Pedobiologia - Journal of Soil Ecology 87-88 (2021) 150751 Contents lists available at ScienceDirect Pedobiologia - Journal of Soil Ecology journal homepage: www.elsevier.com/locate/pedobi Distinct macroinvertebrate soil food webs at one-meter scale in a Mediterranean agroecosystem Carlos Hernández-Castellano a, b, *, Josep Piñol a, b, Xavier Espadaler a a CREAF, Cerdanyola del Vallès 08193, Spain b Univ Autònoma de Barcelona, Cerdanyola del Vallès 08193, Spain A R T I C L E I N F O A B S T R A C T Keywords: Soil macroinvertebrates play a central role in ecosystem processes such as decomposition and nutrient cycling, Stable isotopes but the extent to which macroinvertebrate food web structure varies within ecosystems is basically unknown. δ13C Here, we examined changes in plant and macroinvertebrate community composition at increasing distances from δ15N trees within a citrus grove, following a gradient in organic matter and water availability created by the localized Soil fauna Arthropod community fertilization and irrigation of citrus trees. In addition, we analysed stable isotope signatures (δ13C and δ15N) of Trophic structure soil, plants, and macroinvertebrates to investigate changes in trophic structure within the grove. The macro­ invertebrate community near the tree trunks had a higher number of detritivores and predators than the com­ munity located farther away. After correcting the isotope values of macroinvertebrates by their isotopic baseline, a spatial shift in trophic structure emerged: macroinvertebrates near the trunks had lower δ13C-values than those located farther away (i.e., they apparently relied more on plant- based resources). This pattern is attributable to species only found near the trunks. In contrast, species distributed across the studied area showed no differences in their isotopic signatures across space, suggesting that they are connecting otherwise two compartmentalized food webs. A reduction in organic matter and water availability at increasing distances from trees is the most parsimonious explanation of the observed isotopic gradient in soil and plants, and this may, in turn, be responsible for the observed changes in food web composition and structure. This study shows that spatial variability in macroinvertebrate soil food webs can be present at scales as small as one meter if spatial variability in environmental factors is high enough. 1. Introduction approach considers all the species in an ecosystem as potentially inter­ connected through trophic interactions (community webs sensu Cohen Macroinvertebrates play a crucial role in decomposer food webs. (1977)), but the scale of the study and the scale at which organisms Many species act as ecosystem engineers: structuring the soil, processing experience the environment is not necessarily the same. Even within the large amounts of litter, and producing faeces that stimulate microbial same ecosystem, some spatial variability in resources is expected, which activity and, therefore, decomposition (Hättenschwiler et al., 2005; Cole can produce changes in food web composition and structure at small et al., 2006). Thus, by analysing the spatial variation in macro­ scales. Spatial patterns in relatively uniform ecosystems have been invertebrate food webs and linking observed changes with environ­ found in the microbiota (Ettema and Wardle, 2002), but when larger mental influences, it is possible to identify the key factors of the food components of detrital food webs are included, horizontal patterns may web structure and ecosystem functioning (de Vries et al., 2013). As an disappear (Berg and Bengtsson, 2007). Conversely, in arid ecosystems, example, due to habitat changes, macroinvertebrate communities and where spatial heterogeneity is high, certain microhabitats such as shrubs trophic structure can change, particularly in relation to the trophic and nest mounds harbour more complex macroinvertebrate commu­ guilds or energy pathways present (Seeber et al., 2005; Klarner et al., nities than their surroundings do (Doblas-Miranda et al., 2009). Simi­ 2014). larly, Goncharov et al. (2014) found that different microsites around The studies cited above focused on soil macroinvertebrate food web fallen trees harbour macroinvertebrate communities with different tro­ changes among ecosystems (see Hyodo (2015) for a review). This phic structures. * Corresponding author at: CREAF, Cerdanyola del Vallès 08193, Spain. E-mail address: c.hernandez@creaf.uab.cat (C. Hernández-Castellano). https://doi.org/10.1016/j.pedobi.2021.150751 Received 17 February 2021; Received in revised form 18 June 2021; Accepted 18 June 2021 Available online 23 June 2021 0031-4056/© 2021 The Author(s). Published by Elsevier GmbH. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). C. Hernández-Castellano et al. Pedobiologia - Journal of Soil Ecology 87-88 (2021) 150751 Soil food webs are often divided into grazing and detritus-based arranged in 3 rows and 3 columns. Trees within a row are separated by energy channels (Moore and de Ruiter, 1991). Rather than being 3.5 m and rows are 6 m apart. Since irrigation and fertilizer are applied strictly separated in discreet trophic layers, the position of feeding beneath the tree canopies, we sampled at three distances from each tree: groups of detritivores and predators form a trophic continuum within a Trunk (20 cm to the tree trunk, just beneath the canopy), Middle (150 food web. Many species engage in opportunistic feeding behaviour, with cm distant, at the outer limit of the canopy), and Row (350 cm distant, the result that omnivory is a pervasive feature