Trophic Diversity in a Mediterranean Food Web—Stable Isotope Analysis

Basic and Applied Ecology 13 (2012) 587–596

Trophic diversity in a Mediterranean food web—Stable isotope analysis of an ant community of an organic citrus grove Christian Platnera,b,∗, Josep Pinol˜ c,d, Dirk Sandersa,e, Xavier Espadalerc,d aJ.F. Blumenbach Institute of Zoology and Anthropology, Animal Ecology, Georg August University of Göttingen, Germany bExperimental and Systems Ecology, University of Bielefeld, Germany cCREAF, Cerdanyola del Vallès 08193, Spain dUnitat d’Ecologia, Univ Autònoma Barcelona, Cerdanyola del Vallès 08193, Spain eInstitute of Ecology and Evolution, University of Bern, Balzerstr. 6, 3012 Bern, Switzerland

Received 23 March 2012; accepted 16 September 2012

Abstract

Ants as generalist predators and mutualists of herbivores can play an important role in relative stable agroecosystems like plantations. The categorization of the diverse life strategies and traits into ecological groups like trophic levels is essential for a better understanding of food web structures and a better prediction of changes in communities. Stable isotope technology provides simultaneously detection of trophic levels and the ultimate C source of many species. We studied a highly diverse Mediterranean ant community in an organic citrus grove in Tarragona, NE Spain, and analyzed stable isotope contents of 17 species of ants together with dominating plants and important spider and aphid species to establish trophic guilds and detect seasonal changes. The results revealed significant differences between species spanning over a huge range in δ15N-values of at least 10.67‰ which is only comparable to a Peruvian tropical forest with a much higher species diversity. The trophic levels of ants reflected most of previous knowledge on predaceous vs. plant feeding habits. Messor harvester ants and Camponotus species had the lowest δ15N-values. Aphids, smaller spider species, and most other ant genera, including the dominating species Formica rufibarbis and Lasius grandis, had intermediate δ15N-levels. The large spider Dysdera crocata and the typical Mediterranean ant Pheidole pallidula had higher δ15N-values, but two specialized predatory ants with very tiny workers had the highest trophic level. We found unexpectedly high δ13C-values with a high seasonality for several ground-living ant species. The possible role of soil fauna as a second main food resource besides the most commonly analyzed green food chain is discussed. Our results support the hypothesis that the strong seasonality intrinsic to Mediterranean climate and the high heterogeneity of different plant resources and microclimatic conditions in the organically managed plantation are reflected by a notably high trophic diversity of the ant community.

Zusammenfassung

Ameisen können als generalistische Prädatoren und Symbiosepartner von Pﬂanzenfressern eine wichtige Rolle in relativ sta- bilen Agrar-Ökosystemen, wie z.B. Plantagen und Obstwiesen, spielen. Die Einteilung von Arten mit diversen Lebensstrategien und Eigenschaften in ökologische Gilden, beispielsweise in trophische Gruppen, ist von zentraler Bedeutung für ein besseres

∗Corresponding author. Current address: Experimental and Systems Ecology, University of Bielefeld, Universitätsstr. 25, D-33615 Bielefeld, Germany. Tel.: +49 521 106 5562; fax: +49 521 106 6038. E-mail addresses: [email protected], [email protected] (C. Platner).

1439-1791/$ – see front matter © 2012 Gesellschaft fur¨ Okologie.¨ Published by Elsevier GmbH. All rights reserved. http://dx.doi.org/10.1016/j.baae.2012.09.006 588 C. Platner et al. / Basic and Applied Ecology 13 (2012) 587–596

Verständnisvon Nahrungsnetzen und bessere Voraussagenvon Änderungen in den Lebens-Gemeinschaften. Die Analyse stabiler Isotope bietet die Möglichkeit der gleichzeitigen Aufdeckung trophischer Ebenen und der ursprünglichen Kohlenstoff-Quellen vieler Arten. Wir haben die hoch diverse mediterrane Ameisengemeinschaft einer ökologisch bewirtschafteten Zitrus-Plantage untersucht und die natürlichen Gehalte stabiler Isotope von 17 Ameisenarten zusammen mit dominanten Pflanzen und wichtigen Spinnen- und Blattlausarten gemessen, um trophische Gilden zu etablieren und saisonale Veränderungen zu entdecken. Die Ergebnisse zeigen mit etwa 11‰ eine enorme Bandbreite der δ15N Werte der Ameisen; so viel wie bisher nur für einen Regenwald in Peru mit einer viel größeren Artenzahl beschrieben wurde. Die trophischen Ebenen der Ameisen decken sich gut mit den für viele Arten bereits beschriebenen zoo- und phytophagen Ernährungsweisen. Ernteameisen der Gattung Messor und Camponotus- Arten hatten die niedrigsten δ15N Werte. Blattläuse, die kleineren Spinnenarten und die meisten omnivoren Ameisen wie die dominanten Arten Formica rufibarbis und Lasius grandis hatten mittlere 15N Gehalte. Die große Spinne Dysdera crocata und die im Mittelmeer-Gebiet häufige Ameise Pheidole pallidula hatten deutlich höhere δ15N Werte, doch die höchste trophische Ebene bildeten zwei spezialisierte räuberische Ameisen mit sehr kleinen Arbeiterinnen. Für einige Ameisenarten der Bodenoberfläche fanden wir unerwartet hohe δ13C Werte mit einer starken jahreszeitlichen Änderung. Wir diskutieren die mögliche Rolle der Bodenfauna als zweite Haupt-Nahrungsquelle neben der bisher hauptsächlich analysierten ,,grünen” Nahrungskette. Die Ergebnisse unterstützen die Hypothese, dass sich die starke Saisonalität des mediterranen Klimas und die große Heterogenität vielfältiger Pflanzenarten und mikroklimatischer Bedingungen auf der organisch bewirtschafteten Mandarinenbaum-Wiese in einer bemerkenswert hohen trophischen Vielfalt der Ameisengemeinschaft ausdrückt. © 2012 Gesellschaft fur¨ Okologie.¨ Published by Elsevier GmbH. All rights reserved.

