
helpful comments on the paper.
References
Asshoff, R., Scheu, S., Eisenhauer, N., 2010. Different earthworm ecological groups
interactively impact plant seedling establishment. Eur. J. Soil Biol. 56, 330–334.
Ayanlaja, S.A., Owa, S.O., Adigun, M.O., Senjobi, B.A., Olaleye, A.O., 2001. Leachate
from earthworm castings breaks seed dormancy and preferentially promotes
radicle growth in jute. Hortscience 36, 143–144.
Azcarate, F.M., Peco, B., 2003. Spatial patterns of seed predation by harvester
ants (Messor Forel) in Mediterranean grassland and scrubland. Insect. Soc. 50,
120–126.
Bakker, J., Poschlod, P., Strykstra, R., Bekker, R., Thompson, K., 1996. Seedbanks
and seed dispersal: important topics in restoration ecology. Acta Bot. Neerl. 45,
461–490.
Bekker, R., Bakker, J., Grandin, U., Kalamees, R., Milberg, P., Poschlod, P., Thompson, K.,
Willems, J., 1998. Seed size, shape and vertical distribution in the soil: indicators
of seed longevity. Funct. Ecol. 12, 834–842.
Blanchart, E., Bruand, A., Lavelle, P., 1993. The physical structure of casts of Millsonia
anomala (Oligochaeta: Megascolecidae) in shrub savanna soils (Côte d’Ivoire).
Geoderma 56, 119–132.
Darwin, C.R., 1881. The Formation of Vegetable Mould through the Action of Worms
with Observations on their Habits. John Murray, London.
Decaëns, T., 2000. Degradation dynamics of surface earthworm casts in grasslands
of the eastern plains of Colombia. Biol. Fert. Soils 32, 149–156.
Decaëns, T., Mariani, L., Betancourt, N., Jiménez, J., 2003. Seed dispersion by surface casting activities of earthworms in Colombian grasslands. Acta Oecol. 24,
175–185.
Decaëns, T., Margerie, P., Aubert, M., Hedde, M., Bureau, F., 2008. Assembly rules
within earthworm communities in North-Western France – a regional analysis.
Appl. Soil Ecol. 39, 321–335.
Dray, S., Dufour, A., 2007. The ade4 package: implementing the duality diagram for
ecologists. J. Stat. Softw. 22, 1–20.
Ehrenfeld, J., Ravit, B., Elgersma, K., 2005. Feedback in the plant–soil system. Annu.
Rev. Environ. Resour. 30, 75–115.
Eisenhauer, N., Butenschoen, O., Radsick, S., Scheu, S., 2010. Earthworms as seedling
predators: importance of seeds and seedlings for earthworm nutrition. Soil Biol.
Biochem. 42, 1–8.
Eisenhauer, N., Partsch, S., Parkinson, D., Scheu, S., 2007. Invasion of a deciduous
forest by earthworms: changes in soil chemistry, microflora, microarthropods
and vegetation. Soil Biol. Biochem. 39, 1099–1110.
Eisenhauer, N., Scheu, S., 2008. Invasibility of experimental grassland communities:
the role of earthworms, plant functional group identity and seed size. Oikos 117,
1026–1036.
S165
Eisenhauer, N., Schuy, M., Butenschoen, O., Scheu, S., 2009. Direct and indirect effects
of endogeic earthworms on plant seeds. Pedobiologia 52, 151–162.
Fenner, M., Thompson, K., 2005. The Ecology of Seeds. Cambridge University Press,
Cambridge.
Forey, E., Barot, S., Decaëns, T., Langlois, E., Kam-Rigne, L., Margerie, P., Scheu, S.,
Eisenhauer, N. Importance of earthworm–seed interactions for the composition
and structure of plant communities: a review. Acta Oecol., in press.
Grant, J., 1983. The activities of earthworms and the fates of seeds. In: Satchell,
J.E. (Ed.), Earthworm Ecology: From Darwin to Vermiculture. Chapman & Hall,
London, pp. 107–122.
Jones, C., Lawton, J., Shachak, M., 1994. Organisms as ecosystem engineers. Oikos 69,
373–386.
Laossi, K., Decaëns, T., Jouquet, P., Barot, S., 2010. Can we predict how earthworm
effects on plant growth vary with soil properties? Appl. Environ. Soil Sci. 2010,
1–6, Article ID 784342, http://www.hindawi.com/journals/aess/2010/784342/.