in these communities between rows); thereafter referred as treatments. Thus, we generated 27 (Scheu, 2002). The formalization of such complex networks has tradi­ samples in total. Fig. A.1 shows a detailed layout of the experimental tionally been based on direct observations, but this kind of data is design. difficult to gather in soil communities because of the small size and Samples were taken in July 2013 between 6 a.m. and 10 a.m. At each cryptic habits of the species involved. Besides, occasional observations sampling location, we collected the aerial parts of all plants over an area might not be truly representative of the long-term relationships between of 25 × 25 cm2 and preserved them in a dry paper for further identifi­ species. cation and processing. There was not a clear developed layer of plant The analysis of stable isotope ratios of C and N (δ13C and δ15N) is an litter, but fresh and dried plant material was standing on the soil. For this alternative approach that integrates information over time regarding reason, we directly dug the area to a depth of 15 cm and extracted the sources of energy used by animals, trophic positions, and trophic links bulk of soil. among community members (Tiunov, 2007). Today this technique is The soil was torn apart in small quantities over a white plastic tray, fundamental in the study of soil food webs (Potapov et al., 2019; Quinby all macroinvertebrates were collected with pincers and entomological et al., 2020). Both δ13C and δ15N increase with trophic level as a result of aspirators and immediately preserved in 70 % ethanol. Soil macrofauna fractionation, a process by which consumers become enriched, can be sampled by hand sorting, and taxonomic groups are typically compared to their diet, in the heavy isotope (trophic enrichment). While around 2 mm body width (Swift et al., 1979; Coleman et al., 2004). For enrichment in δ13C is small (< 1‰) and, therefore, useful for inferring this reason, mesofauna was not considered here. Despite most species of potential food sources, the increase in δ15N is larger (approximately mites are associated to mesofauna, three morphospecies of large pred­ 3.4‰) and can be used to estimate trophic position (McCutchan et al., atory mites (1− 3 mm body width) were considered in this study. We 2003; Potapov et al., 2019). Overall, the isotopic variability of a popu­ kept 1 kg of each soil sample for future analysis. lation can be used as a proxy for the width of the species trophic niche We also collected leaves from each sampled citrus tree and samples (Bearhop et al., 2004; Newsome et al., 2007), and has proved useful for of the fertilizer applied over the last three years (2011, 2012, 2013). The elucidating trophic relationships between different components of the fertilizer of 2013 had a different isotopic composition from that of the soil food web (e.g., Scheu and Falca, 2000; Ponsard and Arditi, 2000). other years because of an obligate change of provider. Here we assess the spatial variability in the soil macroinvertebrate food web of a Mediterranean agroecosystem. In particular, we wanted to 2.3. Sample processing and stable isotope analysis understand the influence of spatial variability in environmental factors on the composition of the macroinvertebrate community and its trophic Soil samples were milled and sieved to 0.2 mm and pulverized on a structure. To do so, we took advantage of a pronounced gradient in mortar grinder. We analysed organic matter by oxidation and eliminated organic matter and water availability in a citrus grove caused by local­ inorganic carbon from soil by acidification following Hesse (1971) and ized fertilization and irrigation beneath the tree canopies. Across this Midwood and Boutton (1998), respectively. gradient we i) analysed the plant and macroinvertebrate community Plants were identified using a reference collection of the studied area composition, ii) characterized the C and N stable isotopes of soil, plants, (Kindermann, 2010) and with keys from Bolòs et al. (2005). We selected and macroinvertebrates, and iii) defined trophic groups of species for isotopic analyses those species accounting for 90 % of the total within the delimited food webs. Food web delimitation was based on biomass of each sample and homogenized them to a fine powder with a differences across space in macroinvertebrate community composition mixer mill. and stable isotope signatures. We hypothesized that changes in food web Macroinvertebrates were identified to the species level when composition and structure would emerge at the scale at which changes possible. We selected for isotopic analyses members of edaphic species in resource availability were most important. Specifically, we expect the for which individuals of the same life stage were present at least in two sites beneath the tree canopies to constitute a local refuge for soil fauna samples of a treatment (for ants we considered that a nest was present if in an otherwise semiarid system, driving changes in the identity, there were at least 25 homospecific workers). Analysed species abundance, and trophic relationships between soil organisms in com­ amounted to 48 % of the total species richness, which encompasses 81 % parison to sites located further away. of the total individuals abundance. We weighed 8–10, 1–1.5, and 0.2–2.5 mg of processed soil, plant, 2. Materials and methods and macroinvertebrates, respectively, into tin capsules for stable isotope analyses. If an invertebrate specimen weighed more than 2.5 mg, we 2.1. Study site homogenized it to a fine powder with a pestle. For snails and earth­ worms, we only analysed muscle tissue. Samples were dried for 24− 48 The study was conducted in an organic citrus grove located in La hours at 60 ◦ C. Selva del Camp (Tarragona, NE Spain; 41◦ 13′ 07′ N, 01◦ 08′ 35′ E). The We analysed δ13C and δ15N in all samples at the Stable Isotopes grove consists of ca. 300 clementine trees (Citrus clementina var. clem­ Analysis Lab (LAIE) at the Autonomous University of Barcelona, using a enules) grafted on the hybrid rootstock Carrizo citrange (Poncirus trifo­ continuous-flow Delta V Advantage Isotope Ratio Mass Spectrometer, liata (L.) Raf. x Citrus sinensis (L.) Osb.). Trees are watered twice a week coupled to a Flash 1112 elemental analyser (both from Thermo Fisher during dry periods and fertilized each winter with organic manure. Scientific Inc., Waltham, MA USA). Samples were standardized to IAEA- Grasses and other weeds form a permanent ground cover, which is 600 (caffeine, δ13C: -27.771‰ ± 0.043 SD; δ15N: 1.0‰ ± 0.2 SD) from mowed three or four times a year. More information about the study site the International Atomic Energy Agency (Vienna, Austria). Carbon and and the arthropod assemblages present in the grove can be found in nitrogen isotope ratios were calculated relative to VPDB and atmo­ Piñol et al. (2012). spheric air, respectively, and expressed as ‰. In total, we performed 377 stable isotope analyses (27 of soil and 9 of 2.2. Experimental design and sampling details fertilizer, 79 of plants, including citrus leaves, and 262 of macro­ invertebrates), comprising 18 species of plants and 39 species of We selected for sampling 9 adjacent trees within the citrus grove, macroinvertebrates. 2 C. Hernández-Castellano et al. Pedobiologia - Journal of Soil Ecology 87-88 (2021) 150751 2.4. Data analyses Staphylinid beetles, which may feed on dead arthropods and dung, but also on fungal hyphae (Thayer, 2016). All analyses followed a randomized block design, with treatment We consequently pooled together the treatments showing no differ­ (each distance class to the tree trunk) as a fixed factor and tree as a ences between them in baseline-corrected δ-values. For this new com­ random factor (each tree containing three samples, one for each bination of treatments, we repeated the former statistical test twice, treatment). once for the analysed species present across and therefore shared be­ tween treatments (common species), and once for the analysed species 2.4.1. Plant and macroinvertebrate communities present in a single treatment (unique species). We proceeded in that way To assess spatial variability in food web composition, we compared because common species are likely to forage across the studied area, the plant and macroinvertebrate communities between treatments, therefore integrating the environmental isotopic variability, whereas analysing species composition and abundance of taxonomic groups unique species are likely to have limited foraging ranges, therefore (family or higher). We used biomass (g m− 2) and count data (individuals reflecting the isotopic signature characteristic of a sample. These ana­ m− 2) for plants and macroinvertebrates, respectively. Despite soil sam­ lyses assured that for common species the isotopic baseline of multiple ples consisted in volume rather than surface, we keep our measures treatments is used and that all the isotopic variability from the samples expressed as individuals m-2 because 1) soil fauna abundance is often of the study area in which the species is present is taken into account. expressed in these units and we aim to keep our variable comparable to other similar studies, 2) we analysed the bulk of soil as a whole in 2.4.3. Food web structure relation to macrofauna, and 3) a systematic transformation would not To delimit food webs across space, we followed the foundations of modify the results (i.e. the depth was the same for all samples). For delimiting ecological systems of Post et al. (2007). That is, we consid­ community composition, raw data were square-root transformed, and a ered macroinvertebrates of two treatments to constitute different food permutational multivariate ANOVA was performed using the Bray- webs if they showed differences in community composition and stable Curtis dissimilarity index. If differences among treatments were signif­ isotope signatures. For characterizing each web, we built ellipses of icant, we ran a posteriori pairwise comparisons. For the analyses of the isotopically similar macroinvertebrates (trophic groups) by conducting abundance of taxonomic groups, we conducted a permutational ANOVA pairwise comparisons between species’ isotopic niches (δ13C-δ15N) using Euclidean distances between samples, and Monte Carlo P-values within each trophic guild. We included in this analysis only those species were generated when the number of possible permutations was less than with at least four individuals to ensure the robustness of the test; the 100. P-values were adjusted following the False Discovery Rate (FDR) remaining species were assigned to the closest trophic group, and we method. If differences among treatments were significant, we ran a adjusted the level of significance by setting a conservative threshold of P posteriori pairwise comparisons. < 0.01. The number of estimated trophic levels (TL) for each food web We analysed the relationship between plant and macroinvertebrate was calculated as TL = 2 + (δ15Ntop predator - δ15Nbaseline) / Δδ15N (Post, communities with a Mantel test, using the Bray-Curtis dissimilarity 2002), where 2 is the TL of the primary consumer used to estimate the index between samples and the Pearson correlation. baseline (δ15Nbaseline), δ15Ntop predator is the δ15N of the top predator in To visualize differences in macroinvertebrate community composi­ the food web, and Δδ15N is the increase in δ15N per trophic level (3.4‰; tion among treatments we conducted a non-metric multidimensional McCutchan et al., 2003; Potapov et al., 2019). scaling (NMDS) based on the Bray-Curtis dissimilarity index. To detect Analyses were run with the software PERMANOVA + for PRIMER v. which macroinvertebrate species differed between treatments, we 6 (Anderson et al., 2008) and with the package vegan (Oksanen et al., calculated the contribution of each macroinvertebrate species to the 2019) in R environment (R Core Team, 2019). observed dissimilarity with a SIMPER analysis. 