Advertisement
Browse Subject Areas
?

Click through the PLOS taxonomy to find articles in your field.

For more information about PLOS Subject Areas, click here.

  • Loading metrics

Open Access

Peer-reviewed

Research Article

Root-derived carbon and nitrogen from beech and ash trees differentially fuel soil animal food webs of deciduous forests

  • Sarah L. Zieger ,

    Roles Conceptualization, Formal analysis, Investigation, Methodology, Visualization, Writing – original draft, Writing – review & editing

    szieger@gwdg.de

    Affiliation University of Göttingen, J.F. Blumenbach Institute of Zoology and Anthropology, Animal Ecology, Göttingen, Germany

    ⨯
  • Silke Ammerschubert,

    Roles Data curation, Writing – review & editing

    Affiliation University of Göttingen, Büsgen Institute, Forest Botany and Tree Physiology, Göttingen, Germany

    ⨯
  • Andrea Polle,

    Roles Conceptualization, Funding acquisition, Writing – review & editing

    Affiliation University of Göttingen, Büsgen Institute, Forest Botany and Tree Physiology, Göttingen, Germany

    ⨯
  • Stefan Scheu

    Roles Conceptualization, Funding acquisition, Methodology, Visualization, Writing – original draft, Writing – review & editing

    Affiliations University of Göttingen, J.F. Blumenbach Institute of Zoology and Anthropology, Animal Ecology, Göttingen, Germany, University of Göttingen, Centre of Biodiversity and Sustainable Land Use, Göttingen, Germany

    ⨯

Abstract

Evidence is increasing that soil animal food webs are fueled by root-derived carbon (C) and also by root-derived nitrogen (N). Functioning as link between the above- and belowground system, trees and their species identity are important drivers structuring soil animal communities. A pulse labeling experiment using 15N and 13C was conducted by exposing beech (Fagus sylvatica) and ash (Fraxinus excelsior) seedlings to 13CO2 enriched atmosphere and tree leaves to 15N ammonium chloride solution in a plant growth chamber under controlled conditions for 72 h. C and N fluxes into the soil animal food web of beech, associated with ectomycorrhizal fungi (EMF), and ash, associated with arbuscular mycorrhizal fungi (AMF), were investigated at two sampling dates (5 and 20 days after labeling). All of the soil animal taxa studied incorporated root-derived C, while root-derived N was only incorporated into certain taxa. Tree species identity strongly affected C and N incorporation with the incorporation in the beech rhizosphere generally exceeding that in the ash rhizosphere. Incorporation differed little between 5 and 20 days after labeling indicating that both C and N are incorporated quickly into soil animals and are used for tissue formation. Our results suggest that energy and nutrient fluxes in soil food webs depend on the identity of tree species with the differences being associated with different types of mycorrhiza. Further research is needed to prove the generality of these findings and to quantify the flux of plant C and N into soil food webs of forests and other terrestrial ecosystems.

Citation: Zieger SL, Ammerschubert S, Polle A, Scheu S (2017) Root-derived carbon and nitrogen from beech and ash trees differentially fuel soil animal food webs of deciduous forests. PLoS ONE 12(12): e0189502. https://doi.org/10.1371/journal.pone.0189502

Editor: Mette Vestergård, University of Copenhagen, DENMARK

Received: August 22, 2017; Accepted: November 28, 2017; Published: December 13, 2017

Copyright: © 2017 Zieger et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Data Availability: All data are included within the paper and its Supporting Information files.

Funding: This project was funded by the German Research Foundation [GRK1086/2, project A04].

Competing interests: The authors have declared that no competing interests exist.

Introduction

Soil animal communities of deciduous forest form complex food webs [1,2] of high species diversity [3,4]. They comprise a wide spectrum of trophic levels including primary and secondary decomposers, and first, second and third order predators [5,6]. Mesostigmata live as predators [7,8], while Oribatida and Collembola span over a wide range of trophic levels [9,10]. Diplopoda of deciduous forests mainly live as primary decomposers [11]. Soil food webs are connected to plants and the aboveground system via leaf litter input and root-derived resources, with the role of root-derived carbon (C) in fueling soil animal food webs receiving increased attention [12–15]. Further, it has been suggested recently that also root-derived nitrogen (N) contributes to the nutrition of soil animals [16]. Root-derived C and N enter the soil animal food web via living or dead roots, but also via rhizodeposits [17,18]. Rhizodeposits predominantly consist of low molecular weight carbohydrates and therefore are more easily available for soil microorganisms than recalcitrant litter resources [19,20]. Besides the release of C compounds such as sugars, compounds containing both C and N are released by roots such as amino acids and peptides [21]. In particular for N-fixing plants (e.g., legumes) but also non N-fixing plants (e.g., wheat), it has been shown that root-derived N is transferred into the soil, soil microorganisms and neighboring plants [22].

Rhizodeposits, their transformed products and the elements they contain, enter the soil food web via bacteria and fungi, and are transferred to higher trophic levels via bacterial and fungal feeding soil animals, and via predators high up into the food web [12,13,23]. Mycorrhizal and saprotrophic fungi dominate the fungal community of forest soils and play a central role in C and N cycling [24]. Rhizosphere C and N is rapidly taken up by bacteria and fungi with fungi playing a more important role than previously assumed [25,26]. For soil invertebrates, such as Collembola and Oribatida dominating microarthropods in soils, hyphae of saprotrophic and mycorrhizal fungi act as major source of C and nutrients, and their role in animal nutrition likely outweighs that of leaf litter [13,24,27].

One of the most important factors structuring soil animal communities in forests is tree species identity [28,29]. In deciduous forests, tree species associated with ectomycorrhizal fungi (EMF) and arbuscular mycorrhizal fungi (AMF) co-occur. Both mycorrhizal forms release substantial amounts of C into the rhizosphere, but the flux may differ between EMF and AMF [30,31]. Indeed, it has been shown that beech (Fagus sylvatica) associated with EMF more intensively affects the rhizosphere as compared to ash (Fraxinus excelcior) associated with AMF [28], but the impact of these differences on soil animal nutrition remains elusive.