Keywords: Formicidae; Araneae; Seasonal effects; Generalist predators; Agroecosystem; Clementine orchard; Bird-exclusion

Introduction tropical leaf litter ants functional richness and diversity are closely related to species diversity (Bihn, Gebauer, & Brandl, The categorization of diverse life strategies and traits into 2010). ecological groups like trophic levels is essential for a better Understanding the structure and function of the ant understanding of food web structures and a better predic- community and the niche-separation of many coexisting ant- tion of changes in communities. Ascribing species to trophic species is of fundamental interest (Andersen, 2000). Analysis guilds is a difficult task in arthropod-dominated terrestrial of stable isotope ratios (13C/12C, 15N/14N) has been shown to communities, where generalist predators and omnivores are be a powerful tool for the study of terrestrial and aquatic food commonplace and plant-based and detritus-based food webs webs (Albers, Schaefer, & Scheu, 2006; Post, 2002; Schmidt, are intermixed. Ants as eusocial insects comprise a large Curry, Dyckmans, Rota, & Scrimgeour, 2004). In combina- portion of terrestrial animal biomass and they are often sym- tion with experimental manipulation stable isotope analysis biotic partners of plants, fungi or herbivores (Hölldobler & was useful in disentangling the role of ants in complex food Wilson, 1990). They perform important ecosystem functions webs (Feldhaar, Gebauer, & Blüthgen, 2010; Sanders & and can be keystone species in many food webs. As soil engi- Platner, 2007). Most stable isotope studies have been con- neers ants modulate bottom-up forces for the whole food web ducted in central European or tropical systems but very few by altering the resource supply to other species (Cameraat, in the Mediterranean. In this context it is worth mentioning Williott, Compton, & Incoll, 2002; Frouz, Holec, & Kalcik, the study of Ottonetti, Tucci, Chelazzi, and Santini (2008) in 2003). But they also directly affect above- and belowground an Italian agroecosystem of olive trees in which they inferred populations of different trophic levels including plants, by the trophic role of five species of ants and of some other top-down control via predation, interference and disturbance, organisms of the community using stable isotopes. Here we and tending species of aphids and coccids. Most ant species study a highly diverse Mediterranean ant community in an are omnivores, being able to prey on a wide range of other organic citrus grove in NE Spain with ca. 20 species of ants. invertebrates from the herbivore as well as from the decom- The trophic status of only three of these species has been stud- poser subsystem (Hölldobler & Wilson, 1990; Passera & ied so far using stable isotopes, and only one of them in the Aron, 2005; Platner, 2006; Scheu 2001). They also take up Mediterranean (Fiedler, Kuhlmann, Schlick-Steiner, Steiner, nutrients from plants indirectly by trophobiosis with phloem- & Gebauer, 2007; Ottonetti et al., 2008; Schuch, Platner, & feeding insects (Stadler & Dixon, 2005). Feeding groups Sanders, 2008). In particular, for some of the most important (guilds), consisting of species which depend on similar food ant species in Mediterranean citrus plantations, Lasius gran- resources, are the basic entities in food webs. These guilds are dis, Pheidole pallidula and Plagiolepis pygmaea, there is no assumed to be functional equivalent and therefore exert sim- available information of their relative trophic level within the ilar top-down and bottom-up forces within the community. community. In general the relationship between species and functional The aim of our study is the examination of the trophic diversity is poorly understood (Naeem, 2002), but at least for position of all ant species in a diverse Mediterranean food C. Platner et al. / Basic and Applied Ecology 13 (2012) 587–596 589 web under organic farming using stable isotope analysis. For analyze the main effect of species regardless of bird exclusion most of the studied ant species we provide for the first time and stratum in a second step for all species. data of their natural isotopic contents, together with plants as the base line and other co-occurring members of the commu- Stable isotopic analysis nity (aphids, spiders, and isopods). In particular, we tested the following two hypotheses: (1) a higher number of ant We analyzed specimens captured in late spring (31st May) species will translate into a higher number of trophic groups, and late summer (20th August) of 2008. In the lab, ants revealed by different N and C stable isotopic signatures. In (Hymenoptera: Formicidae) were identified to species level particular, we expect to find a higher number of trophic groups using keys in Collingwood (1978) and Seifert (1992, 2007).A in the studied citrus grove than in the better known ecosys- selected subset of ants, together with some individuals from tems of Central Europe; (2) the strong seasonality intrinsic dominant spider, isopod, and aphid species were dried for to Mediterranean climate will probably cause changes in the ◦ three days at 60 C and weighted into tin capsules in January diets of species, which should be reflected in differences in 2009. Bulk material of leaves (see below) from Citrus trees, their isotopic signature between spring and summer. the grass Hordeum murinum