3. Results 2.4.2. Stable isotopes of soil, plants and macroinvertebrates To assess spatial variability in food web trophic structure, we 3.1. Plant community composition compared the stable isotopes (δ13C and δ15N) of soil, plants and mac­ roinvertebrates among treatments. For soil and plants, we conducted a We collected a total of 27 species of plants from 17 families permutational ANOVA using Euclidean distances between samples. For (Table A.1). Plant community changed across treatments (pseudo-F2,16 plants, we included species as a random factor nested in family (random = 6.14, P < 0.001). Trunk communities were different from those of factor). Middle and Row samples (t = 2.64, P = 0.0059; t = 2.84, P = 0.0024; For macroinvertebrates we ran a permutational ANCOVA on mac­ respectively), but there were no differences between Middle and Row (t roinvertebrate stable isotope signatures with soil δ13C and δ15N as = 1.18, P = 0.23). covariates, that is, macroinvertebrates of a given sample had the soil The vegetation of Trunk samples was dominated by the nitrophilous signature of the same sample as a covariate. This corrected community Parietaria officinalis (Urticaceae) (60 % of all biomass), whereas Middle signature by trophic baseline and then determined if trophic shifts were and Row were dominated by several grass species (76 %), mainly Avena present among treatments. This approach is normally used in soil food barbata, Hordeum murinum, and Cynodon dactylon (the last one is C4). No web studies (Potapov et al., 2019), and in our study is supported by the differences in plant biomass between treatments were found (pseudo- fact that almost all macroinvertebrate signatures are close to those of the F2,16 = 0.74, P = 0.49). soil. We set the trophic guild of macroinvertebrates as a random factor. The trophic guild of macroinvertebrates (detritivores, predators, herbi­ 3.2. Macroinvertebrate community composition vores and omnivores) was defined following the existing literature (Armengol, 1986; Blas, 1987; Altaba and Ros, 1991; Lövei and Sun­ We collected a total of 3838 macroinvertebrate individuals derland, 1996; Deckle and Fasulo, 2001; Zimmer, 2002; Bell et al., 2007; belonging to 82 species and 36 taxonomic levels (family or higher) Gómez and Espadaler, 2007; Krantz and Walter, 2009). This classifica­ (Table A.2). tion allowed us to consider the most important trophic guilds in soils The macroinvertebrate community also changed across treatments food webs, and to relate a priori classification with stable isotope sig­ (pseudo-F2,16 = 5.48, P < 0.001, Fig. 1a). As in plants, Trunk commu­ natures of soil, animals, and plants. Detritivores were defined sensu lato, nities were different from those of Middle and Row samples (t = 2.65, P because despite most species are expected to feed on dead organic = 0.0014; t = 2.75, P = 0.0012; respectively), with only marginally matter, some species may also feed on living components of organic significant differences between Middle and Row (t = 1.48, P = 0.053) matter not analysed here. This might be especially true for some (Fig. 1a). 3 C. Hernández-Castellano et al. Pedobiologia - Journal of Soil Ecology 87-88 (2021) 150751 Fig. 1. (a) Non-metric multidimensional scaling (NMDS) of the macro­ invertebrate community composition at three increasing distances from the cit­ rus trees (Trunk, 20 cm; Middle, 150 cm; Row, 350 cm) (N = 9 per treatment). (b) Interpretation in terms of taxa of the NMDS diagram in (a). For each treat­ ment, the ellipse encompasses all sam­ ples. Species with at least 5% contribution to the dissimilarity among treatments (SIMPER analyses, P < 0.05) are sketched (not to scale; clockwise di­ rection: Messor barbarus, Pheidole pallid­ ula, Formica rufibarbis, Lasius grandis, Armadillidium vulgare, and Aleochar­ inae). Other species contributing less to dissimilarity (amounting up to ~95 % total dissimilarity between treatments) are indicated in the NMDS region in which they exert influence (all species in a list connected to a point are approxi­ mately around this point). Taxonomic groups are indicated in parenthesis (see Fig. 2 for details). Stress (goodness of fit) and k dimensions (axes of the best ordination fit) based on Bray-Curtis dissimilarity are provided in the top- right corner. The macroinvertebrate community composition was weakly corre­ % of total individuals, vs 24.6 % in Trunk). Erythraeidae mites (Acari; lated with the plant community composition (Mantel test, r = 0.18, P = 1− 3 mm body width) and cockroaches (Blattodea) were also more 0.006). abundant in Middle and Row samples. Excluding ants, total macro­ The macroinvertebrate community of Trunk samples was charac­ invertebrate abundance was higher in Trunk samples (1666 ± 260 in­ terized by Aleocharinae staphylinid beetles and the woodlice Arma­ dividuals m− 2) than