Due to the small size of most soil animal species and the opaqueness of the soil system, uncovering food relationships via direct observations is limited calling for alternative approaches such as the analysis of natural variations in stable isotope ratios, lipid analysis and molecular gut content analysis [32,33]. In contrast to previous long-term labeling studies investigating the general importance of root-derived C [13,27], we used a pulse (short-term) labeling approach to follow the incorporation of aboveground fixed C and also N into soil animal food webs. Pulse labeling has been used before to follow the incorporation of C into soil animal food webs [34,35], but soil animals rarely have been analyzed at species level and typically only Collembola and Oribatida were investigated [16,36,37]. In a similar experiment conducted in the field, the C label was too low to allow studying differences between tree species [16]. Except for the latter experiment, incorporation of plant N into the soil animal food web has never been studied.

By pulse labeling plants with 13C and 15N via exposing plant shoots to 13CO2 enriched atmosphere and via immersing plant leaves into 15NH4Cl solution [22], we investigated the flux of root-derived C and N into the soil animal food web. Incorporation of 13C and 15N into soil animal taxa was followed for 5 and 20 days allowing to identify the flux of root-derived C and N into the soil food web. By comparing the flux of C and N into soil animals in the rhizosphere of beech and ash, the role of mycorrhiza type for soil animal nutrition of deciduous forests was investigated.

We hypothesized that (1) incorporation of root-derived C and N into soil animal taxa varies with tree species associated with either EMF (beech) or AMF (ash), with the incorporation in EMF beech exceeding that in AMF ash, and (2) the incorporation rapidly declines with time due to fast turnover rates of root associated microorganisms and microorganism based C and N pools.

Materials and methods

Experimental set up

In May 2012 40 beech and 40 ash naturally regenerated young trees (seedlings) were excavated including intact rhizosphere soil, soil biota and litter from a deciduous forest close to the city of Göttingen (51°35'15.39"N 9°58'57.95"E; 360 m asl). The forest consists of 130–145 year old beech trees interspersed with maple and ash. The main soil type is Leptosol with mull humus on limestone and an average pH of ca. 5.3 [3]. Individual seedlings with undisturbed soil were transferred into planting pots of 23 x 23 cm and a depth of 26 cm. Colonization of beech seedlings by EMF was 92.6 ± 2.2% (SE). Colonization of ash by AMF was not measured, but under similar conditions it was 52.6 ± 1.8% (SE). Seedlings were about 1 m in height (ranging between 73 and 177 cm). Seedlings were kept under the canopy of mature beech trees for two months and then transferred to an outdoor greenhouse for 15N labeling. The seedlings were irrigated regularly and herbs were removed by cutting the shoots at soil surface level.

Labeling

The seedlings were labeled in four batches of ten seedlings of the same species each with a time lapse of 12 days between the batches. For 15N labeling 98 atom % 15N ammonium-chloride (Campro Scientific, Berlin, Germany) was used. Control seedlings were treated with unlabeled ammonium-chloride (Merck, Darmstadt, Germany). In both treatments a 20 mM solution was mixed with sterile distilled water stored at -20°C until usage. At each of three heights of the seedlings (approximately 30, 60 and 90 cm), three leaves of beech and three leaflets of the compound leaf of ash were put in 20 ml scintillation vials containing ammonium-chloride solution for 72 h. To increase label uptake, the leaf surface was abraded with sand paper (Basic Korn 240, LUX, Wermelskirchen, Germany) [38]. The vials were enclosed by using parafilm and placed into plastic bags. To avoid contamination of the soil by leaching of label, planting pots were covered with plastic bags tightened at the stem of the seedlings with Terostat (Teroson Terostat-VII, Henkel, Düsseldorf, Germany). After labeling, leaves and leaflets used for labeling were cut and removed.

After immersion of the leaves into the 15N labeling solution, ten seedlings of the same species were transferred to an air-tight plant growth chamber with a surface area of 95 x 114 cm and a height of 200 cm (S2 Fig). Conditions in the chamber were kept at 1,013 hPa, 20°C and 70% relative humidity, light intensity was 420 μE for 16 h per day. An irrigation system consisting of PVC tubes of an inner diameter of 6 mm (Deutsch & Neumann, Berlin, Germany) fixed with cable connection to the plastic bag (OBO Bettermann GmbH & Co. KG, Menden, Germany) of the planting pots were established. A ventilation system was used for measuring soil respiration within the plastic bags. Pipes from the inside of the plastic bag were connected to 1 M sodium hydroxide solution and opened once a day for 30 min to allow free air exchange.

Seedlings were acclimatized for two days at 400 ppm with unlabeled CO2 before the 13C labeling started by using a 0.5 M solution of sodium carbonate (KMF Laborchemie Handes, Lohmar, Germany). Actual CO2 concentration in the chamber was measured using an infrared gas analyzer (CARBOCAP™ Serie GMM220, Driesen + Kern GmbH, Bad Bramstedt, Germany) attached to a regulator connected to a pump system releasing 5 M lactic acid and sodium carbonate into one flask when the CO2 concentration fell below 400 ppm. The liberated CO2 was pumped into the plant growth chamber. After acclimation seedlings were exposed to 13CO2 for 3 days for 16 h per day with a maximum CO2 concentration of 1800 ppm. For 13CO2 labeling a total of 2.2 and 3 l 0.5 M sodium carbonate solution with 99 atom% 13C sodium carbonate (Sigma-Aldrich, Traufkirchen, Germany) was used for the ten beech and the ten ash seedlings being equivalent to 124 and 155 g sodium carbonate, respectively. To reduce dilution of the 13CO2 by plant derived CO2 at night, CO2 in the chamber was absorbed by pumping the air through 1 M sodium hydroxide solution (see S2 Fig).

