Insect. Soc. DOI 10.1007/s00040-014-0375-1 Insectes Sociaux

RESEARCH ARTICLE

Evaluating the importance of trophobiosis in a Mediterranean community: a stable isotope analysis

K. Brewitt • J. Pin˜ol • C. Werner • W. Beyschlag • X. Espadaler • N. Pe´rez Hidalgo • C. Platner

Received: 30 June 2013 / Revised: 15 October 2014 / Accepted: 16 October 2014 Ó International Union for the Study of Social (IUSSI) 2014

Abstract Trophobiosis between (, Hemi- plantation. We analyzed d13C- and d15N-values of sample ptera) and (Formicidae, ) is consid- sets for fertilized and natural soil, using the fertilizer as ered to provide an important source of nutrition for ants natural isotope label. The results showed trophic relation- by honeydew and aphids themselves used as prey. ships between 18 host plant , 22 aphid species, and 7 However, little is known about nutrient fluxes and the rela- ant species. Direct observation revealed at least 40 different tive importance of trophobiosis for different ant species. plant–aphid combinations and 25 aphid–ant combinations Combining direct contact observations between ants and with a marked range of d15N-values. However, the d13Cand aphids with stable isotope analyses of distinct multitrophic d15N isotope ratios still reflected the trophic levels. A sig- sample sets (soil, plant, aphid, and ant), we aimed at dis- nificant correlation occurred between the isotope ratios of entangling the importance of trophobiosis in a aphids and their host plants. However, no relationship was Mediterranean food web and possible feedbacks on the found between aphids and ants or between plants and ants functional diversity of ants in a species-rich organic Citrus revealing that many ant species do not exhibit a close relationship with their trophobiotic partners. Isotopic data Electronic supplementary material The online version of this allowed us to separate ant species into trophic functional article (doi:10.1007/s00040-014-0375-1) contains supplementary groups and showed the relevance of other food resources. material, which is available to authorized users. The applied fertilizer shifted the isotopic baseline for the whole trophic system. By combining the stable isotope K. Brewitt W. Beyschlag C. Platner Department of Experimental and Systems Ecology, University of analysis with the exact origin of the samples, we avoided a Bielefeld, Universita¨tsstraße 25, 33615 Bielefeld, Germany misleading interpretation of the high isotopic range of species. Thus, we emphasize the importance of considering J. Pin˜ol X. Espadaler a baseline in stable isotope food web studies. CREAF, Cerdanyola del Valle`s, 08193 Barcelona, Spain

J. Pin˜ol X. Espadaler Keywords Functional groups Formicidae Homoptera Universitat Auto`noma Barcelona, Cerdanyola del Valle`s, 08193 Citrus plantation Carbon Nitrogen Barcelona, Spain