in Middle and Row samples (376 ± 46 individuals dillidium vulgare (Fig. 1b). Other species of staphylinid beetles m− 2) (Fig. 2). (Xantholinus sp. and Tachyporinae), woodlice (Porcellio sp.), the spiders Dysdera crocata and Meioneta rurestris, the centipedes Geophilus sp. and 3.3. Stable isotope signatures of soil, plants, and macroinvertebrates Lithobius sp., the earwig Euborellia annulipes, the millipede Polydesmus sp., and earthworms (Amynthas corticis, Dendrobaena sp., and Aporrec­ There were no differences in soil δ13C among treatments (pseudo- todea trapezoides) were also characteristic of Trunk samples (Fig. 1b). F2,16 = 2.03, P = 0.16), but δ15N decreased at increasing distances from The macroinvertebrate community of Middle and Row samples was the tree trunk (pseudo-F2,16 = 104.53, P < 0.001) (Fig. 3a, b). characterized mostly by the presence of several ant species (Messor Soil organic matter content (%) varied similarly, being 7.7 ± 0.6, 4.3 barbarus, Pheidole pallidula, Formica rufibarbis, Lasius grandis, and ± 0.6, and 3.6 ± 0.2 in Trunk, Middle, and Row samples, respectively (N Hypoponera eduardi) and the beetle Oochrotus unicolor, which is indeed a = 4 per treatment). myrmecophilous beetle associated to M. barbarus (Bernard, 1967) Plants had different δ13C and δ15N values among treatments (pseudo- (Fig. 1b). F2,43 = 14.19, P < 0.001; pseudo-F2,43 = 7.88, P = 0.0015; respectively). Trunk samples had higher abundances of staphylinid beetles (Cole­ Trunk plants had the lowest δ13C and the highest δ15N levels, but no optera: Staphylinidae), woodlice (Isopoda), centipedes (Chilopoda) and differences in isotopic signatures were found between Middle and Row millipedes (Diplopoda) (Fig. 2). Earthworms (Oligochaeta) and snails plants (Fig. 3a, b). (Gastropoda) were also relatively more abundant in Trunk samples. Table A.3 contains the complete dataset of stable isotope signatures Conversely, ants were very abundant in Middle and Row samples (80.2 for soil and plants. 4 C. Hernández-Castellano et al. Pedobiologia - Journal of Soil Ecology 87-88 (2021) 150751 Fig. 2. Macroinvertebrate abundance (individuals⋅m− 2) (mean ± SE) at three increasing distances from the citrus trees (Trunk, 20 cm; Middle, 150 cm; Row, 350 cm) (N = 9 per treatment). Key: Col:Sta, Coleoptera: Staphylinidae; Col, Coleoptera (other groups); Iso, Iso­ poda; Chi, Chilopoda; Ara, Araneae; Aca, Acari (1-3 mm body width); Bla, Blattodea; Der, Dermaptera; Oli, Oli­ gochaeta; Dip, Diplopoda; Lep, Lepi­ doptera; Gas, Gastropoda. Different letters indicate significant differences between treatments (P < 0.05). Note the log scale used in the y-axis. Fig. 4. (a) δ13C and (b) δ15N values (mean ± SE) of macroinvertebrates at three increasing distances from the citrus trees (Trunk, 20 cm; Middle, 150 cm; Row, 350 cm). Soil isotopic δ13C and δ15N is used as the isotopic baseline, and therefore included in the models as covariates. Different letters indicate sig­ nificant differences between treatments (P < 0.05). the observed differences in δ13C across treatments vanished (pseudo- F1,124 = 2.13, P = 0.14) (Fig. 5a). In contrast, the unique species to Trunk sites maintained the isotopic differences in δ13C in comparison to unique species to Middle + Row samples (pseudo-F1,75 = 9.11, P = 0.02) (Fig. 5b), as in the entire macroinvertebrate community. No differences in δ15N were found, neither for the common species (pseudo-F1,124 = Fig. 3. (a) δ13C and (b) δ15N values (mean ± SE) of soil and plants at three 3.70, P = 0.057) nor for the unique species (pseudo-F1,75 = 0.43, P = increasing distances from the citrus trees (Trunk, 20 cm; Middle, 150 cm; Row, 0.51) (Fig. 5c, d). Excluding ants from the analysis (unique to Middle + 350 cm). Different letters indicate significant differences between treatments (P Row samples) yielded identical results. < 0.05). Table A.4 contain the complete dataset of stable isotope signatures for macroinvertebrates. Macroinvertebrate isotopic signatures were corrected by their iso­ topic baselines (soil δ13C and δ15N as covariates). Macroinvertebrates 3.4. Food web structure had different δ13C among treatments (pseudo-F2,207 = 7.25; P < 0.001). Trunk macroinvertebrates were depleted in 13C with respect to Middle As the Middle and Row macroinvertebrate communities were similar and Row communities (t = 3.11, P = 0.002; t = 2.17, P = 0.034, in species composition and isotopic signatures, and both differed from respectively), but there were no differences between Middle and Row the communities of the Trunk samples, we delimited two food webs: one macroinvertebrates (t = 0.61, P = 0.56) (Fig. 4a). In contrast, no dif­ for Trunk, at 20 cm from the tree trunks, and one for Middle + Row, at ferences in δ15N were found among treatments (pseudo-F2,207 = 0.22, P 150/350 cm. = 0.81) (Fig. 4b). There was a clear spatial structure in macroinvertebrate soil food We subsequently pooled together Middle and Row samples (Middle webs at one-meter scale, with distinct species near the tree trunks + Row thereafter). Twenty-six per cent of the analysed macro­ showing different isotopic signatures than those species located farther invertebrate species (representing 31 % of the abundance) were common away (Fig. 6). These food webs, though, were partially connected by to Trunk and Middle + Row samples (Fig. A.2). In the common species, common species (26 % of total species), which showed no differences in 5 C. Hernández-Castellano et al. Pedobiologia - Journal of Soil Ecology 87-88 (2021) 150751 whereas there was a gradual pattern of increase in δ15N from detritivores to predators, with overlap among trophic guilds (Fig. A.3). 