L., the herb Parietaria officinalis L. and the C4-grass Cynodon dactylon (L.) Pers. were dried and homogenized to a fine powder prior to analysis. Ratios of 13C and 15N were estimated by a coupled system consist- Material and methods ing of an elemental analyser (NA 1110, Carlo Erba, Milano, Italy) and a gas isotope mass spectrometer (Conflo III Inter- face and Deltaplus, both Finnigan MAT, Bremen, Germany) Study site and sampling details at the University of Göttingen (Centre for Stable Isotope Research and Analysis). Isotope abundances are expressed The study site was a plantation of citrus trees located as relative differences between samples and a reference in at La Selva del Camp (Tarragona, NE Spain; 41◦1307N, delta values (δ‰). For the content of 13C Pee Dee Belemnite 01◦0835E). Climate is Mediterranean, with a rainy spring limestone and for content of 15N atmospheric N were used and autumn and a dry winter and summer. The grove 2 as conventional references. Acetanilide (C H NO, Merck, consisted of ca. 300 Clementine trees grafted on the hybrid 8 9 Darmstadt, Germany) was used for internal calibration with rootstock Carrizo citrange (Poncirus trifoliata (L.) Raf. x Cit- a mean standard deviation <0.1‰. rus sinensis (L.) Osb.). The grove complied with all organic As we did not take samples of plant material in 2008, we agriculture standards, i.e. no pesticides, fungicides or herbi- used plant material sampled in 2009. This potential shortcom- cides were applied and only organic manure was used as ing of the sampling design is probably not important, as plants fertilizer. Trees were regularly watered during dry periods. normally show a clear seasonal variation, but not a between- More details are provided in Pinol,˜ Espadaler, Canellas,˜ and year variation provided that water availability is similar, Perez (2009). which is the case in the irrigated citrus grove. To confirm The animal specimens used for this isotopic study came this hypothesis we analyzed Citrus leaves sampled in June from samples taken in a bird-exclusion experiment carried out 2009 and June 2010 and found no significant differences in in the study site in 2008 and 2009 (Mestre, Pinol,˜ Barrientos, the isotopic values (δ15N = 9.91 in 2009 and 9.80 in 2010; two Cama, & Espadaler, 2012). sample t-test (homogenous variance): t = 0.27, P = 0.80; In the canopy, samples were obtained using beating trays, (df=6) δ13C: −26.09 in 2009 and −25.39 in 2010; Welch two sample captured with entomological aspirators and immediately pre- t-test (inhomogenous variance): t = 0.822, P = 0.47). served in 70% ethanol. In the ground, we put two pitfall traps (df = 3.045) (7 cm depth and 5.5 cm diameter) beneath each tree; pitfalls were opened during 48 h, just after the canopy sample was Tests for effects of bird exclusion, stratum and taken. The pitfalls traps contained 20 mL of water with deter- sample storage gent and after the sampling time concluded 96% ethanol was added to obtain a final ethanol concentration of ca. 70%. The effects of bird exclusion on stable isotope contents of Bird-exclusion had a low impact on the arthropod com- ants were tested by a linear mixed model with bird-exclusion munity, both in tree canopies and in the ground; in particular, as a fixed effect and block as a random effect. This test was the exclusion of birds had no impact on the abundance and done independently for the most common ant species that composition of the ant assemblage in 2008 (Mestre et al., were sampled in sufficient numbers. Bird-exclusion had no 2012). We did not expect to find a different isotopic signa- impact on the isotopic contents of Lasius grandis, Formica ture in individuals from control and from bird-excluded trees, rufibarbis and Pheidole pallidula sampled on the ground and but this hypothesis had to be checked in advance. We tested in canopies neither in spring nor in summer (see Appendix the abundant ant species for these potential effects in a first A: Table S2). A paired t-test for δ15N- and δ13C-values of step to make sure that there were no effects of bird exclusion, ants from Lasius grandis and Formica rufibarbis captured stratum and storage on the isotopic content before we could on the ground and in the canopies showed no significant 590 C. Platner et al. / Basic and Applied Ecology 13 (2012) 587–596 differences between both strata (Appendix A: Table S3). This 20 result allowed us to merge all the sampled ant individuals (A) Spring per species, regardless of the sampling method or if they 18 came from bird-excluded or control trees. In doing so we are assuming that the result we obtained for these three most 16 abundant species can be extended to the rest of the community. For some species it was not possible to sample and 14 N analyze more than a few replicates. We report results from 5 Ppa 1 Tni single replicates from the genera Messor and Camponotus in 12 Lgr Fru Sol the Supplementary Material (Appendix A: Table S4), but we Ppy did not draw any conclusions from this single replicates. 10 Tse Specimens from pitfall traps as well as from beating trays were conserved in 70 vol.% ethanol. A comparison with con- 8 trol samples kept alive for a short period, frozen and dried Messor & Camponotus without any contact to solutions showed no significant dif- 6 -24 -23 -22 -21 -20 -19 -18 ferences (Platner et al., unpublished results). 20 (B) Summer Data analyses 18 Hed Pyr

