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Effects of soil and leaf litter quality on the biomass of two endogeic earthworm species
European Journal of Soil Biology 77 (2016) 9e16
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European Journal of Soil Biology journal homepage: http://www.elsevier.com/locate/ejsobi
Original article
Effects of soil and leaf litter quality on the biomass of two endogeic earthworm species Simone Cesarz a, b, c, *, Dylan Craven a, b, Christoph Dietrich a, b, Nico Eisenhauer a, b a
German Centre for Integrative Biodiversity Research (iDiv) Halle-Jena-Leipzig, Deutscher Platz 5e, 04103 Leipzig, Germany Institute of Biology, Leipzig University, Johannisallee 21, 04103 Leipzig, Germany c €ttingen, Berliner Straße 28, 37073 Go €ttingen, Germany J.F. Blumenbach Institute of Zoology and Anthropology, Georg August University Go b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 9 May 2016 Received in revised form 12 September 2016 Accepted 12 September 2016
Earthworms drive important ecosystem functions like decomposition and nutrient mineralization in many terrestrial ecosystems, which is why factors controlling their mass gain are of great scientific interest. We conducted a microcosm experiment using two common endogeic earthworm species (Aporrectodea caliginosa and Octolasion tyrtaeum) and two different soils (one from a beech-dominated forest and one from a mixed tree species forest in Germany) to test litter quality (different nutrient concentrations) and soil quality effects (low and high) on relative mass gain of earthworms. We hypothesized that mass gain of endogeic earthworms is driven by both soil and litter quality. Soil pH, carbon (C) and nitrogen (N) concentrations were used to characterize soil quality, while leaf litter N, phosphorus (P), calcium (Ca), and magnesium (Mg) concentrations were used as proxies for leaf litter quality. Forest soils were incubated with leaf litter of six common tree species in Central Europe (Fagus sylvatica, Acer platanoides, Acer pseudoplatanus, Carpinus betulus, Tilia spp., and Fraxinus excelsior) that span a gradient in leaf litter quality. In addition, we determined soil microbial biomass C as a potential food source of endogeic earthworms. After three months, relative earthworm mass gain of A. caliginosa and O. tyrtaeum was significantly higher in soil from the mixed tree species forest (high quality soil: þ218% and þ240%, respectively) compared to soil from the beech-dominated forest (low quality soil: þ160% and þ162%, respectively). Relative mass gain of A. caliginosa increased significantly with all leaf litter nutrients in low quality soil, whereas in high quality soil only leaf litter Ca positively affected relative mass gain. Similarly, relative mass gain of O. tyrtaeum increased significantly with increasing concentrations of leaf litter N, Mg, and Ca in the low quality soil. In the high quality soil, only leaf litter Mg significantly increased relative mass gain. Overall, our results indicate that leaf litter quality effects on endogeic earthworm mass gain were more important in low quality soil for both earthworm species. Notably, microbial biomass was significantly higher in high quality soil (506 ± 135 mg C g1 soil dw) compared to low quality soil (217 ± 64 mg C g1 soil dw), but microbial biomass was not significantly affected by leaf litter type and was a poor predictor of relative earthworm mass gain. This finding indicates that endogeic earthworms did not significantly depend on soil microbial biomass, but rather on the quality of dead organic material in the soil and surface leaf litter. As earthworms may prefer feeding on certain microbial taxa, and we only measured total soil microbial biomass, future studies could investigate if leaf litter quality effects on earthworms are mediated by changes in soil microbial community structure, micronutrients, and organic compounds. © 2016 Elsevier Masson SAS. All rights reserved.
Handling Editor: S. Schrader Keywords: Earthworm mass gain Endogeic earthworms Forest soil Leaf litter Nutrient sources Partial least squares path modeling
1. Introduction
* Corresponding author. German Centre for Integrative Biodiversity Research (iDiv) Halle-Jena-Leipzig, Deutscher Platz 5e, 04103 Leipzig, Germany. E-mail addresses: [email protected] (S. Cesarz), [email protected] (D. Craven), [email protected] (C. Dietrich), [email protected] (N. Eisenhauer). http://dx.doi.org/10.1016/j.ejsobi.2016.09.002 1164-5563/© 2016 Elsevier Masson SAS. All rights reserved.
Earthworms play an important role in many terrestrial ecosystems as ecosystem engineers, by modifying soil structure via bioturbation as well as through their contribution to decomposition processes [1e3]. Furthermore, they often comprise, together with Protozoa, the largest biomass of soil fauna [4], and their functional
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significance, i.e., their effects on ecosystem processes, makes earthworms important study organisms in ecological and ecotoxicological research [5,6]. Moreover, some earthworm species are biological invaders in many ecosystems with major impacts on soil chemistry, plant communities, and soil microarthropod communities [7e9]. Consequently, it is vital to identify drivers that control earthworm mass gain. Earthworms can be divided into three ecological groups: epigeic earthworms, which live in and feed on the leaf litter layer on the soil surface; anecic earthworms, which dig large vertical and permanent burrows and feed on surface leaf litter, and endogeic earthworms, which feed on organic matter in the mineral soil and build horizontal burrows in deeper organic and mineral soil layers [10]. Together with anecic earthworms, endogeic earthworms often dominate the biomass of soil invertebrates [2]. Endogeic earthworms have a very low assimilation efficiency (2%), resulting in high consumption rates of their poor quality diet, i.e., soil [11]. It has been suggested that endogeic earthworms feed on stabilized soil particles [12] as well on easily available compounds like glucose [13]. Rajapaksha et al. [14] demonstrated that endogeic earthworms were able to remove soil surface litter, which suggests that leaf litter may serve as potential additional food resource with both stable and soluble compounds. Direct or indirect litter feeding by endogeic earthworm has, however, rarely been investigated. Thus, exploring factors influencing the ecology of endogeic earthworms can help to improve species distribution models [15], understand their influence on the bioavailability of micronutrients [16], and increase our knowledge about their interactions with microorganisms that drive priming effects [17]. The quality of root and leaf litter inputs influence the quality of soil and organic matter and therefore the mass gain of endogeic earthworms [2,18e20]. Generally, low soil pH and low moisture negatively affect earthworm development [21,22], whereas high soil organic matter content positively affects earthworm nutrition and therefore mass gain [11]. Several studies have investigated the importance of soil microorganisms, mainly fungi, for earthworm nutrition but clear evidence is still lacking. Previous studies have suggested that endogeic earthworms consume microorganisms as bycatch during soil ingestion, as helpers to degrade recalcitrant compounds, but also as a direct food resource [11]. Food choice experiments have demonstrated a clear preference of earthworms for leaf litter with low C:N ratio and low concentrations of secondary compounds, and polyphenol content [14,23,24]. In addition, the availability of soil carbon (C) [13,25], and nitrogen (N) [26] have also been shown to increase earthworm mass gain. More recently, high calcium (Ca) concentration in soil was linked to reduced soil acidity and increased fertility, factors which positively affect both earthworm abundance and diversity [18]. While the effects of other leaf litter nutrients, such as phosphorus (P) and magnesium (Mg), on earthworm growth were not studied, a recent study by Resner et al. [27] showed that earthworms reduced the availability of Ca, Mg, K, and P when invading a North American forest. Thus, factors affecting earthworm mass gain have generally been attributed to their feeding preferences and direct food sources. In doing so, effects of leaf litter quality were tested by using mainly anecic and epigeic earthworms, whereas soil quality was tested by using endogeic earthworms. Interactions of leaf litter and soil quality on earthworm mass gain have been largely neglected. Dechaine et al. [28] even suggested ignoring potential feeding relationships of endogeic earthworms on leaf litter material. However, feeding relationships in soil are complex and still poorly understood [29]. Investigating and understanding feeding relationships in soil is of particular importance to appreciate the consequences of anthropogenic environmental changes for species
interactions. The aim of the present study was to explore if the potential dependence of endogeic earthworm species on surface leaf litter material is contingent upon soil nutrient content. While surface leaf litter quality effects on anecic earthworms have already been investigated [11,30], the present study is one of the first investigating potential interaction effects of leaf litter and soil quality on endogeic earthworms. In this study, we investigated drivers of changes in earthworm biomass in soils of contrasting quality (low versus high quality) in combination with leaf litter types (beech, lime, ash, hornbeam, and two maple species) representing a broad gradient in nutrient concentrations and, therefore, leaf litter quality. We explored the relative mass gain of two endogeic earthworm species, Aporrectodea caliginosa and Octolasion tyrtaeum, as this ecological group often dominates the density and biomass of earthworm communities [31] and to determine if two functionally similar species exhibit similar responses to soil and leaf litter quality effects. As detailed above, endogeic species are considered to feed mostly on mineral soil, but leaf litter material could also have indirect effects on earthworm mass gain by altering nutrient availability and/or microbial community biomass and composition [32]. We hypothesized that endogeic earthworm relative mass gain would be significantly higher in high quality soil than in low quality soil (hypothesis 1) due to a higher pH value [2], higher nutrient availability (especially C, N, and Ca) [19], and higher soil microbial biomass [33]. Further, we hypothesized that leaf litter chemistry (C, N, P, Ca, and Mg concentrations) would influence relative mass gain of endogeic earthworms (hypothesis 2). Specifically, we expected increased leaf litter N and Ca concentrations to enhance earthworm biomass [18,34]. We also tested for effects of other leaf litter nutrients, such as P and Mg concentrations, on earthworm relative mass gain as these nutrients have rarely been considered in previous studies and could have significant effects on earthworm mass gain [35,36]. Finally, we hypothesized that negative effects on earthworm mass gain in low quality soil would be compensated by high leaf litter quality (hypothesis 3). Given correlations among different leaf litter quality properties and soil microbial biomass, we used partial least squares path modeling (PLS-PM) to identify the main drivers of earthworm mass gain [37]. 2. Materials and methods 2.1. General design A microcosm experiment was established to study the relative mass gain of the two endogeic earthworm species A. caliginosa and O. tyrtaeum in two different soils (where they naturally occur but in different densities [38]) and in different leaf litter treatments. The experiment was conducted using a two factorial design with two levels for the factor “soil” (low and high quality) and six levels for the factor “leaf litter” (one level for each tree species). Further, we used leaf litter concentrations of N, P, Ca, and Mg as proxies for leaf litter quality, which have been shown to determine essential processes like leaf litter decomposition [39], but other organic compounds like tannins and phenols may also be important for earthworm mass gain [14,23,24]. Every treatment was replicated seven times and the experiment ran for a total of three months. 2.2. Soil and leaf litter Soil and leaf litter were taken from the Hainich National Park (Thuringia, Germany, annual precipitation: 600.5 mm and mean annual temperature: 9.0 C). The soil was taken from a depth of 0e20 cm (without leaf litter layer) in forest plots with different
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Table 1 Properties (mg g1 dry mass) of nitrogen (N), carbon (C), calcium (Ca), magnesium (Mg), phosphorous (P) of leaf litter as well as nutrient ratios of the six tree species used in the present study. Species are sorted according to their C:N.