Sampling

Five seedlings of each batch were harvested after 5 days, the other five after 20 days after start of the 13C labeling, resulting in 10 replicates per tree species and sampling date. Control seedlings were kept in a greenhouse at respective conditions and harvested at the same dates as the labeled seedlings. Litter was collected and soil samples were taken from 0–10 and 10–25 cm soil depth. Animals of the litter and soil were extracted by heat [39] and collected in diethylene glycol-water solution (1:1). Animals were stored at -15°C in 70% ethanol until identification and further processing. Diethylene glycol and ethanol do not affect 15N values and only slightly decrease 13C signatures [40]. As we used labelling typically resulting in marked changes in 13C values and treated all samples in the same way we assume changes due to extraction and storage of animals to be negligible. Fresh fine roots (diameter ≤ 1mm) of beech and ash were sampled after taking soil cores for extracting soil animals. Soil particles were carefully removed from the roots and mycorrhizal root tips were separated from the piece before the tip (lateral root) before samples were freeze-dried, ground in a ball mill (Retsch Schwingmuehle MM400, Retsch GmbH, Haan, Germany) and stored in a desiccator until further analysis.

Stable isotope analysis

For dual 13C and 15N measurement 25–612 μg of animal tissue were transferred into tin capsules and dried at 40°C for 48 h; several individuals of small species had to be pooled to obtain enough tissue material for stable isotope analysis. The animals analyzed were taken from soil except for Tomocerus flavescens, Tomocerus vulgaris and Steganacarus magnus which were taken from litter. Stable isotope ratios were analyzed with a coupled system consisting of an elemental analyzer (for samples > 100 μg: NA 2500, CE-Instruments, Rodano, Milan, Italy; for samples < 100 μg: NA1110, CE-Instruments, Rodano, Milan, Italy) and a mass spectrometer (Delta plus, Finnigan MAT, Bremen, Germany) coupled by a ConFlo III interface (Thermo Electron Corporation, Bremen, Germany) [41]. For stable isotope analysis 1–2 mg dry weight of fine roots, mycorrhizal root tip and lateral root were filled into tin capsules and analyzed with a coupled system consisting of an elemental analyzer NA 1108, Fisons-Instruments, Rodano, Milan, Italy and a mass spectrometer (Delta C, Finnigan MAT, Bremen, Germany) coupled by a ConFlo III interface (Thermo Electron Corporation, Bremen, Germany). Abundances of 13C and 15N are expressed using the δ notation with δsample [‰] = [(Rsample − Rstandard)/Rstandard] × 1000; Rsample and Rstandard represent the 13C/12C and 15N/14N ratios of samples and standard, respectively. For 13C PD Belemnite (PBD) and for 15N atmospheric nitrogen served as the primary standard. Acetanilide (C8H9NO, Merck) was used for internal calibration.

For analyzing the enrichment in 13C and 15N of soil animal taxa we calculated the difference in delta values between animals from labeled and unlabeled trees as Δelement = δlabel—δcontrol, with Δelement representing the Δ13C and Δ15N values. Samples with mean Δ13C and Δ15N in the range of two standard deviations of δ13C and δ15N of respective control samples were assumed not to be enriched and set to zero.

Statistical analysis

Statistical analyses were performed using R v.3.2.4 (R Core Team 2016). Δ15N and Δ13C values of eleven soil animal taxa were analyzed separately using linear mixed effects models [42]. A random effect of tree identity (tree ID) avoiding pseudo-replication of soil animal taxa of the same tree was included. We tested the effect of Tree species (beech and ash) and Sampling date (5 and 20 days after labeling) and their interactions on C and N incorporation into soil animal taxa. Δ15N and Δ13C of the two root compartments (lateral root and mycorrhizal root tip) were analyzed separately using linear mixed effects models. A random effect of root tip identity (root ID) avoiding pseudo-replication of compartments of the same root tip was included. We tested the effect of compartment (lateral root and mycorrhizal root tip) and Sampling date (5 and 20 days after labeling) and their interactions on C and N incorporation into lateral root and mycorrhizal root tip. Δ15N and Δ13C values were log-transformed to improve homogeneity of variance. The model was simplified by stepwise reduction ending up with two models, one for enrichment in C and one for enrichment in N. To inspect differences between animal species for C and N enrichment in beech and ash contrast analyses were performed testing differences within each type of Tree species. Further, individual analyses of variance were performed for each species (see S3 Table). Linear mixed effects models with the intercept set to zero were used to inspect C and N enrichment in animal taxa to be significantly different from zero within each level of Tree species. Data given in text represent means and standard deviations.

Results

Natural abundance

Natural abundance δ13C signatures of soil animal taxa spanned over 5.06 delta units from -25.33 ± 0.4 ‰ in Nothrus palustris (Oribatida) to -20.27 ± 0.73 ‰ in S. magnus (Oribatida), while natural δ15N signatures spanned over 7.44 delta units from -3.00 ± 0.77 ‰ in N. palustris to 4.44 ± 1.34 ‰ in Onychiuridae (Collembola) (S1 Fig).

Carbon

Each of the 11 analyzed soil animal taxa was enriched in 13C (S1 Table) with Δ13C values ranging between 0.75 and 4199 ‰, and differing significantly between species (F10,67 = 84.39, p < 0.001). Incorporation of root-derived C declined in the order Onychiuridae (1748 ± 1456 ‰) > T. vulgaris (722.1 ± 1300 ‰) > juvenile Polydesmidae (451.5 ± 398.0 ‰) > Hypochthonius rufulus (377.2 ± 202.1 ‰) > Veigaia nemorensis (319.2 ± 386.3 ‰) > T. flavescens (120.6 ± 89.56 ‰) > S. magnus (43.45 ± 48.50 ‰) > Uroseius cylindricus (13.53 ± 8.65 ‰) > Trachytes aegrota (9.48 ± 5.14 ‰) > Uropoda cassidea (8.23 ± 3.81 ‰) > N. palustris (6.96 ± 5.06 ‰). The incorporation differed between tree species (significant Animal taxa × Tree species interaction; F10,67 = 4.46, p = < 0.001, S2 Table), but soil animal taxa generally were more enriched in the beech as compared to the ash rhizosphere (Fig 1), which was mainly due to H. rufulus, Onychiuridae, T. aegrota and V. nemorensis (see individual analysis of variance S3 Table). Animal taxa differentially incorporated root-derived C at the two sampling dates (significant Animal taxa × Sampling date interaction; F10,67 = 3.68, p = 0.001), which was mainly due to significant higher enrichment at day 20 after labeling in U. cassidea (10.58 ± 1.28 ‰) as compared to 5 days after labeling (5.88 ± 1.22 ‰). Enrichment in 13C in fine roots of ash exceeded that in beech but differences were only marginally significant (3163 ± 1121 and 2341 ± 1034 ‰ respectively; F1,20 = 3.52, p = 0.075). In beech, ectomycorrhizal root tips were significantly more enriched in 13C (3771 ± 4514 Δ ‰) than lateral roots (2428 ± 2380 Δ ‰, F1,32 = 6.52, p = 0.016).

thumbnail
Download:
Fig 1. Enrichment in 15N and 13C in soil animal species / taxa.