To test for differences in the isotopic signature between 16 ant species we conducted two separate one-way ANOVAs 15 13 of δ N- and δ C-values using ant species as a ﬁxed 14 factor. The statistical analyses were conducted indepen- N Ppa Sol 5 1 dently for each season. To aggregate species into trophically 12 Lgr homogenous subgroups, we calculated subsets of δ15N- and Ppy 13 δ C-signatures which were not statistically different using 10 Fru Tse Tukey’s HSD with a 95% family-wise conﬁdence level. Csy Mst 15 13 To compare the δ N- and δ C-values in a subset of the 8 studied ant species between spring and summer we con- Cae ducted a Welch two-sample t-test. All statistical analyses 6 were conducted with R 2.13.0 (The R Foundation for Sta- -24 -23 -22 -21 -20 -19 -18 tistical Computing, 2011). 13C

Fig. 1. Mean δ15N- and δ13C-values for ant species in late spring (A) and in late summer (B). Error bars indicate 95% confidence intervals Results (species with no error bars were only analyzed once). Abbrevia- tions: Cae (Camponotus aethiops), Csy (Camponotus sylvaticus), The ant community Fru (Formica rufibarbis), Hed (Hypoponera eduardi), Lgr (Lasius grandis), Mst (Messor structor), Ppa (Pheidole pallidula), Ppy (Pla- We found a total of 18 ant species on the ground of the giolepis pygmaea), Pyr (Pyramica membranifera), Sol (Solenopsis citrus grove (Table 1). The most abundant species were Phei- sp.), Tni (Tapinoma nigerrimum), Tse (Tetramorium semilaeve). dole pallidula (53% of all individuals), Formica rufibarbis (13%), Lasius grandis (13%) and Plagiolepis pygmaea (7%). In terms of species importance measured as biomass, the most important species were Formica rufibarbis (47% of total ant General stable isotopic results biomass), Lasius grandis (17%), Pheidole pallidula (13%), Messor barbarus (8%) and M. structor (8%) (Appendix A: We could only conduct stable isotopic analyses of 17 of Table S1). the 18 ant species in the community, as the only sampled Of the 18 species obtained from the ground, seven species specimen of Cardiocondyla mauritanica was too small to be were also found in the canopies (Table 1). The most abundant analyzed alone. The results revealed a huge range in δ15N- species in the canopies were Lasius grandis (44% of the indi- values between species of at least 10.67‰ and a smaller one viduals), Formicarufibarbis (28%), and Plagiolepis pygmaea in δ13C-values of 4.87‰ (Fig. 1). The range of δ15N- and (21%). Measured as biomass, the most important species δ13C-values was higher in late summer than in late spring. were Formica rufibarbis (55% of the ant biomass), Lasius The species effect on isotopic values was highly significant grandis (30%), and Camponotus sylvaticus (9%) (Appendix both for δ15N- and δ13C-values in late spring and in late sum- 13 15 A: Table S1). mer (one-way ANOVA δ Cspring: F8,158 = 52.78; δ Nspring: C. Platner et al. / Basic and Applied Ecology 13 (2012) 587–596 591

Table 1. Abundance measured as number of captured individuals of ants from the ground (using 96 pitfall traps) and from the canopy (using 16 beating trays) in late spring and late summer.

Species Ground (pitfall traps) Canopy (beating trays) Spring Summer Total % Spring Summer Total %

Tapinoma nigerrimum 1 0 1 0.0 0 0 0 0.0 Camponotus aethiops 0 6 6 0.1 6 3 9 1.5 Camponotus foreli 0 1 1 0.0 0 5 5 0.9 Camponotus pilicornis 1 0 1 0.0 0 0 0 0.0 Camponotus sylvaticus 20 25 45 1.0 15 10 25 4.3 Formica ruﬁbarbis 223 383 606 13.3 80 86 166 28.4 Formica subrufa 2 2 4 0.1 0 0 0 0.0 Lasius grandis 479 105 584 12.8 166 88 254 43.5 Plagiolepis pygmaea 234 87 321 7.1 60 61 121 20.7 Cardiocondyla mauritanica 0 1 1 0.0 0 0 0 0.0 Messor barbarus 46 88 134 2.9 0 0 0 0.0 Messor bouvieri 1 0 1 0.0 0 0 0 0.0 Messor structor 55 121 176 3.9 0 0 0 0.0 Pheidole pallidula 760 1660 2420 53.2 1 3 4 0.7 Pyramica membranifera 0 24 24 0.5 0 0 0 0.0 Solenopsis sp. 10 57 67 1.5 0 0 0 0.0 Tetramorium semilaeve 52 99 151 3.3 0 0 0 0.0 Hypoponera eduardi 0 4 4 0.1 0 0 0 0.0

13 15 F8,158 = 103.55; δ Csummer: F11,114 = 19.88; δ Nsummer: Seasonal shift F11,114 = 54.75; all P < 0.0001). Considering only the most common ant species, there is a general shift from spring to summer to more negative δ13C- values (Fig. 2A); however, the shift was only significant for Ant trophic positions F. rufibarbis and P. pygmaea (Table 2). On the contrary, Lasius grandis did not change its δ13C-value, but increased Multiple comparisons following the above significant significantly its δ15N-value from spring to summer. ANOVAs allowed defining five clearly distinct trophic positions in the food web in spring (Fig. 1A; Appendix A: Table S4). Messor barbarus, M. structor, Camponotus sylvaticus and Formica subrufa had both low δ15N- and Comparison of ants with other members of the δ13C-values; three other Camponotus species (C. aethiops, community C. foreli and C. pilicornis), only analyzed once, had 15 13 δ N- and δ C-values that matched this group. Four In late spring, the comparison with simultaneously ana- 15 species showed a medium level of δ N-values, on aver- lyzed plant resources and other animals showed that ants age 2.79‰ higher than the former group: Lasius grandis, with medium δ15N-values are at the same level as the spiders Formica rufibarbis, Tetramorium semilaeve, and Plagi- Pelecopsis mengei (Simon) and Philodromus cespitum (Wal- olepis pygmaea. Of these, Lasius grandis and Formica ckenaer), both inhabiting tree canopies (Fig. 3A). Only the 13 rufibarbis hadalowδ C-value, Tetramorium semilaeve big spider Dysdera crocata C.L. Koch, active on the ground, a medium one, and Plagiolepis pygmaea the highest had clearly higher values of δ15N. Ants from the Messor and one, a single analysis of Tapinoma nigerrimum and of Camponotus group had even slightly lower values than aphids Solenopsis sp. showed an isotopic signature similar to sampled from citrus trees. The isopod Armadillidium sp. had P. pygmaea. Finally, Pheidole pallidula was separated an isotopic signature similar to ants from the lowest trophic 15 from other species by the significantly highest δ N- level (Messor & Camponotus; Fig. 3A). value. In late summer, three spiders commonly found in In late summer there appeared two further species of ants the canopies (Clubiona genevensis L. Koch, Theridion with clearly distinct isotopic signatures: Hypoponera eduardi mystaceum L. Koch, and Philodromus cespitum) had inter- and Pyramica membranifera. Both species had significantly mediate δ15N-values, similar to ants like Formica rufibarbis 15 higher δ N-values than all other species, but the latter had a or Tetramorium semilaeve. The epigaeic spider Dysdera 13 much higher δ C-value than the former (Fig. 1B; Appendix crocata had a δ15N-value higher than the rest of spi- A: Table S3). ders, but still smaller than the ants P. membranifera and 592 C. Platner et al. / Basic and Applied Ecology 13 (2012) 587–596