C. Werner AgroEcoSystem Research, BAYCEER, University of Bayreuth, Introduction Universita¨tsstraße 30, 95447 Bayreuth, Germany Positive interactions between organisms, generally known N. Pe´rez Hidalgo Department of Biodiversity and Environmental Management, as mutualisms, are known as driving forces in terrestrial University of Leo´n, 24071 Leo´n, Spain ecosystems (Ahmadijan and Paracer, 2000; Moran, 2006). For example, trophobiosis between ants and aphids, where Present Address: ants acquire nutrition through sugar-rich honeydew from C. Platner (&) Kampstraße 9, 37217 Witzenhausen, Germany aphids in exchange for providing a protection against pre- e-mail: [email protected] dators and parasitoids, is very important in many ecosys- 123 K. Brewitt et al. tems (Way, 1963; Delabie, 2001; Stadler and Dixon, Michener and Schell, 1994)and3–5% for nitrogen (DeNiro 2005, Blu¨thgen et al., 2006). Large colonies of social insects and Epstein, 1981;MinagawaandWada,1984;Post,2002), like ants are known for their high energy requirements, which allows a categorization of trophic levels (Newton, which can only be satisfied by exploiting energy-rich food 2010). Feldhaar et al. (2010) describe similar average isotopic sources, e.g., honeydew from trophobiosis with aphids. In fractionation of Camponotus floridanus (Buckley 1866) pupae temperate climate zones, the biology of individual ant for nitrogen (DN = 3.0 %)andcarbon(DC = 1.1 %)in species is well known. For instance, a single colony of controlled feeding experiments. Sagers and Goggin (2007) sp. can collect up to 500 kg of honeydew per year found no carbon enrichment between aphids and leaves of (Zoebelein, 1956). 13 % of honeydew dry mass consists of their host plants, but 1.0 % nitrogen enrichment from leaves amino acids and contains all the essential amino acid to aphids and an enrichment of honeydew for carbon (1.9 %) compounds needed for the synthesis of proteins. and for nitrogen (1.4 %) relative to plant leaves. However, Furthermore, some species like Lasius flavus (Fabricius leaf bulk material integrates over the lifetime of leaves, 1782) feed their larvae with root aphids during certain whereas phloem sap d13C-values are generally more dynamic periods of the year (Pontin, 1978; Seifert, 2007). Similarly and reflect seasonal changes in photosynthetic discrimination to the reported predation of Lasius niger (Linnaeus 1758) on (Rascher et al., 2010;Werneretal.,2012). If trophic enrich- Aphis fabae Scopoli, 1763 (e.g., Oliver et al., 2012), we ment between tissues, species, and trophic levels is constant, a observed predation of Aphis sp. by Lasius grandis Forel regression line between two components of sample sets is 1909 in the Citrus canopies in summer. Therefore, aphids expected to have a slope of 1.0 and an intercept equal to the can be an important nutrient source for ants both via hon- degree of fractionation. eydew provision and as direct prey. Cerda´ et al. (1997, Fiedler et al. (2007) studied 43 ant species in the Euro- 1998) and Pekas et al. (2011) describe honeydew and nectar pean temperate zone and gained detailed insight into as the main food resource for some Mediterranean ant isotopic patterns of central European ants showing that species (Lasius grandis, pallidula (Nylander trophobiosis is in fact an important form of nutrition for the 1849), schmitzii Forel 1895, Tapinoma niger- ant genera Formica, Lasius, Camponotus, and Plagiolepis. rimum (Nylander 1856) and several Camponotus species). Ottonetti et al. (2008) successfully identified the food web Thus, trophic relationships between aphids and ants seem to position (d15N signal) and carbon sources (d13C signal) represent an important thread in Mediterranean food webs. from lower level (like Homopteran tending) to high level Roig and Espadaler (2010) adapted and modified the Aus- (with predation) of five ant species in Italy. Platner et al. tralian functional group model from Andersen (1995), (2012) and Mestre et al. (2013) reported for Lasius grandis which is based on habitat requirements and competitive and Formica rufibarbis Fabricius 1793 an enrichment for interactions, to the Mediterranean region. Several functional d15N and d13C of approx. 3 % compared to Aphis spirae- and trophic groups of ants are more important in Mediter- cola Patch 1914, but these samples had to be pooled and, ranean ecosystems than in Central Europe (Platner et al., therefore, lacked a spatial baseline. 2012): granivore harvester ants (Messor spp.), nectarivore To disentangle the importance of trophobiosis for several ants (Camponotus spp.), ant genera Pheidole spp. and Te- ant species and functional groups and to determine the tramorium spp., which feed on plant and animal remains, structure of this food web in an organic Citrus plantation in specialized predators (Hypoponera spp.) and cryptic (i.e., Spain with approximately 100 plant species (herbaceous unknown nutrition and hidden habitat) species (Solenopsis forbs, grasses, trees; Kindermann, 2010), 24 aphid and 22 ant spp.). However, omnivore generalists, often with trophobi- species (Pin˜ol et al., 2008), we analyzed the stable isotope otic feeding behavior (Lasius spp. and Formica spp.), are an composition (d13C and d15N) of soil, host plants, aphids, and abundant trophic group in both biomes. A closer investi- ants. Combining direct field observations of trophobiotic gation of the trophic links within an ant assemblage contacts between ants and aphids with stable isotope anal- provides an ideal model system in which to test how yses of distinctive sample sets (soil, host plant, aphid, and trophobiosis affects the trophic structure of omnivores and tending ant worker; Fig. 1b) allows disentangling of the their positions in food webs. trophobiotic food web and still guarantees connectedness of To disentangle the food web relations between ants, the samples and thereby a correct baseline for consumer associated , and host plants, we analyzed stable trophic positions (Woodcock et al., 2012). isotope ratios (13C/12C, 15N/14N), which has been proven as An organic fertilizer, which was applied in parts of the powerful tool in the analysis of terrestrial and aquatic food orchard, induced a d15N labeling treatment, which provided webs (Blu¨thgen et al., 2003; Schmidt et al., 2004;Albersetal., a modified isotopic baseline of the ecosystem. In a labora- 2006; Hood-Nowotny and Knols, 2007; Tillberg et al., 2010). tory experiment, Schumacher and Platner (2009) showed Typically, become isotopically enriched relative to that the d15N-value of fertilizer spread from plant shoots to their diet by 0–1 % for carbon (DeNiro and Epstein, 1978; aphids and ants. 123 Evaluating the importance of trophobiosis

Fig. 1 The studied system: a non-fertilized and fertilized areas enclosed by a fence: Citrus trees and herbaceous plants in rows are fertilized and irrigated, whereas patches between the rows and beside the Citrus grove are not fertilized and not irrigated (numbers from 50 to 56 refer to the sample codes given in ESM, Table S1); b soil, host plant, aphid, and ant as components from a sample set (black arrows nitrogen flux, dotted arrows carbon flux)