Enrichment in 15N and 13C in soil animal species / taxa in the rhizosphere of beech (Fagus sylvatica) and ash (Fraxinus excelsior). Means and standard error (SE). Note the axis break on the y-axis between 1000 and 2600 ‰.

https://doi.org/10.1371/journal.pone.0189502.g001

Nitrogen

Six of the 11 analyzed soil animal taxa were significantly enriched in 15N with Δ15N values (S1 Table) ranging between zero and 121.0 ‰, and differing significantly between species (F10,77 = 7.55, p < 0.001). Incorporation of root-derived N declined in the order Onychiuridae (27.93 ± 38.92 ‰) > juv. Polydesmidae (7.15 ± 16.04 ‰) > V. nemorensis (6.83 ± 15.39 ‰) > H. rufulus (5.42 ± 4.26 ‰) > T. vulgaris (4.83 ± 12.03 ‰) > T. flavescens (1.27 ± 1.72 ‰) > U. cylindricus (0.60 ± 0.62 ‰) > N. palustris (0.47 ± 0.77 ‰) > S. magnus (0.42 ± 0.83 ‰) > U. cassidea (0.35 ± 0.47 ‰) > T. aegrota (0.22 ± 0.33 ‰). However, the incorporation differed between tree species (significant Animal taxa × Tree species interaction; F10,77 = 2.90, p = 0.004, S2 Table), which was mainly due to significant higher incorporation in Onychiuridae in the beech as compared to the ash rhizosphere (F1,11 = 5.37, p = 0.046; Fig 1). Incorporation of root-derived N did not vary with sampling date (F1,29 = 0.003, p = 0.952) and therefore sampling date was removed from the final model. Enrichment in 15N in fine roots of beech and ash did not differ significantly (F1,20 = 0.28, p = 0.59) being on average 181.9 ± 130.3 ‰. In contrast to 13C, ectomycorrhizal root tips of beech were significantly less enriched in 15N (51.26 ± 76.67 Δ ‰) as compared to lateral roots (134.36 ± 144.49 Δ ‰, F1,32 = 46.21, p = < 0.001).

Discussion

Natural abundance

The classification of the studied soil animal taxa into trophic groups according to natural variations in δ15N signatures resembled earlier findings [6,8–10,43]. High δ15N signatures in Onychiuridae (Collembola) has been reported previously [10,44], however, rather than living on animal prey high 15N signatures may also indicate feeding on mycorrhiza [45] as mycorrhiza sheaths of root tips are enriched in 15N [46].

Incorporation of root-derived C

Each of the analyzed soil animal taxa incorporated root-derived C shortly after labeling indicating that recently plant assimilated C is rapidly transferred into the soil animal food web of beech and ash trees. This reinforces the importance of root-derived C in fueling soil animal food webs [13,15,27]. However, incorporation of root-derived C varied markedly between soil animal taxa as shown previously [15,27]. Under beech, Onychiuridae were most enriched in 13C and 15N with the 13C enrichment exceeding that in fine roots indicating that they fed on root and / or mycorrhizal tissue more enriched in 13C than bulk root tissue. Indeed, mycorrhiza were more enriched in 13C as compared to roots reflecting that recently assimilated C is rapidly transferred to mycorrhiza [47,48]. Supporting the conclusion that Onychiuridae fed on roots and / or mycorrhizal tissue, it has been shown previously that Onychiuridae feed on roots, however, this has only been shown for herbaceous plants [49]. Further, in food choice experiments it has been shown that Collembola feed on mycorrhizal fungi [50]. In line with these findings Collembola have been shown to rapidly incorporate root-derived C in field experiments [34,51–53], with high amounts being incorporated into Onychiuridae [36,37]. Also, the fast tissue turnover of Collembola likely contributed to the high incorporation of root-derived C in Onychiuridae, as previously suggested [34].

In contrast to Onychiuridae, 13C enrichment in juvenile Polydesmidae did not exceed that in fine roots, but they were also highly enriched in 13C and 15N indicating that they also feed on root material. Indeed, it has been shown previously that Polydesmidae feed on root hairs [54] and there is evidence that mixed diets of fungi and plant material increase fertility and growth of Polydesmidae [55]. However, in a root labeling experiment Diplopoda were only moderately enriched in 13C suggesting that they predominantly feed on other resources [14]. As Diplopoda including Polydesmidae are strongly sclerotized slow tissue turnover may have contributed to lower incorporation of root-derived C as compared to the less sclerotized root feeders.

The two predacious mite species studied, H. rufulus (Oribatida) and V. nemorensis (Gamasina), also were highly enriched in 13C indicating that they heavily rely on root resources presumably via feeding on Nematoda or Collembola relying on root-derived C. Indeed, V. nemorensis [7,56] and H. rufulus [57,58] are known to feed on Collembola including Onychiuridae [59]. Further, there is increasing evidence that Gamasina heavily rely on Nematoda prey [60], but see also Kudrin et al. [61].

In the two analyzed Tomocerus species the enrichment in 13C was intermediate, indicating that to some extent root-derived resources contribute to their diet, which is in line with previous studies [36,37]. Low incorporation of root-derived N suggests that it was not based on root feeding but rather on feeding on rhizosphere microorganisms which had incorporated N from root exudates. Tomocerus species are known to feed on a variety of food materials including litter but also fungi and bacteria in the rhizosphere [62], algae [63] as well as Nematoda [64]. Omnivory in Collembola has been proposed previously [65] and mixed diets have been shown to increase growth in Collembola [66]. The high variance in the natural abundance of stable isotopes found in the present study also indicate that the diet of Tomocerus species varies between individuals. Overall, incorporation of root-derived C but little root-derived N likely was due to feeding on mycorrhizal fungi but the intermediate 13C signature suggests that they only form a small fraction of the diet of Tomocerus species.