Table 2. Summary of Welch two-sample t-tests comparing the δ15N- and δ13C-contents of spring and summer samples for selected study species.

Species δ13C δ15N t df Pt df P

Formica ruﬁbarbis 11.21 79.0 <0.0001 1.13 69.9 0.26 Lasius grandis 1.10 26.9 0.28 −3.98 27.4 0.0005 Pheidole pallidula 10.53 47.3 <0.0001 0.53 29.4 0.60 Tetramorium semilaeve 1.47 10.6 0.17 −0.79 9.4 0.45 Plagiolepis pygmaea 1.64 7.5 0.14 −2.19 6.3 0.07 Dysdera crocata −0.47 2.56 0.68 −0.70 3.4 0.53 Armadillidium sp. −2.20 5.9 0.07 0.02 4.9 0.98 Citrus 6.14 4.0 0.004 −1.72 5.1 0.15 Parietaria ofﬁcinalis 0.94 4.7 0.39 −0.52 3.4 0.64

13 Hypoponera eduardi (Fig. 3B). Citrus trees had signiﬁcantly 13 (A) Ants Late spring lower δ C-values than in late spring (Table 2). Late summer

Ppa 12 Discussion

N Isotopic content of ants 5 1 Fru Ppy The ant community of the studied citrus grove covered an 11 15 Lga unexpected range of δ N-values of at least 10.67‰. Based on the assumption of a constant enrichment of 15N by 3.4‰ Tse per trophic level (Minagawa & Wada, 1984; Post, 2002), this difference would correspond to more than three trophic lev- δ15 10 els. Such a high range of N-values within a single ant -24 -23 -22 -21 -20 -19 -18 community has so far been found only in a Peruvian tropical 15 forest (Davidson, Cook, Snelling, & Chua, 2003), but with (B) Other organisms much fewer ant species in the Mediterranean citrus grove than 14 in the tropical forest (17 vs. 112 species). Together with the also relative high range of δ13C-values (4.78‰) these results Dys 13 highlight the high functional diversity of ants in a single community in Mediterranean climate. The organic farming of the

12 citrus grove provides the conditions for a functionally diverse

N community of ants, probably by a very diverse herb layer (>60 5 1 11 species) and a small-scale microclimatic gradient with zones regularly watered and shaded beneath citrus trees to warm

10 and dry soil surface between tree rows (Pinol,˜ Espadaler, & Cit Canellas,˜ 2012). We could analyze for some Messor and Camponotus 9 Arm Par species only a single replicate. However, these results match 8 with more abundant congeneric species. Some other species -30 -28 -26 -24 -22 -20 may have a bias due to undersampling, but we draw conclu- 13 C sions only from significant differences. Specimens sampled by beating were put directly in ethanol to ensure the same Fig. 2. Mean δ15N- and δ13C-values in late spring and in late summer for a selected subset of ants (A) and of other organisms (B). treatment as for pitfall traps. Isotopic values of separately Arrows indicate changes between the two seasons (bold when the sampled arthropods which were frozen without contact to difference is statistically significant for δ15Norδ13C, see Table 2). any solvent and after a short period for clearing the digestive Abbreviations are as in Fig. 1 (ants) and Fig. 3 (other organisms). tract showed no significant differences to samples stored in 70% ethanol for one month. Tillberg, McCarthy, Dolezal, and Suarez (2006) found a shift of δ13C-values after storage in C. Platner et al. / Basic and Applied Ecology 13 (2012) 587–596 593

20 Trophic positions of ants (A) Spring Ants 18 Other animals The analysis of stable isotope contents was able to differ- Spiders entiate several trophic groups of ants in the community. The 16 Plants results for these groups are, in general, in accordance with what was known so far for most ant species (Fiedler et al., 14 Dys 2007; Rojas & Fragoso, 2000). Consequently, these results N

5 for a well studied group of arthropods are good news for 1 12 the study of trophic groups in insects less well known (e.g. Pel Phi Psocoptera) or with highly variable feeding behaviour (e.g. Cit Ago 10 Hemiptera: Miridae; Jung & Lee, 2011). We discuss below Par Arm the characteristics of all identiﬁed ant trophic groups in this 8 Asp study. The analyzed species of the genera Messor and Campono-

6 tus are known as herbivores and reﬂected values at the base of -30 -28 -26 -24 -22 -20 -18 -16 the food web. The granivorous Messor species never climb 20 trees, but forage on the ground beneath trees and between tree rows, feeding on many plant species and therefore prob- (B) Summer 18 ably transferring different isotopic signals horizontally across sites. Camponotus ants are known to feed mainly on sugary