4. Discussion The composition and trophic structure of the macroinvertebrate community of the citrus grove varied spatially, as indicated by the presence of different species assemblages and shifts in isotopic signa­ tures. We were able to distinguish two partially compartmentalized food webs in the study area, which differed in the abundance and identity of detritivores and predators as well as the potential sources of energy used. Our results showed that even within a single ecosystem, at scales as small as one meter, there can exist consistent differences in food web composition and structure. The most parsimonious ecological explana­ tion behind the observed pattern is that spatial variability in organic matter content and water availability drives changes in community composition (both in abundance and identity) and trophic structure (energy pathways and functional groups). 4.1. Spatial variability in environmental factors Fig. 5. (a, b) δ13C and (c, d) δ15N values (mean ± SE) of (a, c) common species Fertilization with organic manure beneath the citrus canopies has (present across treatments) and (b, d) unique species (present in a single two notable effects. First, the soil content of organic matter was higher treatment) of macroinvertebrates between Trunk (20 cm from the citrus trees) there and decreased outwards. Second, as the fertilizer was enriched in and Middle + Row (onwards). Soil isotopic δ13C and δ15N is used as the isotopic 15 baseline, and therefore included in the models as covariates. Different letters N compared to the soil (Fig. 7a), values of δ15N in the soil display the indicate significant differences between treatments (P < 0.05). same pattern as that of the organic matter content. Signatures of δ15N in plants are assumed to reflect those of bulk soil (Marshall et al., 2007) and that is what we found. Plants near the tree trunks had lower δ13C values than plants from the other treatments. This was likely due to the effect of irrigation, as water-stressed plants are generally enriched in 13C because of an increase in water-use efficiency (Marshall et al., 2007). The difference in δ13C between Trunk and Middle/Row plant communities was further broadened by the presence in the latter of Cynodon dactylon, a C4 grass, since C4 plants present higher δ13C values than C3 plants (~ − 14‰ versus ~ − 27‰, respec­ tively, Marshall et al., 2007) (see δ-values weighted by biomass with and without C4 plants in Table A.3). In an agricultural semiarid ecosystem, organic matter and water are expected to be the main limiting factors for soil macrofauna, and therefore the main drivers of small-scale spatial variability in soil food webs. Other soil properties such as structure, porosity and pH, among others, are also important in determining soil communities (van Straalen and Verhoef, 1997v; Bardgett, 2005) but probably play a secondary role here. They might be important in ecosystems where other processes, Fig. 6. The spatial structure of soil food webs at one-meter scale. The Trunk such as animal tramping or local concentration of pesticides, generate food web (20 cm distant from the citrus trees) had distinct macroinvertebrate spatial variability in physicochemical soil properties, eventually leading communities with different isotopic signatures (solid black circles) than the to spatial variability in soil food webs. Middle + Row food webs (150/350 cm distant) (open black circles). Both food webs were partially connected by common species (26 % of total species) (gray 4.2. Spatial variability in macroinvertebrate community composition circles) showing no differences in isotopic signatures between food webs. The macroinvertebrate community was significantly related to the isotopic signatures between food webs (Fig. 6). plant community, but the correlation was low. This result does not imply In all food webs, the vast majority of δ13C signatures of macro­ causality but simply shows that macroinvertebrates and plants experi­ invertebrates were similar to those of soil (Fig. 7a, c), but in Trunk food ence a common spatial variability in environmental conditions. web there was a large group of species with lower δ13C values than the The macroinvertebrate community beneath the canopy differed from soil (Fig. 7a). As a whole, macroinvertebrates spanned more than 4 that located farther away, mainly in having more detritivores (Aleo­ trophic levels both in the Trunk and in the Middle + Row webs (Fig. 7b, charinae staphylinid beetles, woodlice, and earthworms) and predators d). (centipedes). This is likely linked to the fact that decomposer commu­ We established nine trophic groups in Trunk food web and eight nities are most developed in organic soils (Anderson, 1975; Bengtsson trophic groups in Middle + Row food web based on the isotopic signa­ et al., 1998), and benefit from organic farming. Thus, the organic fer­ ture of the macroinvertebrate species (Table A.5). The Trunk food web tilizer applied beneath the tree canopies may benefit faunal decompo­ (Fig. 7b) consisted of five trophic groups of detritivores, one group of sition and predation via a bottom-up effect (Birkhofer et al., 2008). herbivores, one group of omnivores, and two groups of predators. The Hygrophilous millipedes (also detritivorous) and herbivorous snails Middle + Row food web (Fig. 7d) had two trophic groups less of detri­ were also more abundant near the tree trunks. During dry periods, water tivores and one more of predators than the Trunk food web. availability in irrigated areas improves resource quality (plants) and No difference was found in the δ13C signature among trophic groups, therefore boosts herbivore populations (Pérez-Fuertes et al., 2015). 