The Uropodina species studied, U. cassidea, U. cylindricus and T. aegrota, incorporated little root-derived C. Uropodina are slow moving mites which are assumed to feed on Nematoda [64,67,68]. However, at least T. aegrota also has been suggested to feed on fungi [6,69]. The low contribution of root-derived C indicate that their prey relies little on root-derived resources. However, in the long-term U. cassidea has been shown to rely on root-derived C [13]. Presumably, slow tissue turnover contributed to the low incorporation of root-derived C in the present study.

Similar to Uropodina, the Oribatida species N. palustris and S. magnus were little enriched in 13C indicating that they rely little on root-derived resources. This is consistent with their trophic position as primary decomposers suggesting that they predominantly feed on dead organic matter such as leaf litter [9,70]. Higher incorporation of root-derived C in S. magnus than in N. palustris suggests differential incorporation of resources supporting earlier findings on niche differentiation in Oribatida [6,9].

Incorporation of root-derived N

Six of the eleven analyzed soil animal taxa incorporated root-derived N indicating that N in plant leaves also is rapidly transferred to the roots and into the soil animal food web. Although this pathway of N received little attention until today (but see e.g., Brumme et al. [71]), it has been shown recently that root-derived N contributes to the nutrition of soil animals in the field [16]. Notably, incorporation of root-derived N varied significantly between soil animal taxa and, in contrast to C, some species did not incorporate any. Presumably, this is due to the fact that rhizodeposits predominantly consist of C rather than N compounds [72]. However, two secondary decomposers, i.e. juvenile Polydesmidae (Diplopoda) and Onychiuridae (Collembola) incorporated root-derived N presumably via feeding on roots and / or root hairs (see above), supporting our conclusion that they heavily rely on root-derived resources.

Variations with time

As in earlier studies [34], incorporation of root-derived C into soil animal taxa varied with time. However, in contrast to our second hypothesis changes in time were low indicating that the assimilated C was rapidly transported into roots, entered the soil animal food web and stayed there for at least 20 days. Contrasting the general pattern, variation in the incorporation of 13C with time was pronounced in U. cassidea incorporating more C after 20 days than after 5 days, indicating that the prey of this predatory mite species only slowly incorporated root-derived C. Incorporation of root-derived N generally did not vary significantly with time suggesting that N leakage and incorporation into the soil food web stayed rather constant even after the addition of tracer was terminated.

Variations with tree species

The incorporation of root-derived C into the soil animal food web significantly differed between tree species with the incorporation in the beech rhizosphere exceeding that in the ash rhizosphere. This contrasts results of a long-term experiment in which no difference in the flux of root-derived C from beech and ash into soil animals was found after five months [27]. This indicates that in the long-term the low flux of root-derived resources from ash into the soil animal food web is compensated by the provisioning of other root-derived resources, potentially dead roots. The differential incorporation of C with tree species in the present experiment indicates that the resources provided by roots for fueling rhizosphere food webs vary with time. Presumably, differences between tree species in the provisioning of root-derived resources are related to differences in root morphology [73] or the different types of mycorrhiza in beech and ash with the former being associated with EMF and the latter with AMF [31,74]. Selective feeding on EMF has been found for the Protura Acerentomon sp. (S. Zieger, unpubl. data), whereas the species investigated in this study likely fed on mixed diets of root-derived and other resources, which is widespread in soil animals [6,66,75]. However, the high enrichment in 13C and 15N in Onychiuridae in the beech but not in the ash rhizosphere indicates that Onychiuridae feed on EMF or roots associated with EMF. In fact EMF typically forms a dense mantle around roots and likely is ingested by species feeding on roots. The predatory mites H. rufulus, T. aegrota and V. nemorensis also incorporated more 13C in the beech as compared to the ash rhizosphere suggesting that they selectively feed on EMF associated prey species, potentially Onychiuridae and root associated Nematoda.

Conclusions

All soil animal species studied incorporated root-derived C supporting earlier findings that root-derived C plays an important role in fueling soil animal food webs. Notably, not only root-derived C, but also root-derived N was incorporated into the soil animal food web indicating that plant N contributes to the nutrition of soil animals thereby alleviating N deficiency in soil animal food webs. However, in contrast to C not all animal species incorporated root-derived N, presumably as rhizodeposits predominantly consist of C rather than N compounds. Differential incorporation of root C and N suggests that root resources contribute to niche differentiation in soil animal species. Incorporation of root C, but not root N, varied with time suggesting that root-derived C not only contributes to animal metabolism, but to animal body tissue formation and quickly is propagated from low to high trophic levels. Notably, incorporation of root-derived C and N into soil animals varied with tree species, i.e. between beech and ash, indicating that tree identity and mycorrhizal type plays an important role in fueling soil animal food webs. Overall, the results underline the importance of root-derived resources in fueling soil animal food webs and suggest that this not only applies to C but also to N.

Supporting information

S1 Fig. Natural abundance stable isotope signatures.

Natural abundance of δ15N and δ13C signatures of the soil animal species / taxa investigated. Means and standard error (SE) with numbers of replicates in brackets.

https://doi.org/10.1371/journal.pone.0189502.s001

(PPTX)

S2 Fig. Experimental set up.

A: Soil respiration was measured for each of the ten seedlings separately; B: Night filter, to reduce dilution of the applied 13CO2 by plant-derived CO2 at night.

https://doi.org/10.1371/journal.pone.0189502.s002

(TIF)

S1 Table. Enrichment analysis.

Estimate, standard error (SE), t-value and p-value of linear mixed effects models with the intercept set to zero on the enrichment in Δ13C and Δ15N in animal taxa within each level of Tree species (beech and ash).

https://doi.org/10.1371/journal.pone.0189502.s003

(XLSX)

S2 Table. Contrast analysis.

Estimate, standard error (SE), z-value and p-value of contrast analysis of differences in Δ13C and Δ15N values between animal taxa in the rhizosphere of beech and ash (Δ 13C only); full contrast matrix of linear mixed effects models.

https://doi.org/10.1371/journal.pone.0189502.s004

(XLSX)

S3 Table. Individual ANOVA.