16 liquids, like honeydew and nectar. Some previous stable isotope studies showed low trophic levels of Camponotus ants Dys 14 (Blüthgen, Gebauer, & Fiedler, 2003; Fiedler et al., 2007;

N Ottonetti et al., 2008). Ottonetti et al. (2008) argued that 5 1 this pattern is typical for species feeding on N-poor and C- 12 Por Cit Clu Xys rich sources, such as nectar, phloem sap or honeydew. All Camponotus species studied in the present survey had sim- The 10 15 Arm ilar or even lower levels of δ N-values than citrus trees, Par and the aphids Aphis spiraecola and A. gossypii. This result, 8 together with their higher δ13C-value, suggests an additional food resource for Camponotus. Davidson et al. (2003) and 6 -30 -28 -26 -24 -22 -20 -18 -16 Fiedler et al. (2007) discuss the presence or lack thereof of endosymbiotic bacteria as a possible reason for differ- 13C ent isotopic values compared to other ant species feeding on Fig. 3. Mean δ15N- versus mean δ13C-values for the analyzed honeydew, like Lasius spp. and Formica spp. Without addi- species in late spring (A) and in late summer (B). Error bars indi- tional research on other components of the food web as well cate 95% confidence intervals. Only non-ant species are identified as feeding experiments this question could not be answered. with a three-letter abbreviation (points corresponding to ants can be Formica rufibarbis and Lasius grandis are well known identified by comparison with Fig. 1). Abbreviations: spiders: Clu aphid mutualists, but they are also predacious to meet their (Clubiona genevensis), Dys (Dysdera crocata), Pel (Pelecopsis par- nitrogen needs, especially during larval development (Paris allela), Phi (Philodromus cespitum), The (Theridion mystaceum), & Espadaler, 2010; Seifert, 2007). Both species had higher Xys (Xysticus cristatus); Isopoda: Arm (Armadillidium sp.), Por δ15N-values than Messor and Camponotus. This result is sim- (Porcellio sp.); Aphididae: Ago (Aphis gossypii Glover), Asp (Aphis ilar to that reported by Fiedler et al. (2007) for F. rufibarbis spiraecola Patch); plants: Cit (Citrus), Par (Parietaria officinalis). and Camponotus aethiops. Pheidole pallidula and Tetramorium semilaeve had less negative δ13C-values in late spring than F. rufibarbis and L. grandis. Both, P.pallidula and T. semilaeve feed on plant and animal remains, approximately half and half, and their eco- 95% ethanol, whereas Hobson, Gloutney, and Gibbs (1997) logical niches have been so far difficult to tell apart (Retana, and Barrow, Bjorndal, and Reich (2008) found similar to our Cerdá, & Espadaler, 1992). The present isotopic study seems study no differences after treatment with 70% ethanol. How- to provide some clues to differentiate these two species. On 15 ever, there is no influence of ethanol storage on N-content the one hand, P. pallidula had always a higher δ15N-value reported at all, allowing a direct comparison of specimens than T. semilaeve. This result is consistent with observa- δ13 stored in ethanol to other studies. Our C-values are at least tions of Retana et al. (1992), who showed that other ants fully comparable within this study, as all samples were treated are present at a higher percentage in the diet P. pallidula in the same way. than in the diet of T. semilaeve. Our own observations in the 594 C. Platner et al. / Basic and Applied Ecology 13 (2012) 587–596 studied grove confirmed that P. pallidula may predate on, for this observation, but it indicates a trophic differentiation or scavenge, other ant species, as nearly every nest entrance of two otherwise very similar species. F.rufibarbis has larger is marked by remains of arthropod carcasses, mainly of the workers and there are typically several remains of arthro- dominant harvester ant Messor barbarus. On the other hand, pod carcasses around its nests, whereas Lasius grandis is P.pallidula and T. semilaeve had different δ13C-values in late described as mainly trophobiotic. Schuch et al. (2008) found spring but nearly the same level in late summer, so the shift higher δ15N-values in F. rufibarbis than in Lasius niger, in carbon source between seasons is significant only for P. an ecologically similar species to L. grandis. However, we pallidula (Fig. 3). observed L. grandis preying on aphids and Psocoptera in The ant species with the tiniest workers, Plagiolepis pyg- June. With increasing demands of nitrogen due to larval maea (ca. 1–2 mm body length), is generally described as development in early summer, many Lasius species seem to nectarivorous (Bernard, 1967; Forel, 1920) and it also climbs prey to a greater extent on their tended aphids, as described up the citrus trees. It had an intermediate δ15N value between for Lasius flavus by Pontin (1978) and by O’Grady, Schmidt, T. semilaeve and P. pallidula, but was clearly enriched in and Breen (2010). 13C. Under water stress many herbs become more enriched in 13C than many Mediterranean trees with hard leaves and higher water use efficiency (Werner & Máguas, 2010). For Ants vs. other components of the community P. pygmaea Sameshima, Hasegawa, Kitade, Minaka, and Matsumoto (1999) have described endosymbiotic bacteria, When the isotope values of ants are compared with other phylogenetically distant from Blochmannia, which might components of the community, two main characteristics stand exist in any Camponotus species. However, nothing is known out. First, two ant species with tiny workers, Pyramica mem- on the potential role of these bacteria in their host ants’ matter branifera and Hypoponera eduardi, had the highest observed balance (Fiedler et al., 2007). δ15N of all studied species, including several species of spi- Finally, in late summer there were two species (not found in ders. the late spring sample) with very high δ15N-values, Pyram- Second, several ant species had the highest δ13C-values (P. ica membranifera with high δ13C-values, and Hypoponera membranifera, Solenopsis sp., Plagiolepis pygmaea, among eduardi, with intermediate δ13C-values. Their high δ15N- others) of all studied species. Moreover, in general, ants values identify them as specialized predatory ants. It is had higher values than most components of the community known that P. membranifera is, as all Dacetini, a special- (Fig. 3). No plant species is a good candidate as the main ized predator of Collembola and other small soft-bodied soil source for the diet of most ants, the only C4-grass Cyn- arthropods (Wilson, 1954). Similarly, the only found Poneri- odon dactilon with highly enriched δ13C-value (−11.7‰, nae, H. eduardi, is known to be a predator of small hypogaeic Appendix A: Table S3) is not that common to represent arthropods. a likely explanation for the high δ13C-values of most ant species. Isopods showed the highest δ13C-values of the non- ant species of the community (Fig. 3). Some mushrooms from Temporal shift in the isotope content of ants the grove had even δ13C-values of −21.44‰ (Brewitt et al., unpublished results) and soil organic matter is commonly We observed a shift to lower δ13C-values from late spring slightly enriched in 13C relative to plant material (Tiunov, to late summer in most abundant ants (Table 2; Fig. 2A). 2007), so other parts of the soil fauna, not analyzed yet, could This change is probably a consequence of a similar shift play an important role in the diet of this very small sized and in the δ13C-value of citrus trees and, maybe, of other plant mostly subterranean ant species. For example, collembolans species (Table 2; Fig. 2B). A shift in this direction is sur- seem to play an important role in the diet of Central European prising in a Mediterranean climate, as the summer drought ant species (O’Grady et al., 2010; Sanders & Platner, 2007; normally makes the plants to close their stomata and, conse- Schuch et al., 2008). However, the answer to this anomaly of quently, they get enriched, not depleted, in 13C. However, δ13C in ants will have to wait for a more detailed study of the this must not necessarily happen in the study grove as it entire biological community of this citrus grove. is well watered in summer, and evergreen sclerophylls like Citrus generally show minimal or no differences in carbon isotope discrimination between summer and winter (Werner & Máguas, 2010). Acknowledgments The significant shift to higher δ15N-value of Lasius grandis from spring to summer is unique among all studied ant Sincere thanks to Núria Canellas˜ for permission to con- species (Table 2; Fig. 2A). According to their isotope content, duct field research on her land. Klaus Hövemeyer and two Formica rufibarbis and L. grandis seemed to use very similar anonymous referees gave valuable comments and sug- food resources in spring. In late summer, while F. rufibar- gestions. This work has been supported by grants bis followed the general trend of decreasing δ13C-values, L. from MCYT-FEDER (CGL2007-64080-C02-01/BOS and grandis increased instead its δ15N. We have no explanation CGL2010-18182). C. Platner et al. / Basic and Applied Ecology 13 (2012) 587–596 595