Fagus sylvatica Carpinus betulus Tilia spec. Acer platanoides Acer pseudoplatanus Fraxinus excelsior a
C
N
Pa
Caa
Mga
C:N
47.86 46.71 48.10 48.21 46.66 49.03
8.30 11.02 11.89 11.78 11.72 15.65
0.36 0.61 0.63 0.63 0.47 0.69
16.80 21.49 27.47 24.62 30.15 28.83
1.21 2.86 2.58 2.10 3.17 2.87
56.06 40.08 37.93 36.53 36.40 27.60
Data on P, Ca, and Mg concentrations were derived from the same study sites from Jacob et al. [40,41].
species compositions. The beech-dominated forest (low soil quality) had >94% coverage by beech (Fagus sylvatica L.) in the canopy, while the mixed tree species forest (high soil quality) contained beech (~25%), lime (Tilia cordata Mill. and Tilia platyphyllos Scop. in the following named as Tilia spp.), ash (Fraxinus excelsior L.), hornbeam (Carpinus betulus L.), and two species of maple (Acer pseudoplatanus L., and Acer platanoides L.). The soil type is a luvisol with pH ranging from 3.7 to 4.5 in soil from the beech-dominated forest (low quality soil) and 5.7 to 5.9 in soil from mixed tree species forest (high quality soil). The soil was sieved (4 mm) and defaunated (heat extraction at 55 C for seven days) and then stored at room temperature. Leaf litter from beech, lime, ash, hornbeam, Norway maple, and sycamore maple was collected from senescent leaves in the Hainich National Park at plots with high tree diversity. Leaf litter was airdried and stored at room temperature. Sampling leaf litter at one specific location enabled us to use literature data of the chemical composition on the leaf litter (C, N, P, Ca, and Mg) from the same location (site DL 3; see Refs. [40,41]).
2.3. Earthworms Juvenile earthworms were sampled between June and July 2005. To obtain the required number of individuals, earthworms €ttinger were taken from the Hainich National Park and from the Go Forest (Lower Saxony, Germany). Two days before starting the experiment, earthworms were placed in Petri dishes with wet filter paper and kept in a climate chamber at 15 C for 48 h to void their gut content [42]. Afterwards, earthworms were weighed individually, and every microcosm received one earthworm. Earthworm starting mass was 0.164 ± 0.087 g for A. caliginosa and 0.100 ± 0.049 g for O. tyrtaeum, and individuals were randomly assigned to leaf litter and soil treatments. Soil and leaf litter quality had no significant effect on earthworm mortality (P > 0.05 for both species). Average mortality was 16 ± 37% and 34 ± 48% for A. calignosa and O. tyrtaeum, respectively.
2.5. Experimental procedure Large microcosms were filled with 210 g soil (air dried) each, and the smaller ones were filled with 110 g soil. Ensuring that every microcosm received the same amount of carbon via leaf litter addition, C and N content of the different leaf litter species were measured (Table 1). Oven dried leaf litter (70 C, 24 h) was ground, weighed, and placed into tin capsules, which was analyzed by an automated element analyzer (NA 1500, Carlo Erba, Mailand). Every microcosm received 504 mg carbon in the form of leaf litter. Leaf litter was cut into pieces of 1e2 cm2 and was added to the soil surface in each microcosm to simulate leaf fall. The microcosms were watered with 216 ml (large microcosms) and 90 ml (small microcosms) of distilled water, respectively, and incubated for one week before the experiment started. Afterwards, 72 ml and 30 ml of distilled water were added biweekly, respectively. The experiment was terminated after 12 weeks as ash leaf litter had mostly disappeared. Surplus soil water was suctioned by lowering pressure for three days after watering. To estimate earthworm mass at the end of the experiment, microcosms were destructively sampled. The earthworms were placed in Petri dishes as described above to void their gut content for 24 h. The relative change in earthworm mass was used as a proxy for earthworm performance and was calculated as the percent change in mass from the beginning to the end of the experiment. Microbial biomass C was determined using the substrate induced respiration (SIR) method [43], i.e., the respiratory response of microorganisms to glucose addition. Soils used for the measurements were sieved (2 mm) to remove roots and leaf litter. An O2-microcompensation apparatus modified after [44] was used to perform the measurements. We added 8 mg glucose g1 soil dry weight as an aqueous solution to the soil samples. The mean of the lowest three readings within the first 10 h (between the initial peak caused by disturbing the soil and the beginning of microbial growth) was assessed as the maximum initial respiratory response (MIRR; ml O2 g1 soil dry weight), and microbial biomass (mg C g1 soil dry weight) was calculated as 38 MIRR [45].
2.4. Microcosms
2.6. Statistical analysis
The experiment was set up in microcosms consisting of plastic tubes connected to ceramic plates allowing drainage by lowering the atmospheric pressure (0.4e0.6 bar). This system had two different tube sizes; we used large tubes (height 15 cm, ø 6.2 cm) for A. caliginosa and smaller ones (height 15 cm, ø 4.5 cm) for O. tyrtaeum due to differences in body size between the species. As we were interested in treatment effects on relative earthworm mass gain and not in the comparisons of absolute mass gain of the two earthworm species, differences in microcosm size do not affect the conclusions of our study. Microcosms were closed at the top by a lid to prevent earthworms from escaping.