The overall goal of this study was to collect a represen- Citrus tree plantation. The climate in this region is Medi- tative ‘‘snapshot’’ of the trophobiotic partners, to prove the terranean with warm and dry summers and relatively rainy importance of trophobiosis for a diverse agro-ecosystem spring and autumn. The plantation consisted of approx. 300 and to classify the involved ant species into trophic func- clementine trees (Citrus clementina var. clemenules) graf- tional groups. Specifically, we tested the following hypo- ted on the hybrid rootstock Carrizo citrange (Poncirus theses: trifoliata (L.) Raf. 9 Citrus sinensis (L.) Osb.), hereafter referred as ‘‘Citrus’’. Citrus trees were planted in rows with I. Trophobiosis provides important food resources for a distance of 3.5 m between trees, 5 m between rows. aphid tending ant species. This should be detectable by Additionally some other tree species (Corylus avellana, analyses of stable isotopes N and C, and we expect that Prunus dulcis, Prunus persica, Pyrus communis, Olea eu- (a) fertilization with 15N-enriched fertilizer should ropaea, and Punica granatum) exist on this plantation. enhance the d15N values of all interacting components; More detailed information about the site and photographs and (b) a correlation between host plant and aphid are provided by Pin˜ol et al. (2009) and Brewitt (2013). The isotopic signatures with only small enrichment for C fertilizer (Fe Biolo´gico pellet, Agrimartin) and irrigation and N and a correlation between host plant/aphid and water were applied underneath the Citrus tree canopies but ant signatures with an enrichment of approx. 3–4.4 % not in the area between tree rows (Fig. 1a). We defined two for d15N and approx. 1–3 % for d13C depending on the kinds of soil: ‘‘NF’’ = non-fertilized soil (No. 50, 51, and relative contribution of honeydew and predation to the 52 in Fig. 1a) and ‘‘F’’ = fertilized soil (No. 53 to 55 in ants diet. Fig. 1a). As the fertilizer (d15N in 2010: 12.4 % ± 0.18) II. Differences between ant species in specific isotopic had a different nitrogen isotopic signal than the soil (soil on enrichment within a sample set allow classification into plantation without fertilizer: 5.0 % ± 0.4), the application different trophic functional groups. We expect that of fertilizer can be considered a d15N labeling experiment. these different subsets of the ant fauna should regard to the relative contribution that trophobiosis makes up for Sampling their nutrition. We expect to find a relative high specific importance of trophobiosis at least for Lasius grandis Data collection took place in different compartments of the and Formica spp. plantation in La Selva, including all seven tree species, with a total studied area of about 1,500 m2. At the aphid growth peak in June (Pin˜ol et al., 2009), we recorded all visible Methods aphid colonies on different host plants during 7 days in transects in the tree rows. Each day, a further part of plan- Field site tation was studied and every detected aphid colony was checked for foraging ant workers for 5 min at two different Data collection was conducted in La Selva del Camp, Tar- times per day (morning, afternoon). Several individuals of ragona, NE Spain, (41°1300700N, 1°0803500) in an organic aphids, host plant foliage and host plant soil samples