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Available online at www.sciencedirect.com Supplementary Material

Table S1. Biomass (mg dry weight; abundance x mean body weight of the species) of ants captured from the ground (using 96 pitfall traps for 48h) and from the canopy (using 16 beating trays) in late spring (31. May 2008) and late summer (20. August 2008).

Subfamily Species Ground (pitfall traps) Canopy (beating trays) Spring Summer Total % Spring Summer Total % Dolichoderinae Tapinoma nigerrimum 0.1 0.0 0.1 0.0 0.0 0.0 0.0 0.0 Formicinae Camponotus aethiops 0.0 9.3 9.3 0.6 9.3 4.6 13.9 4.0 Camponotus foreli 0.0 1.1 1.1 0.1 0.0 5.5 5.5 1.6 Camponotus pilicornis 1.4 0.0 1.4 0.1 0.0 0.0 0.0 0.0 Camponotus sylvaticus 23.8 29.8 53.6 3.7 17.9 11.9 29.8 8.7 Formica rufibarbis 251.9 432.7 684.6 47.3 90.4 97.1 187.5 54.6 Formica subrufa 2.0 2.0 4.0 0.3 0.0 0.0 0.0 0.0 Lasius grandis 196.3 43.0 239.3 16.5 68.0 36.1 104.1 30.3 Plagiolepis pygmaea 4.2 1.6 5.8 0.4 1.1 1.1 2.2 0.6 Myrmicinae Cardiocondyla mauritanica ------Messor barbarus 41.9 80.1 121.9 8.4 0.0 0.0 0.0 0.0 Messor bouvieri 0.4 0.0 0.4 0.0 0.0 0.0 0.0 0.0 Messor structor 35.1 77.1 112.2 7.7 0.0 0.0 0.0 0.0 Pheidole pallidula 60.3 131.7 191.9 13.3 0.1 0.2 0.3 0.1 Pyramica membranifera 0.0 1.2 1.2 0.1 0.0 0.0 0.0 0.0 Solenopsis sp. 0.4 2.1 2.4 0.2 0.0 0.0 0.0 0.0 Tetramorium semilaeve 6.1 11.7 17.8 1.2 0.0 0.0 0.0 0.0 Ponerinae Hypoponera eduardi 0.0 0.5 0.5 0.0 0.0 0.0 0.0 0.0

Table S2. Summary of the ANOVA tables of δ15N- and δ13C-values of the most common ant species. The ANOVA design was a linear mixed model with bird-exclusion as a fixed factor and block as a random factor. The reported F and P values correspond to the effect of bird-exclusion.