The effects of soil quality (low and high), leaf litter identity (six leaf litter species), and interactions of both treatments were analyzed for relative earthworm mass gain and microbial biomass with 2-way-ANOVA. Homogeneity of variances was tested using the Bartlett test, and normality was checked using Shapiro wilk test. Microbial biomass C was log-transformed to meet the assumptions of ANOVA. The relative change in earthworm mass gain was calculated as percentage change of earthworm mass at the beginning of the experiment (equals 100%) compared to the end of the experiment. Partial least squares path modeling was performed in R using the ‘plspm’ package [37] to infer direct and indirect
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effects of leaf litter quality (using leaf litter N, P, Ca, and Mg as mg g1 dry mass), litter C (mg g1 dry mass), and microbial biomass C (mg C g1 dry soil weight) on earthworm relative mass gain of both earthworm species separately. Leaf litter quality was treated as a latent variable consisting of leaf litter N, P, Ca, and Mg as manifest variables. As leaf litter C concentrations are strongly associated with other structural measures of leaf litter, such as lignin and cellulose [46], it was treated as a separate latent variable, thereby ignoring possible effects of soluble C compounds [47]. Lastly, microbial biomass C was treated as a latent variable. We expected that leaf litter quality and soil microbial biomass C would increase relative mass gain of both earthworm species and that leaf litter C concentrations would decrease earthworm relative mass gain. The statistical significance of path coefficients for models were assessed using 1000 bootstrap replicates to determine if they differed from zero, as were the contributions of individual manifest variables to their corresponding latent variable. All analyses were performed using R 3.2.2 [48]. 3. Results In the high quality soil, the relative mass gain of A. caliginosa (mean ± SD: 218 ± 55%; P ¼ 0.0052) and O. tyrtaeum (240 ± 75%; P < 0.0001) was significantly higher than in the low quality soil (Fig. 1a; 160 ± 80% and þ162 ± 67%, respectively). Leaf litter identity and interactions with soil quality had no significant effects on relative mass gain (P > 0.05). Further, soil microbial biomass C was significantly higher in the high quality soil for both A. caliginosa (450 ± 149 mg C g1 soil dry weight; P < 0.0001) and O. tyrtaeum (547 ± 112 mg C g1 soil dry weight; P < 0.0001), compared to low quality soil (191 ± 58 and 239 ± 63 mg C g1 soil dry weight, respectively; Fig. 1b). Leaf litter identity and interactions had no significant effects on microbial biomass (P > 0.05). The relative mass gain of A. caliginosa and O. tyrtaeum was
significantly positively correlated to the quality of the leaf litter in the low quality soil (Fig. 2a,c; Table 2). In the high quality soil, leaf litter quality effects on earthworm mass gain were markedly weaker and non-significant for O. tyrtaeum (Fig. 2d, Table 2), and the relative mass gain of A. caliginosa was slightly negatively but not significantly correlated with leaf litter quality (Fig. 2b, Table 2). While Ca, Mg, N, and P concentrations determined leaf litter quality effects on the relative mass gain of A. caliginosa in low quality soil, Ca concentration was the only significant determinant of leaf litter quality effects in the high quality soil (Fig. 2a,b; Table 3). Ca, Mg, and N concentrations determined leaf litter quality effects on the relative mass gain of O. tyrtaeum in the low quality soil, whereas Mg and N concentrations determined leaf litter quality effects in the high quality soil (Fig. 2c,d; Table 3). Leaf litter C concentrations had no significant effects on the relative mass gain of either earthworm species in both low and high quality soils. However, leaf litter C concentrations significantly increased microbial biomass in the high quality soil in the presence of O. tyrtaeum (Fig. 2d; Table 2), but not in the other treatments. Generally, the influence of microbial biomass C on the relative mass gain of both earthworm species was positive but non-significant in both soils. Microbial biomass C was not significantly affected by leaf litter quality (Fig. 2; Table 2). 4. Discussion Exploring the nutrition of ecosystem engineers, such as earthworms, is fundamental for understanding how environmental changes influence the composition and functioning of ecosystems. The objective of the present study was to assess soil quality effects and to identify leaf litter traits determining endogeic earthworm relative mass gain in different contexts. As expected, earthworm relative mass gain was higher in high quality soil. Beyond this soil effect, we found that the relative mass gain of the two endogeic earthworm species tested was determined by leaf litter nutrient concentrations in the low quality soil, which was acidic, nutrientpoor, and low in soil microbial biomass. Other studies have shown previously significant effects of leaf litter Ca [18], soil Mg [35], leaf litter and soil N [34], and C:P ratios [36] on biomass, density, and species richness of total and epigeic earthworms. This study, for the first time, investigated interactive effects of soil quality and leaf litter concentrations of C, N, P, Ca, and Mg on endogeic earthworms. Our findings suggest that leaf litter N, P, Ca, and Mg may be major determinants of endogeic earthworm relative mass gain. As both endogeic earthworm species were able to gain nutrients from soil surface litter, future studies could investigate if endogeic earthworms actively switch to a more leaf litterbased nutrition in low quality soils or if leaching or increased microbial decomposition of high quality leaf litter affect earthworm mass gain. 4.1. Soil quality
Fig. 1. Effect of soil quality (low and high) in a microcosm experiment with the two endogeic earthworms Aporrectodea caliginosa and Octolasion tyrtaeum on mean ± SD of a) relative earthworm mass gain and b) microbial biomass C after an experimental duration of three months. Asterisks represent ANOVA results (log-transformed for microbial biomass) of soil effect with *** ¼ P < 0.001 and ** ¼ P < 0.01.
In line with our expectations, we found that earthworm mass gain was significantly greater in high quality soil, i.e., soil with higher pH, nutrient availability, and microbial biomass. Our results suggest that soil pH was an important driver of these soil quality effects [18] because soil pH from the beech-dominated forest (3.7e4.5) was at the lower limit of the tolerable range of A. caliginosa [49]. However, recent studies have highlighted that soil pH does not directly affect the density of soil organisms, but rather indirectly drives chemical processes related to nutrient availability [36]. For instance, P forms insoluble complexes with Ca at alkaline pH values, and with Al and Fe at acidic pH values [50]. In our study, we found N, P, Ca, and Mg to be important for earthworm relative mass gain at low soil quality with acidic soil pH, which suggests
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Fig. 2. Partial least squares path models of the drivers of earthworm relative mass gain. Best fitting models for direct and indirect effects of leaf litter quality, leaf litter C concentrations, and microbial biomass C relationships on earthworm relative mass gain of Aporrectodea caliginosa (a,b) and Octolasion tyrtaeum (c,d) in soils of a beech-dominated forest (low quality soil, i.e., low soil pH, low nutrient availability) and a mixed tree species forest (high quality soil, i.e., high soil pH, high nutrient availability). Leaf litter quality data were derived by concentrations of leaf litter N, P, Ca, and Mg, leaf litter C concentration by measuring carbon in leaf litter (C), microbial biomass C was informed by data derived from substrateinduced respiration (mC) and earthworm relative mass gain by data about earthworm mass gain (mg). Numbers on arrows are path coefficients indicating a positive (solid line) or negative effect (dashed line). Numbers above manifest variables indicate the proportional contribution of a manifest variable to its corresponding latent variable. In the case of leaf litter C concentrations and microbial biomass C only one manifest variable contributed to the analysis resulting in a weight of one which is not shown. Significant paths are given in bold. Percentages in shaded circles are R2 values and indicate the variance explained by the model. Goodness of fit: a) 0.476); b) 0.401; c) 0.372; d) 0.397.