F- and p-values of the effect of Tree species (beech and ash) and Sampling after labeling (5 and 20 days) on the enrichment in 13C in animal taxa and the effect of Tree species (beech and ash) on the enrichment in 15N.

https://doi.org/10.1371/journal.pone.0189502.s005

(XLSX)

S4 Table. Dataset.

13C and 15N stable isotope values of soil animal taxa (Animal taxa) from soil or litter samples (Layer) of control and labeled (Treatment) beech and ash trees (Tree species) sampled after 5 and 20 days (sampling date) after 13C labeling.

https://doi.org/10.1371/journal.pone.0189502.s006

(XLSX)

Acknowledgments

We are grateful to Prof. Dr. Ammer for study site advice. We thank Tobias Lauermann and Janine Sommer for help excavating trees from the forest. Bernd Kopka, Thomas Klein from the Labor für Radioisotope (LARI, University of Göttingen) for technical advice and help in using the plant growth chamber and the Kompetenzzentrum Stabile Isotope (KOSI, University of Göttingen) for measuring stable isotopes.

References

  1. 1. Ehnes RB, Pollierer MM, Erdmann G, Klarner B, Eitzinger B, Digel C, et al. Lack of energetic equivalence in forest soil invertebrates. Ecology. 2014; 95: 527–537. pmid:24669745
  2. 2. Digel C, Curtsdotter A, Riede J, Klarner B, Brose U. Unravelling the complex structure of forest soil food webs: Higher omnivory and more trophic levels. Oikos. 2014; 123: 1157–1172.
  3. 3. Schaefer M. The soil fauna of a beech forest on limestone: trophic structure and energy budget. Oecologia. 1990; 82: 128–136. pmid:28313148
  4. 4. Anderson JM. The enigma of soil animal diversity. In: Vaněk J, editor. Progress in Soil Zoology Proceedings of the Fifth International Colloquium of Soil Zoology. Prague: Academia; 1975. pp. 51–58. https://doi.org/10.1007/978-94-010-1933-0
    • 5. Ponsard S, Arditi R. What can stable isotopes (δ15N and δ13C) tell about the food web of soil macro-invertebrates? Ecology. 2000; 81: 852–864.
    • 6. Scheu S, Falca M. The soil food web of two beech forests (Fagus sylvatica) of contrasting humus type: stable isotope analysis of a macro- and a mesofauna-dominated community. Oecologia. 2000; 123: 285–296. pmid:28308733
    • 7. Koehler HH. Predatory mites (Gamasina, Mesostigmata). Agric Ecosyst Environ. 1999; 74: 395–410.
    • 8. Klarner B, Maraun M, Scheu S. Trophic diversity and niche partitioning in a species rich predator guild–Natural variations in stable isotope ratios (13C/12C, 15N/14N) of mesostigmatid mites (Acari, Mesostigmata) from Central European beech. Soil Biol Biochem. 2013; 57: 327–333.
    • 9. Schneider K, Migge S, Norton RA, Scheu S, Langel R, Reineking A, et al. Trophic niche differentiation in soil microarthropods (Oribatida, Acari): evidence from stable isotope ratios (15N/14N). Soil Biol Biochem. 2004; 36: 1769–1774.
    • 10. Chahartaghi M, Langel R, Scheu S, Ruess LR. Feeding guilds in Collembola based on nitrogen stable isotope ratios. Soil Biol Biochem. 2005; 37: 1718–1725.
    • 11. Semenyuk II, Tiunov A V. Isotopic signature (15N/14N and 13C/12C) confirms similarity of trophic niches of millipedes (Myriapoda, Diplopoda) in a temperate deciduous forest. Biol Bull. 2011; 38: 283–291.
    • 12. Ruf A, Kuzyakov Y, Lopatovskaya O. Carbon fluxes in soil food webs of increasing complexity revealed by 14C labelling and 13C natural abundance. Soil Biol Biochem. 2006; 38: 2390–2400.
    • 13. Pollierer MM, Langel R, Körner C, Maraun M, Scheu S. The underestimated importance of belowground carbon input for forest soil animal food webs. Ecol Lett. 2007; 10: 729–736. pmid:17594428
    • 14. Gilbert KJ, Fahey TJ, Maerz JC, Sherman RE, Bohlen P, Dombroskie JJ, et al. Exploring carbon flow through the root channel in a temperate forest soil food web. Soil Biol Biochem. 2014; 76: 45–52.
    • 15. Scheunemann N, Digel C, Scheu S, Butenschoen O. Roots rather than shoot residues drive soil arthropod communities of arable fields. Oecologia. 2015; 179: 1135–1145. pmid:26267404
    • 16. Zieger SL, Holczinger A, Sommer J, Rath M, Kuzyakov Y, Polle A, et al. Beech trees fuel soil animal food webs via root-derived nitrogen. Basic Appl Ecol. 2017; Forthcoming.
    • 17. Kuzyakov Y, Gavrichkova O. Time lag between photosynthesis and carbon dioxide efflux from soil: a review of mechanisms and controls. Glob Chang Biol. 2010; 16: 3386–3406.
    • 18. Högberg P, Högberg MN, Göttlicher SG, Betson NR, Keel SG, Metcalfe DB, et al. High temporal resolution tracing of photosynthate carbon from the tree canopy to forest soil microorganisms. New Phytol. 2008; 177: 220–228. pmid:17944822
    • 19. van Hees PAW, Jones DL, Finlay R, Godbold DL, Lundström US. The carbon we do not see—the impact of low molecular weight compounds on carbon dynamics and respiration in forest soils: a review. Soil Biol Biochem. 2005; 37: 1–13.
    • 20. Glanville H, Rousk J, Golyshin P, Jones DL. Mineralization of low molecular weight carbon substrates in soil solution under laboratory and field conditions. Soil Biol Biochem. 2012; 48: 88–95.
    • 21. Bais HP, Weir TL, Perry LG, Gilroy S, Vivanco JM. The role of root exudates in rhizosphere interactions with plants and other organisms. Annu Rev Plant Biol. 2006; 57: 233–266. pmid:16669762