LATE SPRING δ13C δ 15N Species habitat F df P F df P Pheidole pallidula Ground 0.26 1. 37 0.62 2.16 1. 37 0.15 Formica rufibarbis Ground 1.12 1. 36 0.30 0.08 1. 35 0.78 Formica rufibarbis Canopy 0.01 1. 22 0.92 0.00 1. 21 0.96 Lasius grandis Ground 0.00 1.18 0.98 0.43 1. 18 0.52 Lasius grandis Canopy 3.38 1.13 0.09 0.00 1. 13 0.96

LATE SUMMER δ 13C δ 15N Species habitat F df P F df P Pheidole pallidula Ground 1.59 1, 21 0.22 2.33 1, 21 0.14 0.15 Formica rufibarbis Ground 0.04 1, 31 0.83 0.88 1, 31 0.36 0.78 Formica rufibarbis Canopy 0.70 1, 10 0.42 0.11 1, 10 0.75 0.96 Lasius grandis Ground 2.46 1, 10 0.15 0.07 1, 10 0.80 0.52 Lasius grandis Canopy 0.10 1, 5 0.77 1.58 1, 5 0.26 0.96

1 Table S3. Summary of paired t-test of δ15N- and δ13C-values of ants of two of the most abundant species captured in the canopy (beating tray) or in the ground (pitfall trap) of the same tree.

LATE SPRING δ 13C δ 15N Species t df P T Df P Formica rufibarbis 1.45 22 0.16 1.58 20 0.13 Lasius grandis 0.19 13 0.86 0.27 13 0.79

LATE SUMMER δ 13C δ 15N Species t df P T df P Formica rufibarbis -0.59 11 0.57 -0.14 11 0.89 Lasius grandis 1.87 3 0.16 -0.23 3 0.83

Table S4. Mean δ15N- and mean δ13C-values for the analyzed species in late spring and in late summer, significant differences between isotopic values of ant species within a season are represented by different letters (Tukey’s HSD test, P < 0.06). CI 95% = confidence interval for the mean.

LATE SPRING δ 13C δ 15N Group Species Mean CI 95% Mean CI 95% n Ants Camponotus aethiops -22.43 --- 8.66 --- 1 Camponotus foreli -22.40 --- 8.07 --- 1 Camponotus pilicornis -21.78 --- 7.91 --- 1 Camponotus sylvaticus cd -22.15 1.15 c 8.41 0.07 4 Formica rufibarbis d -22.35 0.11 b 11.11 0.13 62 Formica subrufa cd -22.35 2.22 c 8.86 17.60 2 Lasius grandis d -22.63 0.19 b 11.06 0.10 37 Messor barbarus cd -22.13 2.90 c 7.74 0.54 4 Messor bouvieri -21.39 --- 8.18 --- 1 Messor structor cd -22.55 0.07 c 7.82 1.19 3 Pheidole pallidula b -20.12 0.24 a 12.25 0.11 40 Plagiolepis pygmaea a -19.15 1.10 b 11.18 0.42 8 Solenopsis sp. -19.02 --- 11.52 --- 1 Tapinoma nigerrimum -19.35 --- 11.84 --- 1 Tetramorium semilaeve c -20.51 1.78 b 10.63 0.54 8 Spiders Dysdera crocata -22.42 1.11 13.56 1.16 4 Pelecopsis mengei -25.54 0.14 10.90 1.00 6 Philodromus cespitum -23.44 0.40 10.60 1.54 5 Aphids Aphis spiraecola -25.14 0.91 8.65 0.47 4 Aphis gossypii -24.52 0.58 9.32 0.76 4 Isopoda Armadillidium sp. -22.88 1.58 8.86 0.65 4 Plants Citrus -26.09 0.23 9.91 0.62 4 Parietaria officinalis -28.29 0.84 8.71 0.74 4 Hordeum murinum -27.77 7.86 5.16 7.45 2

2 Table S4 (cont.)

LATE SUMMER δ 13C δ 15N Group Species Mean CI 95% Mean CI 95% n

Ants Camponotus aethiops c -22.89 0.50 d 7.63 0.89 4 Camponotus foreli -23.49 --- 8.55 --- 1 Camponotus sylvaticus c -22.80 0.78 d 8.25 1.81 4 Formica rufibarbis c -23.49 0.17 c 10.95 0.25 46 Formica subrufa c -23.68 7.37 bc 10.82 4.32 2 Hypoponera eduardi bc -21.83 0.46 a 16.78 3.48 3 Lasius grandis c -22.90 0.48 bc 11.56 0.24 21 Messor barbarus -22.69 24.78 6.94 9.21 2 Messor structor bc -21.90 2.18 d 8.40 1.26 3 Pheidole pallidula c -22.18 0.32 b 12.16 0.31 24 Plagiolepis pygmaea ab -20.53 1.95 bc 11.99 0.92 5 Pyramica membranifera a -18.96 0.91 a 18.30 2.80 3 Solenopsis sp. a -18.81 3.85 b 12.62 1.39 4 Tetramorium semilaeve bc -22.06 0.91 c 10.97 1.10 7 Spiders Dysdera crocata -21.94 4.04 14.07 2.67 3 Clubiona genevensis -25.63 0.37 10.76 1.03 4 Xysticus cristatus -23.80 0.55 10.71 0.70 4 Theridion mystaceum -24.11 0.95 10.52 0.53 4 Isopoda Armadillidium sp. -21.24 1.77 8.85 1.07 4 Porcellio sp. -21.69 2.27 12.04 0.90 4 Plants Citrus -27.27 0.57 10.54 0.98 4 Parietaria officinalis -28.80 1.54 9.20 2.96 4 Cynodon dactylon -11.73 0.26 8.02 1.74 4