that those nutrients are limiting earthworm nutrition in soil from a beech-dominated forest but can be derived from leaf litter. Importantly, we could not determine if nutrients were less
available due to a generally lower input to soil or due to the formation of insoluble complexes at low pH, but our results suggest that earthworm mass gain is limited by lower nutrient availability
Table 2 Path coefficients of latent variables for partial least squares path fitted for two endogeic earthworm species (Aporrectodea caliginosa and Octolasion tyrtaeum) in soils of two different qualities, i.e., from a beech-dominated forest (low soil quality, i.e., low soil pH and low nutrient availability) and a mixed tree species forest (high quality soil, i.e., high soil pH and high nutrient availability). Significant relationships are given in bold. PC ¼ path coefficient; CI ¼ confidence interval. Earthworm species
Soil quality
Paths
PC
Lower 95% CI
Upper 95% CI
Aporrectodea caliginosa
low
leaf litter quality / microbial biomass C leaf litter C concentrations / microbial biomass C leaf litter quality / earthworm relative mass gain leaf litter C concentrations / earthworm relative mass gain microbial biomass C / earthworm relative mass gain
0.041 0.232 1.118 0.566 0.197
0.862 0.453 0.664 0.032 0.217
0.869 0.927 1.672 1.152 0.534
high
leaf litter quality / microbial biomass C leaf litter C concentrations / microbial biomass C leaf litter quality / earthworm relative mass gain leaf litter C concentrations / earthworm relative mass gain microbial biomass C / earthworm relative mass gain
0.796 0.606 0.237 0.108 0.243
0.089 0.036 1.347 1.239 0.630
1.289 1.323 0.744 0.837 0.940
low
leaf litter quality / microbial biomass C leaf litter C concentrations / microbial biomass C leaf litter quality / earthworm relative mass gain leaf litter C concentrations / earthworm relative mass gain microbial biomass C / earthworm relative mass gain
0.523 0.219 0.693 0.424 0.136
0.246 0.504 0.185 0.254 0.210
1.112 0.863 1.194 0.985 0.504
high
leaf litter quality / microbial biomass C leaf litter C concentrations / microbial biomass C leaf litter quality / earthworm relative mass gain leaf litter C concentrations / earthworm relative mass gain microbial biomass C / earthworm relative mass gain
0.686 0.632 0.491 0.067 0.064
0.051 0.014 0.096 0.566 0.290
1.242 1.157 1.086 0.690 0.423
Octolasion tyrtaeum
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Table 3 Contribution of manifest variables (concentrations of N, P, Ca, and Mg of leaf litter) to the latent variable ‘leaf litter quality’ in partial least squares path models fitted for two endogeic earthworm species (Aporrectodea caliginosa and Octolasion tyrtaeum) in soils of two different qualities, i.e., from a beech-dominated forest (low soil quality, i.e., low soil pH and low nutrient availability) and a mixed tree species forest (high quality soil, i.e., high soil pH and high nutrient availability). Weights indicate the proportional contribution of a manifest variable to its corresponding latent variable. CI ¼ confidence interval. Significant relationships, i.e., those that do not overlap zero, are given in bold. Earthworm species
Soil quality
Manifest variable
Weights
Lower 95% CI
Upper 95% CI
Aporrectodea calignosa
low
leaf leaf leaf leaf
0.342 0.216 0.274 0.287
0.255 0.112 0.149 0.195
0.472 0.299 0.372 0.449
high
leaf litter N leaf litter Mg leaf litter Ca leaf litter P
0.252 0.236 0.317 0.270
0.107 0.167 0.061 0.405
0.424 0.709 0.747 0.605
low
leaf litter N leaf litter Mg leaf litter Ca leaf litter P
0.250 0.462 0.285 0.087
0.059 0.187 0.142 0.301
0.382 0.784 0.401 0.334
high
leaf litter N leaf litter Mg leaf litter Ca leaf litter P
0.343 0.255 0.176 0.336
0.221 0.045 0.129 0.002
0.476 0.498 0.407 0.603
Octolasion tyrtaeum
litter litter litter litter
N Mg Ca P
in soil. This nutrient shortage was relaxed in the high quality soil as indicated by the lower impact of leaf litter nutrients on earthworm mass gain. In the case of A. caliginosa, only Ca positively affected its mass gain, whereas mass gain of O. tyrtaeum increased with leaf litter N and Mg, indicating differences between the two endogeic earthworm species. During decomposition, tannin content of leaf litter, especially that of the low quality beech leaf litter, may further constrain earthworm mass gain by reducing soil nutrient availability through the formation of mineralization-resistant proteintannin complexes [51e53]. We further hypothesized that soil microbial biomass would drive earthworm growth. Although we found microbial biomass to be higher in the high quality soil compared to the low quality soil, PLS path models showed no significant direct relationship between microbial biomass and earthworm growth. Feeding choice experiments have shown that earthworms prefer specific microorganisms, mainly fungi [54e56], suggesting that it is not biomass per se that affects earthworm nutrition but rather the presence of specific taxa of the microbial community. Further, we suggest that microbial communities differed between the two soils mainly due to differences in soil properties like pH and nutrient availability [57]. Endogeic earthworms need litter material to be ‘pre-decomposed’ by microorganisms in order to access nutrients and future studies could identify the most important microbial groups or taxa mediating leaf litter effects on endogeic earthworms. 4.2. Leaf litter nutrient concentrations and interactions with soil quality Leaf litter N, P, Ca, and Mg concentrations showed positive correlations with earthworm relative mass gain in the low quality soil. Endogeic earthworm species are thought to feed primarily on organic material in the mineral soil [58]. However, there is some evidence that A. caliginosa is not restricted to mineral soil as its only food source, but that it can also directly feed on leaf litter [11]. Further, it was shown that endogeic earthworms selectively feed on enriched organic matter [14,59] indicating that they are able to directly benefit from leaf litter material. The stronger increase of earthworm relative mass gain with higher leaf litter nutrient concentrations in the low quality soil suggests, in addition to unfavorable abiotic soil conditions, that litter micronutrient content and P also affected earthworm relative mass gain. Low quality leaf litter, such as that of beech, is high in
tannin content [60] and decreased earthworm relative mass gain in this study. That the leaf litter quality effects were weaker in the high quality soil supports the idea that endogeic earthworms rely more on soil organic material. However, in soils with low amounts of organic material, such as those found in beech-dominated forest, leaf litter effects significantly affected the relative mass gain of both species studied. This may also indicate that endogeic species can switch feeding behavior in response to soil and leaf litter quality. PLS path models revealed that in microcosms with O. tyrtaeum and high quality soil, leaf litter C concentration significantly affected microbial biomass, whereas in all other cases no significant relationship was found for this path. This may indicate that distinct earthworm species may significantly affect leaf litter effects on microbial communities or vice versa. We further suggest the need to go beyond the consideration of solely C and N concentrations and investigate micronutrients and organic molecules, such as phenols, tannins, and lignin, to further current understanding of earthworm nutrition [61,62]. Our study confirmed the significant role of leaf litter Ca concentrations in driving earthworm communities found in a tree plantation in Poland [18]. In the study by Mueller et al. [35] at the same field site in Poland, the presence and abundance of the anecic earthworm species Lumbricus terrestris, as well as the species richness of earthworms, were strongly positively correlated with soil pH and the amount of base cations (mostly Ca and Mg) in leaf litter and soils, likely due to the high Ca requirements of some earthworm species [18,63]. Many earthworm species produce calcite granules in specialized calciferous glands, which may explain high Ca requirements of A. caliginosa. Their function is not known but it is suggested that they regulate CO2 respiration, excrete excess Ca, and neutralize acidic gut pH [64]. We have no information about Mg requirements of earthworms but due to similar chemical traits as Ca, there might be similarities in reactions with P as stated above. P was shown to be important at all trophic levels in forest ecosystems [36] and should also be included in standard measurements to test the generality of our findings. In the present study, both endogeic earthworm species gained more mass in high quality soil, with higher pH and nutrient concentrations, and consistently depended on litter quality in low quality soil. Our results indicate that endogeic earthworms can benefit from nutrient-rich leaf litter in nutrient-poor soils and that leaf litter effects are not always mediated via soil microbial biomass. Future studies could investigate microbial community