123 K. Brewitt et al.

(approx. 5–10 g of the top 5 cm soil) were collected for all were directly air-dried for approx. 2 days. Finally, all aphid colonies on individual plants. In case of observation samples were oven-dried at 70 °C for 48 h. Plant and soil of direct contacts between aphids and ants, the respective material was milled to a fine powder, and weighted into tin ant workers were sampled as well. Connected samples of capsules (approx. 2 mg). Ants and aphids were analyzed soil, host plant individuals, aphid colonies, and tending ant entirely; samples from small species include several indi- worker form distinctive sample sets (Fig. 1b). We observed viduals when necessary. We analyzed d13C and d15N from in total 123 colonies, from which 58 were all samples at the University of Bielefeld, using a continu- attended by ants. 73 sample sets from independent plant ous-flow Isotope Ratio Mass Spectrometer (IRMS, individuals were analyzed, 55 of them were attended by IsoPrime, Elementar, Hanau, Germany) coupled to an ele- ants. From some trees 16 additional aphid species from mental analyzer (EuroVector EA, Hekatech, Wegberg, different colonies were sampled and analyzed, these were Germany). Samples were standardized to IAEA-N2 not visited by ants and the connectedness of samples were (ammonium sulfate, d15N: 20.3 % ± 0.2 SD) and IAEA- considered in the correlation analysis. Analyzed ant–aphid CH-4 (wood, d13C: -24.0 % ± 0.6 SD) from International pairs were several meters apart to reduce the chance that Atomic Energy Agency (Vienna, Austria). Carbon and attending workers came from the same ant colony. Ants nitrogen isotope ratios were calculated relative to VPDBee were collected with entomological exhauster and before and atmospheric air, respectively, and expressed as % foraging on honeydew, to avoid an influence of the current (Table 1). food in the gaster on the isotope values. Whole bodies of workers were analyzed to make results comparable to Data analyses aphids and avoiding an artificial effect on the mass balance of the whole organism by removal of the abdomen, which To illustrate and analyze the diversity of all involved plant, contains the majority of lipid storage in insects (DeNiro and aphid, and ant species, a network of all observed contacts Epstein, 1978; Tillberg et al., 2010). We analyzed few For- were plotted using the R package Bipartite 2.18 (Dormann mica subrufa Roger 1859 workers with large crops after et al., 2008). The number of observed contacts (ESM Tab. attending aphid colonies to control the effect of our proce- S1) differs in some cases from number of stable isotope dure and found a mean depletion of -0.5 % in d13C and an analysis samples (Table 3), since not every sample was enrichment of ?0.3 % in d15N compared to workers from analyzed. The effect of fertilization and food web compo- the same colony before attending aphids. The C/N ratio nent (i.e., trophic level) on d15N and d13C-values were increased from 4.5 to 5.5 %. Blu¨thgen et al. (2003) reported tested by two-factorial analysis of variance, the effect of a similar depletion in d13C between honeydew-filled gaster fertilization on stable isotope contents of soil by one-fac- and remaining body of Oecophylla smaragdina (Fabricius torial ANOVA. Thereafter differences within each food web 1775). Careful observation and sampling of ant workers component between samples from F (fertilized) and NF before food intake seems to be appropriate to control for gut (non-fertilized) patches were compared using Welch’s t test. contents and avoid ‘‘overtreatment’’ for ant species with d13C- and d15N composition between the directly connected small crops. Additionally, root samples from Avena barbata food web components were compared by correlation and (Poaceae) were collected to look for the presence of root regression analysis. Pearson’s product moment correlations aphids. In these samples, both root aphids and ants were were statistically tested for all combinations of interacting found, but no direct contacts between them could be trophic components. The slopes of the regression lines and observed. Plant material consisted of foliage and stems from their R2 were calculated separately for samples from F and herbs and foliage from trees. The goal was to collect a NF patches. ANOVAs were carried out with R 2.15.3 (R representative cross-section of the trophobiotic system in development Core Team), t test and correlations were the orchard. Host plant parallels were preserved as dried conducted with SigmaPlot 11.0 (Systat Software Inc., San copies in a herbarium and subsequently identified. From Jose, CA, USA). every ant and aphid sample, a second sample was stored in ethanol (70 %) for species identification (Collingwood, 1978;Go´mez and Espadaler, 2007; Nieto Nafrı´a and Mier Results Durante, 1998, Nieto Nafrı´a et al., 2003, 2005; Blackman and Eastop, 2013). Diversity of tritrophic interactions

Sample preparation We found one Coccidae (scale ) species and 22 aphid species, 8 of them with no observed ant interactions. These Ants and aphids were placed in paper bags and deep frozen Hemiptera formed colonies on 18 host plants species, (-18 °C, 24–48 h). Leaves of host plants and soil samples including seven tree species and the Poaceae Avena 123 vlaigteiprac ftrophobiosis of importance the Evaluating Table 1 Mean C/N, d13C, and d15N values ± SE (standard error of the mean) of all species involved in studied tritrophic interactions, soil and fertilizer from not fertilized (NF) and fertilized (F) plots in and beside the Citrus grove in La Selva del Camp, Spain Species C/N d13C(%) d15N(%) n Mean Mean SE Mean SE

Soil Out of Citrus grove NF 11.1 -20.26 4.71 4.48 1.57 2 Border of Citrus grove NF 11.5 -25.80 0.37 4.90 0.39 12 Between tree rows NF 9.8 -25.30 1.32 6.38 1.41 2 Within fertilized rows F 9.5 -24.50 0.28 8.24 0.17 41 Direct under fert. trees F 9.5 -24.37 0.09 8.74 0.28 13 Direct under fert. patches F -24.61 10.56 1 Fertilizer 2010 F 9.5 -25.01 0.10 12.35 0.18 3 Plants Asteraceae NF 28.6 -29.21 0.18 3.51 0.28 2 F 24.1 -30.20 0.32 6.95 0.49 2 Avena barbata Pott ex Link, 1800 NF 30.2 -27.67 0.90 6.30 3.23 2 F 29.6 -27.84 1.31 7.50 1.67 3 A. barbata, roots F 26.6 -29.25 0.35 7.40 1.06 3 Carduus pycnocephalus L., 1763 NF 31.4 -29.24 0.65 4.28 0.53 4 Chaerophyllum sp. L., 1753 F 10.2 -30.23 0.53 11.17 1.29 4 Citrus clementina hort. ex Tan. F 17.9 -25.39 0.80 9.80 0.49 5 Corylus avellana L., 1753 F 21.8 -24.49 0.38 4.04 0.16 2 Foeniculum vulgare Miller, 1768 NF 14.4 -29.96 5.73 1 Hirschfeldia incana (L.) Lagreze-Fossat, 1847 NF 17.8 -29.15 6.82 1 Malva parviflora L., 1753 NF 7.3 -29.82 12.01 1 F 15.8 -28.84 0.75 9.11 1.65 6 Olea europaea L., 1753 NF 32.4 -24.84 0.07 1.55 0.09 2 Parietaria officinalis L., 1753 F 7.9 -30.38 11.54 1 Plantago sp. L., 1753 NF 18.8 -29.91 0.31 2.76 0.36 4 Prunus dulcis D.A.Webb, 1967 F 22.7 -26.43 8.21 1 Prunus persica (L.) Batsch, 1801 F 14.2 -26.90 0.06 8.28 1.08 2 Punica granatum L., 1753 Pyrus communis L., 1753 NF 23.4 -28.13 3.44 1 F 22.8 -27.94 0.20 5.84 2.40 2 Sonchus tenerrimus L., 1753 NF 31.6 -28.68 7.93 1 F 17.9 -28.18 0.35 10.60 0.44 18