    • 22. Wichern F, Eberhardt E, Mayer J, Joergensen RG, Müller T. Nitrogen rhizodeposition in agricultural crops: Methods, estimates and future prospects. Soil Biol Biochem. 2008; 40: 30–48.
    • 23. Lemanski K, Scheu S. Fertilizer addition lessens the flux of microbial carbon to higher trophic levels in soil food webs of grassland. Oecologia. 2014; 176: 487–496. pmid:25147053
    • 24. Leake JR, Donnelly DP, Boddy L. Interactions Between Ecto-mycorrhizal and Saprotrophic Fungi. In: van der Heijden MGA, Sanders IR, editors. Mycorrhizal Ecology. Berlin: Springer; 2002. pp. 345–372. https://doi.org/10.1007/978-3-540-38364-2
      • 25. Johnson D, Leake JR, Ostle N, Ineson P, Read DJ. In situ 13CO2 pulse-labelling of upland grassland demonstrates a rapid pathway of carbon flux from arbuscular mycorrhizal mycelia to the soil. New Phytol. 2002; 153: 327–334.
      • 26. Lemanski K, Scheu S. Incorporation of 13C labelled glucose into soil microorganisms of grassland: Effects of fertilizer addition and plant functional group composition. Soil Biol Biochem; 2014; 69: 38–45.
      • 27. Eissfeller V, Beyer F, Valtanen K, Hertel D, Maraun M, Polle A, et al. Incorporation of plant carbon and microbial nitrogen into the rhizosphere food web of beech and ash. Soil Biol Biochem. 2013; 62: 76–81.
      • 28. Cesarz S, Fender A-C, Beyer F, Valtanen K, Pfeiffer B, Gansert D, et al. Roots from beech (Fagus sylvatica L.) and ash (Fraxinus excelsior L.) differentially affect soil microorganisms and carbon dynamics. Soil Biol Biochem; 2013; 61: 23–32.
      • 29. Tedersoo L, Bahram M, Cajthaml T, Põlme S, Hiiesalu I, Anslan S, et al. Tree diversity and species identity effects on soil fungi, protists and animals are context dependent. ISME J. 2016; 10: 346–362. pmid:26172210
      • 30. Phillips RP, Fahey TJ. Patterns of rhizosphere carbon flux in sugar maple (Acer saccharum) and yellow birch (Betula allegheniensis) saplings. Glob Change Biol. 2005; 11: 983–995.
      • 31. Phillips RP, Fahey TJ. Tree species and mycorrhizal associations influence the magnitude of rhizosphere effects. Ecology. 2006; 87: 1302–1313. pmid:16761608
      • 32. Brose U, Scheu S. Into darkness: unravelling the structure of soil food webs. Oikos. 2014; 123: 1153–1156.
      • 33. Traugott M, Kamenova S, Ruess LR, Seeber J, Plantegenest M. Empirically characterising trophic networks: What merging DNA-based methods, stable isotope and fatty acid analyses can offer. Advances in Ecological Research. 2013; pp. 177–224.
      • 34. Ostle NJ, Briones MJI, Ineson P, Cole L, Staddon P, Sleep D. Isotopic detection of recent photosynthate carbon flow into grassland rhizosphere fauna. Soil Biol Biochem. 2007; 39: 768–777.
      • 35. Högberg MN, Briones MJI, Keel SG, Metcalfe DB, Campbell C, Midwood AJ, et al. Quantification of effects of season and nitrogen supply on tree below-ground carbon transfer to ectomycorrhizal fungi and other soil organisms in a boreal pine forest. New Phytol. 2010; 187: 485–493. pmid:20456043
      • 36. Potapov AM, Goncharov AA, Tsurikov SM, Tully T, Tiunov A V. Assimilation of plant-derived freshly fixed carbon by soil collembolans: Not only via roots? Pedobiologia. 2016; 59: 189–193.
      • 37. Fujii S, Mori AS, Kominami Y, Tawa Y, Inagaki Y, Takanashi S, et al. Differential utilization of root-derived carbon among collembolan species. Pedobiologia. 2016; 59: 225–227.
      • 38. Hertenberger G, Wanek W. Evaluation of methods to measure differential 15N labeling of soil and root N pools for studies of root exudation. Rapid Commun Mass Spectrom. 2004; 18: 2415–2425. pmid:15386635
      • 39. Kempson D, Lloyd M, Ghelardi R. A new extractor for woodland litter. Pedobiologia. 1963; 3: 1–21.
      • 40. Ponsard S, Amlou M. Effects of several preservation methods on the isotopic content of Drosophila samples. Comptes Rendus l’Academie des Sci—Ser III. 1999; 322: 35–41.
      • 41. Langel R, Dyckmans J. Combined (13) C and (15) N isotope analysis on small samples using a near-conventional elemental analyzer/isotope ratio mass spectrometer setup. Rapid Commun Mass Spectrom. 2014; 28: 1019–1022. pmid:24677523
      • 42. Pinheiro J, Bates D, DebRoy S, Sarkar D. nlme: Linear and Nonlinear Mixed Effects Models. R package ver. 3.1–120. R package; 2013.
        • 43. Maraun M, Erdmann G, Fischer BM, Pollierer MM, Norton RA, Schneider K, et al. Stable isotopes revisited: Their use and limits for oribatid mite trophic ecology. Soil Biol Biochem. 2011; 43: 877–882.
        • 44. Semenina EE, Tiunov A V. Trophic fractionation (Δ15N) in Collembola depends on nutritional status: A laboratory experiment and mini-review. Pedobiologia. 2011; 54: 101–109.
        • 45. Pollierer MM, Langel R, Scheu S, Maraun M. Compartmentalization of the soil animal food web as indicated by dual analysis of stable isotope ratios (15N/14N and 13C/12C). Soil Biol Biochem. 2009; 41: 1221–1226.
        • 46. Högberg P, Högbom L, Schinkel H, Högberg M, Johannisson C, Wallmark H. 15N abundance of surface soils, roots and mycorrhizas in profiles of European forest soils. Oecologia. 1996; 108: 207–214. pmid:28307831
        • 47. Johnson D, Leake JR, Read DJ. Transfer of recent photosynthate into mycorrhizal mycelium of an upland grassland: short-term respiratory losses and accumulation of 14C. Soil Biol Biochem. 2002; 34: 1521–1524.