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composition to test if certain soil microbial taxa mediate soil surface leaf litter effects on endogeic earthworms. Acknowledgements We acknowledge funding by the German Research Foundation in the frame of the Emmy Noether research group (Ei 862/2). Further support came from the German Centre for Integrative Biodiversity Research (iDiv) Halle-Jena-Leipzig, funded by the German Research Foundation (FZT 118). The study was part of the Research Training Group 1086 funded by the German Research Foundation. Simone Cesarz thanks Sonja Migge-Kleian for supervision and support and Matthias Schaefer for providing all the necessary resources and support to conduct this study. We also thank two reviewers for helpful comments on earlier versions of this paper. References [1] G.G. Brown, I. Barois, P. Lavelle, Regulation of soil organic matter dynamics and microbial activity in the drilosphere and the role of interactions with other edaphic functional domains, Eur. J. Soil Biol. 36 (2000) 177e198. [2] C.A. Edwards, Earthworm Ecology, second ed., CRC Press, Columbus, Ohio, 2004. [3] M. Blouin, M.E. Hodson, E.A. Delgado, G. Baker, L. Brussaard, K.R. Butt, J. Dai, L. Dendooven, G. Peres, J.E. Tondoh, D. Cluzeau, J.J. Brun, A review of earthworm impact on soil function and ecosystem services, Eur. J. Soil Sci. 64 (2013) 161e182. [4] M. Bonkowski, M. Schaefer, Interactions between earthworms and soil protozoa: a trophic component in the soil food web, Soil Biol. Biochem. 29 (1997) 499e502. [5] R. Latif, M. Malek, H. Mirmonsef, Cadmium and lead accumulation in three endogeic earthworm species, Bull. Environ. Contam. Toxicol. 90 (2013) 456e459. [6] I.M. Lubbers, K.J. van Groenigen, S.J. Fonte, J. Six, L. Brussaard, J.W. van Groenigen, Greenhouse-gas emissions from soils increased by earthworms, Nat. Clim. Chang. 3 (2013) 187e194. [7] N. Eisenhauer, S. Partsch, D. Parkinson, S. Scheu, Invasion of a deciduous forest by earthworms: changes in soil chemistry, microflora, microarthropods and vegetation, Soil Biol. Biochem. 39 (2007) 1099e1110. [8] N.A. Fisichelli, L.E. Frelich, P.B. Reich, N. Eisenhauer, Linking direct and indirect pathways mediating earthworms, deer, and understory composition in Great Lakes forests, Biol. Invasions 15 (2012) 1057e1066. [9] P.F. Hendrix, M.A. Callaham, J.M. Drake, C.-Y. Huang, S.W. James, B.A. Snyder, W. Zhang, Pandora's box contained bait: the global problem of introduced earthworms, Annu. Rev. Ecol. Evol. Syst. 39 (2008) 593e613. , Strategies lombriciennes, Ecol. Bull. 25 (1977) 122e132. [10] M.B. Bouche [11] J.P. Curry, O. Schmidt, The feeding ecology of earthworms e a review, Pedobiologia 50 (2007) 463e477. [12] O. Ferlian, S. Cesarz, S. Marhan, S. Scheu, Carbon food resources of earthworms of different ecological groups as indicated by 13C compound-specific stable isotope analysis, Soil Biol. Biochem. 77 (2014) 22e30. [13] A.V. Tiunov, S. Scheu, Carbon availability controls the growth of detritivores (Lumbricidae) and their effect on nitrogen mineralization, Oecologia 138 (2004) 83e90. [14] N.S.S. Rajapaksha, K.R. Butt, E.I. Vanguelova, A.J. Moffat, Earthworm selection of Short rotation forestry leaf litter assessed through preference testing and direct observation, Soil Biol. Biochem. 67 (2013) 12e19. € der, Modelling distribution patterns of [15] J. Palm, N.L.M.B. van Schaik, B. Schro anecic, epigeic and endogeic earthworms at catchment-scale in agro-ecosystems, Pedobiologia 56 (2013) 23e31. [16] N. Bityutskii, P. Kaidun, K. Yakkonen, Can earthworms alleviate nutrient disorders of plants subjected to calcium carbonate excess? Appl. Soil Ecol. 98 (2015) 20e29. [17] T. Eck, M. Potthoff, J. Dyckmans, F. Wichern, R.G. Joergensen, Priming effects of Aporrectodea caliginosa on young rhizodeposits and old soil organic matter following wheat straw addition, Eur. J. Soil Biol. 70 (2015) 38e45. [18] P.B. Reich, J. Oleksyn, J. Modrzynski, P. Mrozinski, S.E. Hobbie, D.M. Eissenstat, J. Chorover, O.A. Chadwick, C.M. Hale, M.G. Tjoelker, Linking litter calcium, earthworms and soil properties: a common garden test with 14 tree species, Ecol. Lett. 8 (2005) 811e818. [19] A. Guckland, M. Jacob, H. Flessa, F.M. Thomas, C. Leuschner, Acidity, nutrient stocks, and organic-matter content in soils of a temperate deciduous forest with different abundance of European beech (Fagus sylvatica L.), J. Plant Nutr. Soil Sci. 172 (2009) 500e511. [20] S. Cesarz, A. Fender, F. Beyer, K. Valtanen, B. Pfeiffer, D. Gansert, A. Polle, R. Daniel, S. Scheu, Roots from beech (Fagus sylvatica L.) and ash (Fraxinus excelsior L.) differentially affect soil microorganisms and carbon dynamics, Soil Biol. Biochem. 61 (2013) 23e32.
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Contents lists available at ScienceDirect
European Journal of Soil Biology journal homepage: http://www.elsevier.com/locate/ejsobi
Original article
Effects of soil and leaf litter quality on the biomass of two endogeic earthworm species Simone Cesarz a, b, c, *, Dylan Craven a, b, Christoph Dietrich a, b, Nico Eisenhauer a, b a
German Centre for Integrative Biodiversity Research (iDiv) Halle-Jena-Leipzig, Deutscher Platz 5e, 04103 Leipzig, Germany Institute of Biology, Leipzig University, Johannisallee 21, 04103 Leipzig, Germany c €ttingen, Berliner Straße 28, 37073 Go €ttingen, Germany J.F. Blumenbach Institute of Zoology and Anthropology, Georg August University Go b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 9 May 2016 Received in revised form 12 September 2016 Accepted 12 September 2016
Earthworms drive important ecosystem functions like decomposition and nutrient mineralization in many terrestrial ecosystems, which is why factors controlling their mass gain are of great scientific interest. We conducted a microcosm experiment using two common endogeic earthworm species (Aporrectodea caliginosa and Octolasion tyrtaeum) and two different soils (one from a beech-dominated forest and one from a mixed tree species forest in Germany) to test litter quality (different nutrient concentrations) and soil quality effects (low and high) on relative mass gain of earthworms. We hypothesized that mass gain of endogeic earthworms is driven by both soil and litter quality. Soil pH, carbon (C) and nitrogen (N) concentrations were used to characterize soil quality, while leaf litter N, phosphorus (P), calcium (Ca), and magnesium (Mg) concentrations were used as proxies for leaf litter quality. Forest soils were incubated with leaf litter of six common tree species in Central Europe (Fagus sylvatica, Acer platanoides, Acer pseudoplatanus, Carpinus betulus, Tilia spp., and Fraxinus excelsior) that span a gradient in leaf litter quality. In addition, we determined soil microbial biomass C as a potential food source of endogeic earthworms. After three months, relative earthworm mass gain of A. caliginosa and O. tyrtaeum was significantly higher in soil from the mixed tree species forest (high quality soil: þ218% and þ240%, respectively) compared to soil from the beech-dominated forest (low quality soil: þ160% and þ162%, respectively). Relative mass gain of A. caliginosa increased significantly with all leaf litter nutrients in low quality soil, whereas in high quality soil only leaf litter Ca positively affected relative mass gain. Similarly, relative mass gain of O. tyrtaeum increased significantly with increasing concentrations of leaf litter N, Mg, and Ca in the low quality soil. In the high quality soil, only leaf litter Mg significantly increased relative mass gain. Overall, our results indicate that leaf litter quality effects on endogeic earthworm mass gain were more important in low quality soil for both earthworm species. Notably, microbial biomass was significantly higher in high quality soil (506 ± 135 mg C g1 soil dw) compared to low quality soil (217 ± 64 mg C g1 soil dw), but microbial biomass was not significantly affected by leaf litter type and was a poor predictor of relative earthworm mass gain. This finding indicates that endogeic earthworms did not significantly depend on soil microbial biomass, but rather on the quality of dead organic material in the soil and surface leaf litter. As earthworms may prefer feeding on certain microbial taxa, and we only measured total soil microbial biomass, future studies could investigate if leaf litter quality effects on earthworms are mediated by changes in soil microbial community structure, micronutrients, and organic compounds. © 2016 Elsevier Masson SAS. All rights reserved.
Handling Editor: S. Schrader Keywords: Earthworm mass gain Endogeic earthworms Forest soil Leaf litter Nutrient sources Partial least squares path modeling
1. Introduction
* Corresponding author. German Centre for Integrative Biodiversity Research (iDiv) Halle-Jena-Leipzig, Deutscher Platz 5e, 04103 Leipzig, Germany. E-mail addresses: [email protected] (S. Cesarz), [email protected] (D. Craven), [email protected] (C. Dietrich), [email protected] (N. Eisenhauer). http://dx.doi.org/10.1016/j.ejsobi.2016.09.002 1164-5563/© 2016 Elsevier Masson SAS. All rights reserved.