123 Urtica sp. L., 1753 F 12.2 -28.65 0.74 14.42 0.42 5 123 Table 1 continued Species C/N d13C(%) d15N(%) n Mean Mean SE Mean SE

Aphid Aphididae NF 9.3 -25.59 0.50 7.83 1.57 3 Aphis sp. L., 1758 F 6.3 -29.73 0.08 13.88 1.38 2 Aphis fabae Scopoli, 1763 NF 6.7 -29.89 0.55 4.49 0.17 4 F 5.1 -29.79 11.04 1 Aphis gossypii Glover, 1877 NF 9.1 -27.06 0.40 9.14 0.60 5 F 7.9 -28.37 0.83 11.58 1.18 4 Aphis spiraecola Patch, 1914 NF 8.9 -28.45 0.38 4.89 0.36 3 F 8.2 -27.21 0.59 11.96 0.68 8 Aphis umbrella (Bo¨rner, 1950) NF 4.2 -29.42 11.16 1 F 6.0 -30.21 0.56 10.01 0.72 5 Baizongia pistaciae (L., 1767) F Brachycaudus amygdalinus (Schouteden, 1905) F 6.7 -26.83 0.25 11.03 0.35 4 Brachycaudus cardui (L., 1758) NF 5.2 -29.15 0.68 5.41 0.41 4 Brachycaudus persicae (Passerini, 1860) F 6.9 -27.13 0.33 11.45 0.12 4 Brevicoryne brassicae (L., 1758) NF 5.1 -27.87 8.32 1 Corylobium avellanae (Schrank, 1801) F 6.2 -27.65 6.58 1 Dysaphis plantaginea (Passerini, 1860) NF 6.5 -26.91 6.85 1 F 8.2 -27.62 0.31 5.56 0.36 8 Geum utricularia (Passerini, 1860) F Hyalopterus amygdali (Blanchard, 1840) F 7.2 -26.35 11.92 1 lactucae (L., 1758) NF 6.3 -28.43 9.12 1 F 8.3 -28.70 0.31 11.99 0.64 14 Myzocallis coryli (Goeze, 1778) F 5.3 -27.77 5.57 1 Myzus persicae (Sulzer, 1776) F 5.9 -25.12 8.51 1 Sitobion avenae (Fabricius, 1775) NF 7.2 -26.58 3.26 1 F 6.4 -27.96 0.36 6.56 0.28 3 sp. Mordvilko, 1914 NF 4.7 -29.26 5.29 1 F 7.0 -29.83 1.24 7.11 0.95 2 Uroleucon sonchi (L., 1767) F 7.2 -28.72 0.60 8.48 2.54 2 Scale Ceroplastes sp. Gray, 1828 NF 6.4 -25.72 0.37 3.01 0.30 2 F 33.7 -25.31 8.80 1 al. et Brewitt K. Evaluating the importance of trophobiosis

n barbata, and were tended by seven ant species. We observed 66 different combinations of observed contacts between the participating species (Fig. 2), 40 combinations of host plants and aphids and 26 of aphids and ants. The most abundant aphid species were Aphis spiraecola and Aphis gossypii Glover 1877 (tended by ants, most abundant on Citrus trees) and Hyperomyzus lactucae (never observed to be tended by ants) and Aphis fabae (five host plant spe- )

% cies, but not observed to be tended by ants in our system). t considered in statistical analyses

N( Additionally, we found two species of root aphids (Bai- 15

d zongia pistaciae and Geoica utricularia) forming colonies. The herbaceous host plants with highest number of aphid colonies were Sonchus tenerrimus (24 colonies) and Avena barbata (17 colonies; Fig. 2). The ant species Lasius grandis, Formica subrufa, and Pheidole pallidula showed most of the ant–aphid interactions (Fig. 2; Table S1).