        • 48. Valtanen K, Eissfeller V, Beyer F, Hertel D, Scheu S, Polle A. Carbon and nitrogen fluxes between beech and their ectomycorrhizal assemblage. Mycorrhiza. 2014. 24: 645–650. pmid:24756632
        • 49. Endlweber K, Ruess LR, Scheu S. Collembola switch diet in presence of plant roots thereby functioning as herbivores. Soil Biol Biochem. 2009; 41: 1151–1154.
        • 50. Shaw PJA. A consistent hierarchy in the fungal feeding preferences of the Collembola Onychiurus armatus. Pedobiologia. 1988; 31: 179–187.
        • 51. Albers D, Schaefer M, Scheu S. Incorporation of plant carbon into the soil animal food web of an arable system. Ecology. 2006; 87: 235–245. pmid:16634314
        • 52. Larsen T, Gorissen A, Krogh PH, Ventura M, Magid J. Assimilation dynamics of soil carbon and nitrogen by wheat roots and Collembola. Plant Soil. 2007; 295: 253–264.
        • 53. Scheunemann N, Pausch J, Digel C, Kramer S, Scharroba A, Kuzyakov Y, et al. Incorporation of root C and fertilizer N into the food web of an arable field: Variations with functional group and energy channel. Food Webs. 2016; 9: 39–45.
        • 54. Gunn A, Cherrett JM. The Exploitation of Food Resources by Soil Meso-Invertebrates and Macro-Invertebrates. Pedobiologia. 1993; 37: 303–320.
        • 55. David J-F, Célérier M-L. Effects of yeast on the growth and reproduction of the saprophagous millipede Polydesmus angustus (Diplopoda, Polydesmidae). Biol Fertil Soils. 1997; 24: 66–69.
        • 56. Walter DE, Proctor HC. Mites: Ecology, Evolution & Behaviour. Dordrecht: Springer; 2013. https://doi.org/10.1007/978-94-007-7164-2
          • 57. Behan-Pelletier V, Hill S. Feeding habitat of sixteen species of Oribatei (Acari) from an acid peat bog, Glenamoy Ireland. Rev d’Écologie Biol du Sol. 1983; 20: 221–267.
          • 58. Riha G. Zur Ökologie der Oribatiden in Kalksteinböden. Zool Jahrbuecher Abteilung fuer Syst Oekologie und Geogr der Tiere. 1951; 80: 407–450.
          • 59. Hurlbutt HW. Coexistence and Anatomical Similarity in Two Genera of Mites, Veigaia and Asca. Syst Biol. 1968; 17: 261–271.
          • 60. Heidemann K, Ruess LR, Scheu S, Maraun M. Nematode consumption by mite communities varies in different forest microhabitats as indicated by molecular gut content analysis. Exp Appl Acarol. 2014; 64: 49–60. pmid:24705854
          • 61. Kudrin AA, Tsurikov SM, Tiunov A V. Trophic position of microbivorous and predatory soil nematodes in a boreal forest as indicated by stable isotope analysis. Soil Biol Biochem. 2015; 86: 193–200.
          • 62. Pollierer MM, Dyckmans J, Scheu S, Haubert D. Carbon flux through fungi and bacteria into the forest soil animal food web as indicated by compound-specific 13C fatty acid analysis. Funct Ecol. 2012; 26: 978–990.
          • 63. Wolters V. Resource allocation in Tomocerus flavescens (Insecta, Collembola): a study with C-14-labelled food. Oecologia. 1985; 65: 229–235. pmid:28310670
          • 64. Heidemann K, Hennies A, Schakowske J, Blumenberg L, Ruess LR, Scheu S, et al. Free-living nematodes as prey for higher trophic levels of forest soil food webs. Oikos. 2014; 123: 1199–1211.
          • 65. Filser J. The role of Collembola in carbon and nitrogen cycling in soil. Pedobiologia. 2002; 46: 234–245.
          • 66. Scheu S, Simmerling F. Growth and reproduction of fungal feeding Collembola as affected by fungal species, melanin and mixed diets. Oecologia. 2004; 139: 347–353. pmid:15021980
          • 67. Buryn R, Brandl R. Are the morphometrics of chelicerae correlated with diet in mesostigmatid mites (Acari)? Exp Appl Acarol. 1992; 14: 67–82.
          • 68. Muraoca M, Ishibashi N. Nematode-Feeding Mites and Their Feeding Behavior. Appl Entomol Zool. 1976; 11: 1–7.
          • 69. Constantinescu IC, Cristescu C. Studies on the trophic preferences of certain Uropodina mites (Acarina: Anactinotrichida, Uropodina) for some taxa of imperfect fungi. Analele Ştiinţifice ale Univ „Al I Cuza” Iaşi, s Biol Anim. 2007; 83–87.
          • 70. Rihani M, Cancela da Fonseca JP, Kiffer E. Decomposition of beech leaf litter by microflora and mesofauna. II. Food preferences and action of oribatid mites on different substrates. Eur J Soil Biol. 1995; 31: 67–79. 35400005491585.0020
          • 71. Brumme R, Leimcke U, Matzner E. Interception and uptake of NH4 and NO3 from wet deposition by above-ground parts of young beech (Fagus silvatica L.) trees. Plant Soil. 1992; 142: 273–279.
          • 72. Merbach W, Mirus E, Knof G, Remus R, Ruppel S, Russow R, et al. Release of carbon and nitrogen compounds by plant roots and their possible ecological importance. J Plant Nutr Soil Sci. 1999; 162: 373–383.
          • 73. Kubisch P, Hertel D, Leuschner C. Do ectomycorrhizal and arbuscular mycorrhizal temperate tree species systematically differ in root order-related fine root morphology and biomass? Front Plant Sci. 2015; 6. pmid:25717334
          • 74. Brzostek ER, Greco A, Drake JE, Finzi AC. Root carbon inputs to the rhizosphere stimulate extracellular enzyme activity and increase nitrogen availability in temperate forest soils. Biogeochemistry. 2012; 115: 65–76.
          • 75. Raubenheimer D, Simpson SJ. Organismal stoichiometry: Quantifying non-independence among food components. Ecology. 2004; 85: 1203–1216.