Earthworms play an important role in many terrestrial ecosystems as ecosystem engineers, by modifying soil structure via bioturbation as well as through their contribution to decomposition processes [1e3]. Furthermore, they often comprise, together with Protozoa, the largest biomass of soil fauna [4], and their functional
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significance, i.e., their effects on ecosystem processes, makes earthworms important study organisms in ecological and ecotoxicological research [5,6]. Moreover, some earthworm species are biological invaders in many ecosystems with major impacts on soil chemistry, plant communities, and soil microarthropod communities [7e9]. Consequently, it is vital to identify drivers that control earthworm mass gain. Earthworms can be divided into three ecological groups: epigeic earthworms, which live in and feed on the leaf litter layer on the soil surface; anecic earthworms, which dig large vertical and permanent burrows and feed on surface leaf litter, and endogeic earthworms, which feed on organic matter in the mineral soil and build horizontal burrows in deeper organic and mineral soil layers [10]. Together with anecic earthworms, endogeic earthworms often dominate the biomass of soil invertebrates [2]. Endogeic earthworms have a very low assimilation efficiency (2%), resulting in high consumption rates of their poor quality diet, i.e., soil [11]. It has been suggested that endogeic earthworms feed on stabilized soil particles [12] as well on easily available compounds like glucose [13]. Rajapaksha et al. [14] demonstrated that endogeic earthworms were able to remove soil surface litter, which suggests that leaf litter may serve as potential additional food resource with both stable and soluble compounds. Direct or indirect litter feeding by endogeic earthworm has, however, rarely been investigated. Thus, exploring factors influencing the ecology of endogeic earthworms can help to improve species distribution models [15], understand their influence on the bioavailability of micronutrients [16], and increase our knowledge about their interactions with microorganisms that drive priming effects [17]. The quality of root and leaf litter inputs influence the quality of soil and organic matter and therefore the mass gain of endogeic earthworms [2,18e20]. Generally, low soil pH and low moisture negatively affect earthworm development [21,22], whereas high soil organic matter content positively affects earthworm nutrition and therefore mass gain [11]. Several studies have investigated the importance of soil microorganisms, mainly fungi, for earthworm nutrition but clear evidence is still lacking. Previous studies have suggested that endogeic earthworms consume microorganisms as bycatch during soil ingestion, as helpers to degrade recalcitrant compounds, but also as a direct food resource [11]. Food choice experiments have demonstrated a clear preference of earthworms for leaf litter with low C:N ratio and low concentrations of secondary compounds, and polyphenol content [14,23,24]. In addition, the availability of soil carbon (C) [13,25], and nitrogen (N) [26] have also been shown to increase earthworm mass gain. More recently, high calcium (Ca) concentration in soil was linked to reduced soil acidity and increased fertility, factors which positively affect both earthworm abundance and diversity [18]. While the effects of other leaf litter nutrients, such as phosphorus (P) and magnesium (Mg), on earthworm growth were not studied, a recent study by Resner et al. [27] showed that earthworms reduced the availability of Ca, Mg, K, and P when invading a North American forest. Thus, factors affecting earthworm mass gain have generally been attributed to their feeding preferences and direct food sources. In doing so, effects of leaf litter quality were tested by using mainly anecic and epigeic earthworms, whereas soil quality was tested by using endogeic earthworms. Interactions of leaf litter and soil quality on earthworm mass gain have been largely neglected. Dechaine et al. [28] even suggested ignoring potential feeding relationships of endogeic earthworms on leaf litter material. However, feeding relationships in soil are complex and still poorly understood [29]. Investigating and understanding feeding relationships in soil is of particular importance to appreciate the consequences of anthropogenic environmental changes for species
interactions. The aim of the present study was to explore if the potential dependence of endogeic earthworm species on surface leaf litter material is contingent upon soil nutrient content. While surface leaf litter quality effects on anecic earthworms have already been investigated [11,30], the present study is one of the first investigating potential interaction effects of leaf litter and soil quality on endogeic earthworms. In this study, we investigated drivers of changes in earthworm biomass in soils of contrasting quality (low versus high quality) in combination with leaf litter types (beech, lime, ash, hornbeam, and two maple species) representing a broad gradient in nutrient concentrations and, therefore, leaf litter quality. We explored the relative mass gain of two endogeic earthworm species, Aporrectodea caliginosa and Octolasion tyrtaeum, as this ecological group often dominates the density and biomass of earthworm communities [31] and to determine if two functionally similar species exhibit similar responses to soil and leaf litter quality effects. As detailed above, endogeic species are considered to feed mostly on mineral soil, but leaf litter material could also have indirect effects on earthworm mass gain by altering nutrient availability and/or microbial community biomass and composition [32]. We hypothesized that endogeic earthworm relative mass gain would be significantly higher in high quality soil than in low quality soil (hypothesis 1) due to a higher pH value [2], higher nutrient availability (especially C, N, and Ca) [19], and higher soil microbial biomass [33]. Further, we hypothesized that leaf litter chemistry (C, N, P, Ca, and Mg concentrations) would influence relative mass gain of endogeic earthworms (hypothesis 2). Specifically, we expected increased leaf litter N and Ca concentrations to enhance earthworm biomass [18,34]. We also tested for effects of other leaf litter nutrients, such as P and Mg concentrations, on earthworm relative mass gain as these nutrients have rarely been considered in previous studies and could have significant effects on earthworm mass gain [35,36]. Finally, we hypothesized that negative effects on earthworm mass gain in low quality soil would be compensated by high leaf litter quality (hypothesis 3). Given correlations among different leaf litter quality properties and soil microbial biomass, we used partial least squares path modeling (PLS-PM) to identify the main drivers of earthworm mass gain [37]. 2. Materials and methods 2.1. General design A microcosm experiment was established to study the relative mass gain of the two endogeic earthworm species A. caliginosa and O. tyrtaeum in two different soils (where they naturally occur but in different densities [38]) and in different leaf litter treatments. The experiment was conducted using a two factorial design with two levels for the factor “soil” (low and high quality) and six levels for the factor “leaf litter” (one level for each tree species). Further, we used leaf litter concentrations of N, P, Ca, and Mg as proxies for leaf litter quality, which have been shown to determine essential processes like leaf litter decomposition [39], but other organic compounds like tannins and phenols may also be important for earthworm mass gain [14,23,24]. Every treatment was replicated seven times and the experiment ran for a total of three months. 2.2. Soil and leaf litter Soil and leaf litter were taken from the Hainich National Park (Thuringia, Germany, annual precipitation: 600.5 mm and mean annual temperature: 9.0 C). The soil was taken from a depth of 0e20 cm (without leaf litter layer) in forest plots with different
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Table 1 Properties (mg g1 dry mass) of nitrogen (N), carbon (C), calcium (Ca), magnesium (Mg), phosphorous (P) of leaf litter as well as nutrient ratios of the six tree species used in the present study. Species are sorted according to their C:N.
Fagus sylvatica Carpinus betulus Tilia spec. Acer platanoides Acer pseudoplatanus Fraxinus excelsior a
C
N
Pa
Caa
Mga
C:N
47.86 46.71 48.10 48.21 46.66 49.03
8.30 11.02 11.89 11.78 11.72 15.65
0.36 0.61 0.63 0.63 0.47 0.69
16.80 21.49 27.47 24.62 30.15 28.83
1.21 2.86 2.58 2.10 3.17 2.87
56.06 40.08 37.93 36.53 36.40 27.60
Data on P, Ca, and Mg concentrations were derived from the same study sites from Jacob et al. [40,41].
species compositions. The beech-dominated forest (low soil quality) had >94% coverage by beech (Fagus sylvatica L.) in the canopy, while the mixed tree species forest (high soil quality) contained beech (~25%), lime (Tilia cordata Mill. and Tilia platyphyllos Scop. in the following named as Tilia spp.), ash (Fraxinus excelsior L.), hornbeam (Carpinus betulus L.), and two species of maple (Acer pseudoplatanus L., and Acer platanoides L.). The soil type is a luvisol with pH ranging from 3.7 to 4.5 in soil from the beech-dominated forest (low quality soil) and 5.7 to 5.9 in soil from mixed tree species forest (high quality soil). The soil was sieved (4 mm) and defaunated (heat extraction at 55 C for seven days) and then stored at room temperature. Leaf litter from beech, lime, ash, hornbeam, Norway maple, and sycamore maple was collected from senescent leaves in the Hainich National Park at plots with high tree diversity. Leaf litter was airdried and stored at room temperature. Sampling leaf litter at one specific location enabled us to use literature data of the chemical composition on the leaf litter (C, N, P, Ca, and Mg) from the same location (site DL 3; see Refs. [40,41]).
2.3. Earthworms Juvenile earthworms were sampled between June and July 2005. To obtain the required number of individuals, earthworms €ttinger were taken from the Hainich National Park and from the Go Forest (Lower Saxony, Germany). Two days before starting the experiment, earthworms were placed in Petri dishes with wet filter paper and kept in a climate chamber at 15 C for 48 h to void their gut content [42]. Afterwards, earthworms were weighed individually, and every microcosm received one earthworm. Earthworm starting mass was 0.164 ± 0.087 g for A. caliginosa and 0.100 ± 0.049 g for O. tyrtaeum, and individuals were randomly assigned to leaf litter and soil treatments. Soil and leaf litter quality had no significant effect on earthworm mortality (P > 0.05 for both species). Average mortality was 16 ± 37% and 34 ± 48% for A. calignosa and O. tyrtaeum, respectively.
2.5. Experimental procedure Large microcosms were filled with 210 g soil (air dried) each, and the smaller ones were filled with 110 g soil. Ensuring that every microcosm received the same amount of carbon via leaf litter addition, C and N content of the different leaf litter species were measured (Table 1). Oven dried leaf litter (70 C, 24 h) was ground, weighed, and placed into tin capsules, which was analyzed by an automated element analyzer (NA 1500, Carlo Erba, Mailand). Every microcosm received 504 mg carbon in the form of leaf litter. Leaf litter was cut into pieces of 1e2 cm2 and was added to the soil surface in each microcosm to simulate leaf fall. The microcosms were watered with 216 ml (large microcosms) and 90 ml (small microcosms) of distilled water, respectively, and incubated for one week before the experiment started. Afterwards, 72 ml and 30 ml of distilled water were added biweekly, respectively. The experiment was terminated after 12 weeks as ash leaf litter had mostly disappeared. Surplus soil water was suctioned by lowering pressure for three days after watering. To estimate earthworm mass at the end of the experiment, microcosms were destructively sampled. The earthworms were placed in Petri dishes as described above to void their gut content for 24 h. The relative change in earthworm mass was used as a proxy for earthworm performance and was calculated as the percent change in mass from the beginning to the end of the experiment. Microbial biomass C was determined using the substrate induced respiration (SIR) method [43], i.e., the respiratory response of microorganisms to glucose addition. Soils used for the measurements were sieved (2 mm) to remove roots and leaf litter. An O2-microcompensation apparatus modified after [44] was used to perform the measurements. We added 8 mg glucose g1 soil dry weight as an aqueous solution to the soil samples. The mean of the lowest three readings within the first 10 h (between the initial peak caused by disturbing the soil and the beginning of microbial growth) was assessed as the maximum initial respiratory response (MIRR; ml O2 g1 soil dry weight), and microbial biomass (mg C g1 soil dry weight) was calculated as 38 MIRR [45].