Effect of fertilizer ) % The d15N isotope values varied by 12.9 % (1.5–14.4 %) for C( 24.4524.4621.78 0.71 0.03 11.21 9.99 12.77 0.88 1.05 4 2 1 23.0324.8023.8820.53 0.1521.57 0.21 0.23 0.35 11.87 0.22 9.45 12.18 12.75 0.34 12.67 0.38 0.46 0.68 17 0.58 7 10 2 7 24.37 6.76 1 23.2821.61 10.74 12.21 1 1 13 ------d plants, 10.6 % (3.3–13.9 %) for aphids and 6.0 % (6.8 % Formica subrufa in NF–12.8 % Pyramica membranifera in F) for ants. Aphid species also exhibited a large range in d13C-values (5 %). The ant species had similarly variable d13C values (Dd13C = 4.3 %) with low values in Lasius 13

Mean Mean SE Mean SE grandis (d C =-24.8 %) and high in Pheidole pallidula (d13C =-20.5 %). Mean d13C- and d15N- values of indi- vidual components of the sample sets (Fig. 3) showed a clear separation into trophic levels from soil as a baseline via host plants as producers followed by herbivorous aphids F 7.1 FF 4.8 4.0 and then ants at the highest trophic level. Differences in carbon and nitrogen isotopic composition were highly sig- nificant between the components of the food web (Table 2). The effect of fertilizer on nitrogen isotopic values was highly significant for soil and the whole food web (Table 2), but this effect was dependent on the components (significant interaction fertilizer 9 component): fertilized soil had 3.4 % higher d15N-values than non-fertilized soil (Fig. 3). Host plants grown in F soils had 5.2 % higher mean d15N- ) F 4.5

(Emery, 1869)values F than plants 4.3 from NF soil. Compared with soils, mean 15 (Latreille, 1798) F 5.1

(Latreille, 1798) NFd N ofplants 5.1 was enriched by 1.8 % in NF and 2.3 % in F. Fabricius, 1793 F 5.4 (Nylander, 1849) NFAphids 4.1 showed 3.5 % higher mean d15N-values in colonies Roger, 1859 NF 5.4

Forel 1909 NFfromfertilized 4.8 host plants. Ant workers as the most mobile components in the multitrophic system also had higher d15N-values in fertilized patches, but the difference between Iberoformica subrufa NF and F subsystems was only 1.3 % and not statistically Pyramica membranifera Lasius grandis Pheidole pallidula Plagiolepis pygmaea (syn. Formica rufibarbis Formica subrufa Camponotus aethiops Speciessignificant C/N (Table 3).

Relationship between isotopic compositions across the trophic levels continued

There was a significant positive correlation between d15N- number of analyzed samples; for all observed contacts see ESM, Table S1. Stable isotope contents of Pomegranate tree and two root aphid species were no n Ant Table 1 values of host plants and their soil (Fig. 4a; Table 4) and 123 K. Brewitt et al.

Fig. 2 Quantitative tritrophic host plant–aphid–ant food web (65 row). Linkage width indicates number of trophic interaction, bar sizes different combinations of observed contacts in approximately 140 indicates total interaction frequencies. White parts of bars aphid observed sample sets). Horizontal bars represent abundance of host colonies without observed ant-contact; some species names are plants (bottom row), Hemiptera (middle row), and Formicidae (upper abbreviated, for full species names see Table 1 between aphids and their host plants (Fig. 4c) in fertilized aphids studied shows a classification of ant species patches. We further found a highly significant relationship (Fig. 5; Table 5) into more trophobiotic species (Formica between d13C-values of aphids and their host plants, also in subrufa: Dd13C =?3.1 %, Lasius grandis: ?3.1 %, Pla- fertilized patches (Fig. 4d; Table 4). There was no signifi- giolepis pygmaea: ?3.3 %) and less trophobiotic species cant correlation between ants and their tended aphids and with large differences in d13C to their tended aphids ants and plants, neither in d13C nor in d15N values (Fig. 4e– (Pheidole pallidula: Dd13C =?8.1 %, Pyramica mem- f; Table 4). Ants were significantly more enriched in d13C branifera: ?7.3 %). and d15N than their tended aphids (Dd13C =?4.9; Dd15N =?2.3) and plants (Dd13C =?5.2; Dd15N = ?3.1; Tukey’s test: adj. P \ 0.0001 for all four compari- Discussion sons). In contrast, the slight enrichment between aphids and their host plants was not significant (Dd13C =?0.2, Trophic diversity P = 0.602; Dd15N =?0.8, P = 0.138). Our observations assigned many aphid species directly Trophic functional groups of ants within the trophobiotic system in the Mediterranean area. The ‘‘snapshot’’ of 1 week in June confirms the high The three most abundant ant species in the study were diversity of trophobiotic food system recognizable to the mostly observed in contact with Dysaphis plantaginea and high number of observed contacts and combinations (met- Brachycaudus persicae (Lasius grandis), Aphis spiraecola rics of a tripartite network analysis in Table S2 in EMS). and Aphis gossypii (Formica subrufa) and Aphis umbrella Our Stable isotope analysis indicates a relative high trophic (Pheidole pallidula). A species-specific calculation of the level diversity in this Mediterranean agro-ecosystem. Mean d13C differences between different species of ants and d15N-values of workers of most abundant species and

123 Evaluating the importance of trophobiosis

only 0.8 % in d15N and 1.9 % in d13C within the fertilizer treatment. The harvester ant Messor barbarus (L. 1767), which had the lowest trophic level in the study by Platner et al. (2012), was never observed collecting honeydew or preying on aphids and was, therefore, not analyzed in this study. In tropical rainforests in Australia Blu¨thgen et al. (2003) found 50 ant species covering a range of 7.1 % in d15N and 6.8 % in d13C and Davidson et al. (2003) reported from Peru 112 ant species covering a range of 9.5 % in d15N. Both studies concluded that ant communities represent a continuum from herbivores to predators, with pronounced omnivory, and that honeydew and nectar are important diets of many canopy ants.