Laossi, K., Noguera, D., Bartolomé-Lasa, A., Mathieu, J., Blouin, M., Barot, S., 2009.
Effects of an endogeic and an anecic earthworm on the competition between four
annual plants and their relative fecundity. Soil Biol. Biochem. 41, 1668–1673.
Lattaud, C., Locati, S., Mora, P., Rouland, C., Lavelle, P., 1998. The diversity of digestive
systems in tropical geophagous earthworms. Appl. Soil Ecol. 9, 189–195.
Lavelle, P., 1997. Faunal activities and soil processes: adaptive strategies that determine ecosystem function. Adv. Ecol. Res. 27, 93–132.
Lavelle, P., Bignell, D., Lepage, M., Wolters, W., Roger, P., Ineson, P., Heal, O., Dhillion,
S., 1997. Soil function in a changing world: the role of invertebrate ecosystem
engineers. Eur. J. Soil Biol. 33, 159–193.
Lavelle, P., Decaëns, T., Aubert, M., Barot, S., Blouin, M., Bureau, F., Margerie, P., Mora,
P., Rossi, J., 2006. Soil invertebrates and ecosystem services. Eur. J. Soil Biol. 42,
S3–S15.
Lee, K., 1985. Earthworms: Their Ecology and Relationships with Soils and Land Use.
Academic Press, Sydney, Australia.
McRill, M., Sagar, G., 1973. Earthworms and seeds. Nature 243, 482.
Milcu, A., 2006. The role of earthworms for plant performance and ecosystem functioning in a plant diversity gradient. PhD Thesis. Darmstadt.
Milcu, A., Schumacher, J., Scheu, S., 2006. Earthworms (Lumbricus terrestris)
affect plant seedling recruitment and microhabitat heterogeneity. Ecology 20,
261–268.
Pirk, G.I., De Casenave, J.L., 2011. Seed Preferences of three harvester ants of the
genus Pogonomyrmex (Hymenoptera: Formicidae) in the Monte desert: are they
reflected in the diet? Ann. Entomol. Soc. Am. 104, 212–220.
R Development Core Team, 2004. R: A Language and Environment for Statistical
Computing. R Foundation for Statistical Computing, Vienna, Austria.
Regnier, E., Harrison, S., Liu, J., Schmoll, J., Edwards, C., Arancon, N., Holloman, C.,
2008. Impact of an exotic earthworm on seed dispersal of an indigenous US
weed. J. Appl. Ecol. 45, 1621–1629.
SAS, P., 2008. JMP 8 Introductory Guide. Sas Institute Inc., Cary, NC, USA.
Satchell, J.E., 1967. Lumbricidae. In: Burge, A., Raw, F. (Eds.), Soil Biology. Academic
Press, London, pp. 259–322.
Scheu, S., 2003. Effects of earthworms on plant growth: patterns and perspectives.
The 7th International Symposium on Earthworm Ecology – Cardiff Wales – 2002.
Pedobiologia 47, 846–856.
Shumway, D., Koide, R., 1994. Seed preferences of Lumbricus terrestris L. Appl. Soil
Ecol. 1, 11–15.
Tenenhaus, M., Young, F., 1985. An analysis and synthesis of Multiple Correspondence Analysis, optimal scaling, homogeneity analysis and other methods for
quantifying categorical multivariate data. Psychometrika 50, 91–119.
Thompson, K., Green, A., Jewels, A.M., 1994. Seeds in soil and worm casts from a
neutral grassland. Funct. Ecol. 8, 29–35.
Willems, J., Huijsmans, K., 1994. Vertical seed dispersal by earthworms: a quantitative approach. Ecography 17, 124–130.
Zaller, J.G., Arnone III, J.A., 1999. Interactions between plant species and earthworm casts in a calcareous grassland under elevated CO2 . Ecology 80,
873–881.
Zaller, J.G., Saxler, N., 2007. Selective vertical seed transport by earthworms:
implications for the diversity of grassland ecosystems. Eur. J. Soil Biol. 43,
86–91.
Zaykin, D.V., Zhivotovsky, L.A., Westfall, P.H., Weir, B.S., 2002. Truncated product
method for combining P-values. Genet. Epidemiol. 22, 170–185.