6 C. Hernández-Castellano et al. Pedobiologia - Journal of Soil Ecology 87-88 (2021) 150751 Fig. 7. δ13C and δ15N (mean ± SE) values of macroinvertebrates, plants, soil, and other environmental items for the (a) Trunk (20 cm distant from the citrus trees) and (c) Middle + Row (150/350 cm distant) food webs. Vertical (for mean δ13C-values) and horizontal (for mean δ15N-values) lines are plotted for soil (solid) and plant (dashed) signatures. Note the thinner vertical dashed line for leaves of citrus trees. (b, d) The same food webs, with macroinvertebrates of a given trophic guild regrouped into ellipses of isotopically similar species, representing trophic groups of detritivores (solid line), herbivores (dotted line), omnivores (dot-dash line), and predators (dashed line). Each trophic group is labelled (e.g., d1 = trophic group 1 of detritivores). δ15N value for each trophic level (TL) is indicated on the y-axis. Key for graphics b) and d): solid black circle, Araneae; solid black square, Isopoda; solid black triangle, Staphylinidae; solid black diamond, Gastropoda; open black circle, Chilopoda; open black square, Diplopoda; open black triangle, Blattodea; open black diamond, Oligochaeta; solid grey circle, Formicidae; solid grey square, Der­ maptera; solid grey triangle, Coleoptera; Acari (1-3 mm body width) not visible, pointed with an arrow. Conversely, ants, which are typically found in relatively dry environ­ near the tree trunk enhance plant quality, fostering the exploitation of ments (Hölldobler and Wilson, 1990), were more abundant farther the grazing energy channel. In turn, beneath the canopy, the presence of away. citrus leaves and the tree root system may foster the consumption of other food components not analysed here, such as decomposed plant material, root exudates and mycorrhizal fungi, which have higher δ13C 4.3. Stable isotope signatures of macroinvertebrates signatures than fresh plant material (Hobbie et al., 2001; Boström et al., 2008, Pollierer et al., 2009) and are exploited by multiple soil organisms The isotopic composition of the environment affects the isotopic (Chahartaghi et al., 2005; Klarner et al., 2013). composition at the base of the food web, which in turn affects the entire The analysis of bulk stable isotopes of soil animals provides infor­ community. Therefore, when comparing food webs, it is necessary to mation about potential food resources, that is, we can infer their diet distinguish between differences in isotopic signatures due to shifts in based on the isotopic composition of the other components of the food trophic structure from those due to changes in the isotopic baseline itself web (see the isotopic map in Potapov et al., 2019). Whilst the interpre­ (Post, 2002). While the isotope signatures of less-mobile organisms may tation of the stable isotopic values of macroinvertebrates near the tree closely reflect those of their immediate environment, an increase in the trunk is consistent with our hypotheses (i.e., they might be feeding on size of an organism’s foraging area is expected to lead to the buffering of plants or fungi), a definitive proof would require advanced techniques some of the environmental isotopic variability (Bearhop et al., 2004; such as isotope labelling or analyses of compound-specific stable iso­ Brewitt et al., 2015). topes of fatty acids (Pollierer et al., 2007, 2012). By correcting isotopic signatures of macroinvertebrates by their isotopic baseline, we were able to determine that macroinvertebrates near the tree trunks were depleted in 13C in comparison to those located 4.4. Food webs in space: delimitation and main characteristics farther away. This difference was mediated by unique species, that is, species only present near the trunk. In contrast, species foraging across We defined two food webs at the study site. The composition of the the studied area integrated the spatial isotopic variability. This suggests macroinvertebrate community near the tree trunks was different from that changes in macroinvertebrate community composition (mainly that located above one meter away, largely because of a higher abun­ through the addition of new species) near the tree trunk drive the dance of detritivores and predators. In addition, Trunk communities exploitation of an energy channel which is not exploited by the more seem to rely more on plant-based resources, as demonstrated by lower distant organisms. We hypothesize that nutrient and water subsidies baseline-corrected δ13C-values in the macroinvertebrate community. 7 C. Hernández-Castellano et al. Pedobiologia - Journal of Soil Ecology 87-88 (2021) 150751 The two food webs were thus compartmentalized, albeit partially con­ overlap between trophic groups (p1-p2) and trophic niches of species nected by the 26 % of total species common to both communities (rep­ (mainly spiders and chilopods). Besides, the mean 15N enrichment in resenting 31 % of the abundance). predators is 3.1‰ that of detritivores (versus 2‰ in Trunk). The similarity in δ13C values between trophic guilds and their close relation to the isotopic soil signature in the two food webs suggests the 4.6. Concluding remarks presence of a generalized pathway of energy flux from soil organic matter to detritivores to predators (e.g., Halaj et al., 2005). Enrichment In the studied agroecosystem, the gradient in soil organic matter in 13C relative to plant material is a typical feature in soil food webs (also content (from organic fertilizer) and water