Importance of trophobiosis

Here, we analyze trophobiosis with stable isotopes in a Mediterranean agro-ecosystem. We could trace the unique d15N of fertilizer from the soil up to the level of aphids. 13 15 Fig. 3 Mean values (±SE). d C and d N of different components Schumacher and Platner (2009) and Lescarno et al. (2012) (soil, host plants, aphids, and ants) in fertilized (black diamond) and demonstrated that plants, associated aphids, and ants reflect non-fertilized (gray diamond) areas, additionally the mean value 15 (±SE) of fertilizer (black), data and statistics shown in Table 3 the d N signal of soil and fertilizer. We used this effect as a d15N labeling experiment expecting that all other trophic levels of the community would be influenced. The signifi- Table 2 Effect of fertilization (degrees of freedom: df = 1), food web cant fertilizer effect (Table 2) confirmed our expectation. component (i.e., trophic level; df = 2), and their interaction (df = 2) 15 on d15N- and d13C-values of plants, aphids, and ants, tested by two- The effect of fertilizer on d N was detected at all levels, but factorial ANOVA (error df = 211) the significant interaction between fertilizer and food web component indicated that the enrichment effect of fertilizer d 13C d15N was not significant for ants. The low influence of 15N from F value PFvalue P fertilizer on ants could be a hint that ants may obtain smaller Fertilizer 3.82 0.052* 87.58 \0.001*** amounts of nitrogen by trophobiosis as in tropical forests Component 195.86 \0.001*** 22.31 \0.001*** (Blu¨thgen et al., 2003; Davidson et al., 2003) or temperate Fertilizer 9 component 1.14 0.321 5.21 0.006** systems (e.g., Pontin, 1978). However, the lack of a fertilizer effect on ants may be likewise explained through their larger mobility. Effect of fertilizer and isotopic variance between genera in Central Europe typically span a range of approx. the species was greatest for the immobile plants, slightly 3 % in diverse dry grasslands (Sanders and Platner, 2007; reduced for the mostly immobile aphids and lowest for the O´ Grady et al., 2010) and other habitats (Fiedler et al., 2007). mobile ants. Without more detailed studies on nutritional Similarly, in the dry grasslands mean d13C-values of ecology of the ubiquitous and more specialized Mediterra- workers also span a relatively small range of approx. 2 %. nean ant species, the amount of nitrogen obtained from Ottonetti et al. (2008) documented the isotopic values of the trophobiosis could not be quantified. five most common species of ants in a Mediterranean olive Plants had a higher enrichment in 15N than the soil in orchard with similar ranges, whereas Platner et al. (2012) fertilized patches (?5.2 and ?3.4 % compared to NF, found within and directly below the Citrus tree crowns in respectively), indicating an efficient utilization of the the plantation used for this study a range of 10.7 % for d15N organic fertilizer, resulting in a shift of the herbaceous plant and 4.8 % for d13C. Even the five most common species in community toward more nitrophilic species like Parietaria spring had significant differences of 4.5 and 3.6 % in their officinalis. Aphids get their nitrogen directly from the d15N- and d13C-values, respectively. Three of the species, phloem sap of their host plants and thus reflecting to a great Lasius grandis, Pheidole pallidula, and Plagiolepis pyg- extent the signal of their host plant and the fertilizer maea, were also commonly observed in this study tending (Schumacher and Platner, 2009; Wilson et al., 2011). Nitro- aphid colonies in fertilized patches (i.e., in and underneath gen content of phloem sap is generally low, so sap suckers Citrus tree crowns). Their interspecific differences were did not show a strong trophic enrichment because discri- 123 K. Brewitt et al.

Table 3 Mean isotopic values for components from non-fertilized and fertilized patches d13C(%) d15N(%) NF nF nP NF nF nP