2.4. Microcosms
2.6. Statistical analysis
The experiment was set up in microcosms consisting of plastic tubes connected to ceramic plates allowing drainage by lowering the atmospheric pressure (0.4e0.6 bar). This system had two different tube sizes; we used large tubes (height 15 cm, ø 6.2 cm) for A. caliginosa and smaller ones (height 15 cm, ø 4.5 cm) for O. tyrtaeum due to differences in body size between the species. As we were interested in treatment effects on relative earthworm mass gain and not in the comparisons of absolute mass gain of the two earthworm species, differences in microcosm size do not affect the conclusions of our study. Microcosms were closed at the top by a lid to prevent earthworms from escaping.
The effects of soil quality (low and high), leaf litter identity (six leaf litter species), and interactions of both treatments were analyzed for relative earthworm mass gain and microbial biomass with 2-way-ANOVA. Homogeneity of variances was tested using the Bartlett test, and normality was checked using Shapiro wilk test. Microbial biomass C was log-transformed to meet the assumptions of ANOVA. The relative change in earthworm mass gain was calculated as percentage change of earthworm mass at the beginning of the experiment (equals 100%) compared to the end of the experiment. Partial least squares path modeling was performed in R using the ‘plspm’ package [37] to infer direct and indirect
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S. Cesarz et al. / European Journal of Soil Biology 77 (2016) 9e16
effects of leaf litter quality (using leaf litter N, P, Ca, and Mg as mg g1 dry mass), litter C (mg g1 dry mass), and microbial biomass C (mg C g1 dry soil weight) on earthworm relative mass gain of both earthworm species separately. Leaf litter quality was treated as a latent variable consisting of leaf litter N, P, Ca, and Mg as manifest variables. As leaf litter C concentrations are strongly associated with other structural measures of leaf litter, such as lignin and cellulose [46], it was treated as a separate latent variable, thereby ignoring possible effects of soluble C compounds [47]. Lastly, microbial biomass C was treated as a latent variable. We expected that leaf litter quality and soil microbial biomass C would increase relative mass gain of both earthworm species and that leaf litter C concentrations would decrease earthworm relative mass gain. The statistical significance of path coefficients for models were assessed using 1000 bootstrap replicates to determine if they differed from zero, as were the contributions of individual manifest variables to their corresponding latent variable. All analyses were performed using R 3.2.2 [48]. 3. Results In the high quality soil, the relative mass gain of A. caliginosa (mean ± SD: 218 ± 55%; P ¼ 0.0052) and O. tyrtaeum (240 ± 75%; P < 0.0001) was significantly higher than in the low quality soil (Fig. 1a; 160 ± 80% and þ162 ± 67%, respectively). Leaf litter identity and interactions with soil quality had no significant effects on relative mass gain (P > 0.05). Further, soil microbial biomass C was significantly higher in the high quality soil for both A. caliginosa (450 ± 149 mg C g1 soil dry weight; P < 0.0001) and O. tyrtaeum (547 ± 112 mg C g1 soil dry weight; P < 0.0001), compared to low quality soil (191 ± 58 and 239 ± 63 mg C g1 soil dry weight, respectively; Fig. 1b). Leaf litter identity and interactions had no significant effects on microbial biomass (P > 0.05). The relative mass gain of A. caliginosa and O. tyrtaeum was
significantly positively correlated to the quality of the leaf litter in the low quality soil (Fig. 2a,c; Table 2). In the high quality soil, leaf litter quality effects on earthworm mass gain were markedly weaker and non-significant for O. tyrtaeum (Fig. 2d, Table 2), and the relative mass gain of A. caliginosa was slightly negatively but not significantly correlated with leaf litter quality (Fig. 2b, Table 2). While Ca, Mg, N, and P concentrations determined leaf litter quality effects on the relative mass gain of A. caliginosa in low quality soil, Ca concentration was the only significant determinant of leaf litter quality effects in the high quality soil (Fig. 2a,b; Table 3). Ca, Mg, and N concentrations determined leaf litter quality effects on the relative mass gain of O. tyrtaeum in the low quality soil, whereas Mg and N concentrations determined leaf litter quality effects in the high quality soil (Fig. 2c,d; Table 3). Leaf litter C concentrations had no significant effects on the relative mass gain of either earthworm species in both low and high quality soils. However, leaf litter C concentrations significantly increased microbial biomass in the high quality soil in the presence of O. tyrtaeum (Fig. 2d; Table 2), but not in the other treatments. Generally, the influence of microbial biomass C on the relative mass gain of both earthworm species was positive but non-significant in both soils. Microbial biomass C was not significantly affected by leaf litter quality (Fig. 2; Table 2). 4. Discussion Exploring the nutrition of ecosystem engineers, such as earthworms, is fundamental for understanding how environmental changes influence the composition and functioning of ecosystems. The objective of the present study was to assess soil quality effects and to identify leaf litter traits determining endogeic earthworm relative mass gain in different contexts. As expected, earthworm relative mass gain was higher in high quality soil. Beyond this soil effect, we found that the relative mass gain of the two endogeic earthworm species tested was determined by leaf litter nutrient concentrations in the low quality soil, which was acidic, nutrientpoor, and low in soil microbial biomass. Other studies have shown previously significant effects of leaf litter Ca [18], soil Mg [35], leaf litter and soil N [34], and C:P ratios [36] on biomass, density, and species richness of total and epigeic earthworms. This study, for the first time, investigated interactive effects of soil quality and leaf litter concentrations of C, N, P, Ca, and Mg on endogeic earthworms. Our findings suggest that leaf litter N, P, Ca, and Mg may be major determinants of endogeic earthworm relative mass gain. As both endogeic earthworm species were able to gain nutrients from soil surface litter, future studies could investigate if endogeic earthworms actively switch to a more leaf litterbased nutrition in low quality soils or if leaching or increased microbial decomposition of high quality leaf litter affect earthworm mass gain. 4.1. Soil quality
Fig. 1. Effect of soil quality (low and high) in a microcosm experiment with the two endogeic earthworms Aporrectodea caliginosa and Octolasion tyrtaeum on mean ± SD of a) relative earthworm mass gain and b) microbial biomass C after an experimental duration of three months. Asterisks represent ANOVA results (log-transformed for microbial biomass) of soil effect with *** ¼ P < 0.001 and ** ¼ P < 0.01.
In line with our expectations, we found that earthworm mass gain was significantly greater in high quality soil, i.e., soil with higher pH, nutrient availability, and microbial biomass. Our results suggest that soil pH was an important driver of these soil quality effects [18] because soil pH from the beech-dominated forest (3.7e4.5) was at the lower limit of the tolerable range of A. caliginosa [49]. However, recent studies have highlighted that soil pH does not directly affect the density of soil organisms, but rather indirectly drives chemical processes related to nutrient availability [36]. For instance, P forms insoluble complexes with Ca at alkaline pH values, and with Al and Fe at acidic pH values [50]. In our study, we found N, P, Ca, and Mg to be important for earthworm relative mass gain at low soil quality with acidic soil pH, which suggests
S. Cesarz et al. / European Journal of Soil Biology 77 (2016) 9e16
13
Fig. 2. Partial least squares path models of the drivers of earthworm relative mass gain. Best fitting models for direct and indirect effects of leaf litter quality, leaf litter C concentrations, and microbial biomass C relationships on earthworm relative mass gain of Aporrectodea caliginosa (a,b) and Octolasion tyrtaeum (c,d) in soils of a beech-dominated forest (low quality soil, i.e., low soil pH, low nutrient availability) and a mixed tree species forest (high quality soil, i.e., high soil pH, high nutrient availability). Leaf litter quality data were derived by concentrations of leaf litter N, P, Ca, and Mg, leaf litter C concentration by measuring carbon in leaf litter (C), microbial biomass C was informed by data derived from substrateinduced respiration (mC) and earthworm relative mass gain by data about earthworm mass gain (mg). Numbers on arrows are path coefficients indicating a positive (solid line) or negative effect (dashed line). Numbers above manifest variables indicate the proportional contribution of a manifest variable to its corresponding latent variable. In the case of leaf litter C concentrations and microbial biomass C only one manifest variable contributed to the analysis resulting in a weight of one which is not shown. Significant paths are given in bold. Percentages in shaded circles are R2 values and indicate the variance explained by the model. Goodness of fit: a) 0.476); b) 0.401; c) 0.372; d) 0.397.