availability (from irrigation) known and “detrital shift”) and is thought to be related to the acquisition from the tree trunks outwards was reflected in the isotopic signatures of of 13C-enriched microbial biomass (saprotrophic fungi and bacteria) both soil and plants. This spatial variability in environmental factors is from soil organic matter (Potapov et al., 2019). However, the wide range thought to be the main driver of changes in food web structure. These of δ13C (5.0‰ ± 0.2 for the two food webs) entails greater compart­ subsidies of detritus and water may benefit decomposers and predators mentalization stemming from multiple potential pathways between via a bottom-up effect and also through the exploitation of plant-based primary production and processed soil organic matter (Schmidt et al., resources near the tree trunks, resulting in the creation of a food web 2004; Pollierer et al., 2009). that is distinct and predominantly independent from that located just In contrast, there was a pattern of 15N enrichment from detritivores one meter away. to predators. This means that classifying macroinvertebrates into a priori Thus, the present study shows that trophic guilds is useful for understanding food web structure. However, there was a large degree of intraguild variability, as reflected by the δ15N a) spatial variability in macroinvertebrate soil food webs can be present range in detritivores (4.9 ± 0.1‰) and predators (5.6 ± 1.2‰). Thus, at a scale as small as one meter if spatial variability in environmental such classification is only useful when the taxonomic resolution is high factors is high enough, and (e.g., Mestre et al., 2013). The high intraguild variability and the overlap b) within-habitat heterogeneity should be taken into account when between trophic guilds corroborates the idea that soil food webs are determining sampling scales and food-web-related properties and composed of a trophic continuum rather than of well-defined trophic ecosystem processes. levels (Scheu and Falca, 2000; Ponsard and Arditi, 2000), and contain functional groups within trophic guilds (Oelbermann and Scheu, 2010). Declaration of Competing Interest 4.5. Food webs in space: trophic groups of detritivores and predators The authors declare that they have no known competing financial Loboptera decipiens (Blattodea) appears to be the primary decom­ interests or personal relationships that could have appeared to influence poser at the base of both food webs (d1; Fig. 7b and d), with isotopic the work reported in this paper. values like those of soil. Instead, the remaining detritivores have higher δ15N -values, seeming to act as secondary decomposers, that is, feeding Acknowledgments on microbially processed organic matter (d2–d5; Fig. 7b and d). Among these, staphylinid beetles had the lowest δ13C values (below soil δ13C) We thank Tancredi Caruso and two anonymous reviewers for their (d3-d4, Fig. 7b, d), suggesting that they may rely on mycorrhizal fungi, constructive comments. We thank the following people for their assis­ which are enriched in 13C and 15N relative to fresh plant material tance in animal identification: JA Barrientos and L Mestre (Arachnida), (Hobbie et al., 2001). While in the Trunk food web this distinctive en­ MJI Briones (Oligochaeta), V Bros (Gastropoda), J Marshall (Blattodea), ergy channel is well developed (Fig. 7b), in Middle + Row food web it is A Serra (Myriapoda), and J Muñoz-Batet (Coleoptera). Pau Comes pro­ marginal (Fig. 7d). The remaining detritivores had higher δ13C values vided valuable assistance with the isotopic analyses. We thank MJ and were mainly represented by millipedes and earthworms (only in Anderson for advice with PERMANOVA analyses, and L Higgins for Trunk food web; d3 and d5, respectively, Fig. 7b) and isopods (d2, d5, revising the English version. We are grateful to Núria Cañellas for Fig. 7b; d2, d3, Fig. 7d), potentially feeding on humified soil organic permission to conduct field research on her land. This study was funded matter and 15N -enriched detritus such as faecal material, which is also by the Ministerio de Ciencia y Tecnología - FEDER (Spain) (CGL2010- enriched in 13C through microbial processing (DeNiro and Epstein, 18182/BOS) and supported by a collaboration grant from the Ministerio 1978). 13C enrichment in millipedes and woodlice can also be related to de Educación, Cultura y Deporte (Spain) (8031397). the presence of inorganic carbon in their cuticle (Maraun et al., 2011; Data is available upon request. Potapov et al., 2019). There were two distinct groups of predators in the Trunk food web Appendix A. Supplementary data (Fig. 7b). The first group (p1) was composed of predatory staphylinids beetles and lyniphiid spiders (average body length 2 mm; Nentwig et al., Supplementary material related to this article can be found, in the 2016), potentially exploiting the distinctive energy channel preying online version, at doi:https://doi.org/10.1016/j.pedobi.2021.150751. upon other staphylinids. The second group (p2) is formed of chilopods and bigger spiders (average body length 10 mm; Nentwig et al., 2016), References potentially preying on secondary decomposers below them. Since the predator group p2 is one trophic level above p1, intraguild predation Altaba, C.R., Ros, J. (Eds.), 1991. Invertebrats no artròpodes. Història Natural dels Països must also be considered (McNabb et al., 2001; Mestre et al., 2013), with Catalans, vol. 8. Enciclopèdia Catalana, Barcelona. Anderson, J.M., 1975. The enigma of soil animal species diversity. In: Vanek, J. 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