that those nutrients are limiting earthworm nutrition in soil from a beech-dominated forest but can be derived from leaf litter. Importantly, we could not determine if nutrients were less
available due to a generally lower input to soil or due to the formation of insoluble complexes at low pH, but our results suggest that earthworm mass gain is limited by lower nutrient availability
Table 2 Path coefficients of latent variables for partial least squares path fitted for two endogeic earthworm species (Aporrectodea caliginosa and Octolasion tyrtaeum) in soils of two different qualities, i.e., from a beech-dominated forest (low soil quality, i.e., low soil pH and low nutrient availability) and a mixed tree species forest (high quality soil, i.e., high soil pH and high nutrient availability). Significant relationships are given in bold. PC ¼ path coefficient; CI ¼ confidence interval. Earthworm species
Soil quality
Paths
PC
Lower 95% CI
Upper 95% CI
Aporrectodea caliginosa
low
leaf litter quality / microbial biomass C leaf litter C concentrations / microbial biomass C leaf litter quality / earthworm relative mass gain leaf litter C concentrations / earthworm relative mass gain microbial biomass C / earthworm relative mass gain
0.041 0.232 1.118 0.566 0.197
0.862 0.453 0.664 0.032 0.217
0.869 0.927 1.672 1.152 0.534
high
leaf litter quality / microbial biomass C leaf litter C concentrations / microbial biomass C leaf litter quality / earthworm relative mass gain leaf litter C concentrations / earthworm relative mass gain microbial biomass C / earthworm relative mass gain
0.796 0.606 0.237 0.108 0.243
0.089 0.036 1.347 1.239 0.630
1.289 1.323 0.744 0.837 0.940
low
leaf litter quality / microbial biomass C leaf litter C concentrations / microbial biomass C leaf litter quality / earthworm relative mass gain leaf litter C concentrations / earthworm relative mass gain microbial biomass C / earthworm relative mass gain
0.523 0.219 0.693 0.424 0.136
0.246 0.504 0.185 0.254 0.210
1.112 0.863 1.194 0.985 0.504
high
leaf litter quality / microbial biomass C leaf litter C concentrations / microbial biomass C leaf litter quality / earthworm relative mass gain leaf litter C concentrations / earthworm relative mass gain microbial biomass C / earthworm relative mass gain
0.686 0.632 0.491 0.067 0.064
0.051 0.014 0.096 0.566 0.290
1.242 1.157 1.086 0.690 0.423
Octolasion tyrtaeum
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S. Cesarz et al. / European Journal of Soil Biology 77 (2016) 9e16
Table 3 Contribution of manifest variables (concentrations of N, P, Ca, and Mg of leaf litter) to the latent variable ‘leaf litter quality’ in partial least squares path models fitted for two endogeic earthworm species (Aporrectodea caliginosa and Octolasion tyrtaeum) in soils of two different qualities, i.e., from a beech-dominated forest (low soil quality, i.e., low soil pH and low nutrient availability) and a mixed tree species forest (high quality soil, i.e., high soil pH and high nutrient availability). Weights indicate the proportional contribution of a manifest variable to its corresponding latent variable. CI ¼ confidence interval. Significant relationships, i.e., those that do not overlap zero, are given in bold. Earthworm species
Soil quality
Manifest variable
Weights
Lower 95% CI
Upper 95% CI
Aporrectodea calignosa
low
leaf leaf leaf leaf
0.342 0.216 0.274 0.287
0.255 0.112 0.149 0.195
0.472 0.299 0.372 0.449
high
leaf litter N leaf litter Mg leaf litter Ca leaf litter P
0.252 0.236 0.317 0.270
0.107 0.167 0.061 0.405
0.424 0.709 0.747 0.605
low
leaf litter N leaf litter Mg leaf litter Ca leaf litter P
0.250 0.462 0.285 0.087
0.059 0.187 0.142 0.301
0.382 0.784 0.401 0.334
high
leaf litter N leaf litter Mg leaf litter Ca leaf litter P
0.343 0.255 0.176 0.336
0.221 0.045 0.129 0.002
0.476 0.498 0.407 0.603
Octolasion tyrtaeum
litter litter litter litter
N Mg Ca P
in soil. This nutrient shortage was relaxed in the high quality soil as indicated by the lower impact of leaf litter nutrients on earthworm mass gain. In the case of A. caliginosa, only Ca positively affected its mass gain, whereas mass gain of O. tyrtaeum increased with leaf litter N and Mg, indicating differences between the two endogeic earthworm species. During decomposition, tannin content of leaf litter, especially that of the low quality beech leaf litter, may further constrain earthworm mass gain by reducing soil nutrient availability through the formation of mineralization-resistant proteintannin complexes [51e53]. We further hypothesized that soil microbial biomass would drive earthworm growth. Although we found microbial biomass to be higher in the high quality soil compared to the low quality soil, PLS path models showed no significant direct relationship between microbial biomass and earthworm growth. Feeding choice experiments have shown that earthworms prefer specific microorganisms, mainly fungi [54e56], suggesting that it is not biomass per se that affects earthworm nutrition but rather the presence of specific taxa of the microbial community. Further, we suggest that microbial communities differed between the two soils mainly due to differences in soil properties like pH and nutrient availability [57]. Endogeic earthworms need litter material to be ‘pre-decomposed’ by microorganisms in order to access nutrients and future studies could identify the most important microbial groups or taxa mediating leaf litter effects on endogeic earthworms. 4.2. Leaf litter nutrient concentrations and interactions with soil quality Leaf litter N, P, Ca, and Mg concentrations showed positive correlations with earthworm relative mass gain in the low quality soil. Endogeic earthworm species are thought to feed primarily on organic material in the mineral soil [58]. However, there is some evidence that A. caliginosa is not restricted to mineral soil as its only food source, but that it can also directly feed on leaf litter [11]. Further, it was shown that endogeic earthworms selectively feed on enriched organic matter [14,59] indicating that they are able to directly benefit from leaf litter material. The stronger increase of earthworm relative mass gain with higher leaf litter nutrient concentrations in the low quality soil suggests, in addition to unfavorable abiotic soil conditions, that litter micronutrient content and P also affected earthworm relative mass gain. Low quality leaf litter, such as that of beech, is high in
tannin content [60] and decreased earthworm relative mass gain in this study. That the leaf litter quality effects were weaker in the high quality soil supports the idea that endogeic earthworms rely more on soil organic material. However, in soils with low amounts of organic material, such as those found in beech-dominated forest, leaf litter effects significantly affected the relative mass gain of both species studied. This may also indicate that endogeic species can switch feeding behavior in response to soil and leaf litter quality. PLS path models revealed that in microcosms with O. tyrtaeum and high quality soil, leaf litter C concentration significantly affected microbial biomass, whereas in all other cases no significant relationship was found for this path. This may indicate that distinct earthworm species may significantly affect leaf litter effects on microbial communities or vice versa. We further suggest the need to go beyond the consideration of solely C and N concentrations and investigate micronutrients and organic molecules, such as phenols, tannins, and lignin, to further current understanding of earthworm nutrition [61,62]. Our study confirmed the significant role of leaf litter Ca concentrations in driving earthworm communities found in a tree plantation in Poland [18]. In the study by Mueller et al. [35] at the same field site in Poland, the presence and abundance of the anecic earthworm species Lumbricus terrestris, as well as the species richness of earthworms, were strongly positively correlated with soil pH and the amount of base cations (mostly Ca and Mg) in leaf litter and soils, likely due to the high Ca requirements of some earthworm species [18,63]. Many earthworm species produce calcite granules in specialized calciferous glands, which may explain high Ca requirements of A. caliginosa. Their function is not known but it is suggested that they regulate CO2 respiration, excrete excess Ca, and neutralize acidic gut pH [64]. We have no information about Mg requirements of earthworms but due to similar chemical traits as Ca, there might be similarities in reactions with P as stated above. P was shown to be important at all trophic levels in forest ecosystems [36] and should also be included in standard measurements to test the generality of our findings. In the present study, both endogeic earthworm species gained more mass in high quality soil, with higher pH and nutrient concentrations, and consistently depended on litter quality in low quality soil. Our results indicate that endogeic earthworms can benefit from nutrient-rich leaf litter in nutrient-poor soils and that leaf litter effects are not always mediated via soil microbial biomass. Future studies could investigate microbial community
S. Cesarz et al. / European Journal of Soil Biology 77 (2016) 9e16
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