Ant -23.35 ± 0.45 11 -23.26 ± 0.23 44 0.869 10.64 ± 0.53 11 11.91 ± 0.24 44 0.112 Aphid -28.08 ± 0.33 25 -28.17 ± 0.19 64 0.823 6.70 ± 0.48 25 10.22 ± 0.38 64 <0.001 Plant -28.73 ± 0.39 19 -28.12 ± 0.26 54 0.203 4.57 ± 0.64 19 9.74 ± 0.41 54 <0.001 Soil -25.15 ± 0.74 16 -24.47 ± 0.21 55 0.504 5.01 ± 0.40 16 8.40 ± 0.15 55 <0.001 P values from Welch two sample t test (bold highly significant differences between NF and F). Fertilizer: d13C: -25.01 % ± 0.10; d15N: 12.35 % ± 0.18; n = 3 mination against d15N is lower if the entire resource has neither in d15N nor in d13C. The most abundant ants had an to be exploited. In aphids living on non-fertilized plants enrichment in d15Nof?1.4 to ?4.0 % compared to their with a mean nitrogen content of 1.8 % N the mean enrich- tended aphids. Sagers and Goggin (2007) documented a ment of d15N was ?2.1 % compared to their host plants d15N-enrichment from plant leaves to honeydew of ?1.4 % what was clearly higher as the enrichment of aphids and to aphids of ?1.0 %. The expected enrichment of ants (?0.5 %) living on fertilized plants with a nitrogen content compared to their diet is ?3 % (Feldhaar et al., 2010). In of 2.3 % N. Wilson et al. (2011) described a similar isotopic non-fertilized patches ants had a mean d15N-enrichment separation of aphid and host plants, which depends on from ?5.0 % compared to plants. Blu¨thgen et al. (2003) nitrogen availability. The separation of our aphids and their reported up to 7.1 % d15N-enrichment between the trophic host plants was lower as described by Wilson et al. (2011), levels in aphids and ants. Aphid tending ants in La Selva but we clearly confirmed the described negative correlation were observed preying on other arthropods including pre- between plant nitrogen content and aphid enrichment. As dators (e.g., spiders) and they did not reflect the fertilizer plants are immobile and directly connected to the substrate, signal from plants and aphids, indicating the use of other N they reflect the d15N signal of soil (Ho¨gberg, 1997; Bey- sources besides honeydew. The mean differences in the schlag et al., 2009). Thus, soil values are often used as a d13C signal between ants and aphids were remarkably large baseline of a studied system in food web studies with d15N (NF: Dd13C =?4.73 %;F:Dd13C =?4.91 %) and isotopes (Gibb and Cunningham, 2011). Generally it is higher than expected, indicating that trophobiosis provides advantageous to combine direct observations with isotopic not only the important items in the diet of aphid tending analysis to detect the influence of fertilizer on the isotopic ants. Many other ant species in this agro-ecosystem seem to baseline and to enable correlations between compartments. also use carbon sources not provided by the plant-herbivore This highlights the importance of considering the baseline food chain, as they are highly enriched in 13C (Platner et al., when analyzing food webs and could help to avoid false 2012). The only C4-grass in the study system, Cynodon interpretation of high isotopic variances (Feldhaar et al., dactylon with highly enriched d13C-value (Platner et al., 2010; Woodcock et al., 2012). Parallel to the large and 2012) was never observed with aphid colonies and is even significant effect of the fertilizer on plant d15N, we found a during the summer not that common to represent a likely highly significant correlation within fertilized and a mar- explanation for the high d 13C-values of most ant species. ginally significant correlation within non-fertilized Only Messor harvester ant species were observed actively treatments. The d13C-values of soil and host plants were feeding at Cynodon seeds in September, but Platner et al. also significantly correlated in non-fertilized areas. The (2012) reported for these ant species similarly low d 13C- d13C signal of the soil is inversely determined by carbon values as for Citrus trees and Lasius grandis, which were input by plants (root exudates and dead plant material), significantly lower than d 13C-values of Pheidole pallidula storage and turnover by microorganism, and the degree of and Plagiolepis pygmaea in late spring. Blu¨thgen et al. irrigation. In non-fertilized soils, carbon flow from plants to (2003) reported a high enrichment in 13C of tropical ants and mycorrhiza is usually higher than in fertilized soils (Giar- other arthropods compared to plants, in particular for dina et al., 2004; Johnson, 2010), which could possibly predatory species with high d15N-values like several Phei- explain why the correlation between soil and plant d13Cis dole species and Thomisid spiders. In many ecosystems, significant only for non-fertilized soils. carbon from the detritus-based food chain could be impor- As expected, we found a strong correlation between tant, in particular for ant species like Pheidole pallidula and fertilized host plants and aphids for stable isotopes d13C and Pyramica membranifera. Soil fauna species in La Selva like d15N. However, the isotopic signals of ant workers were not Isopoda (Armadillium sp.: d13C =-22.4 % ± 0.29; d15N correlated to isotopic signals of aphids and host plants, = 7.6 % ± 1.96) and Collembola (d13C =-24.2 %,

123 Evaluating the importance of trophobiosis

δ15N δ13C 20 ef-15

15 -20

10

N (‰) -25 C (‰) 15 13 δ δ

5 -30

0 -35 0 5 10 15 20 -35 -30 -25 -20 -15 15 13 20 δ N (‰) δ C (‰) c -15 d

y = 0,7789x + 2,8379 15 R² = 0,6569 -20 Pearson = 0.811

y = 0,6888x - 9,0363 10 -25 R² = 0,5904 N (‰) C (‰) Pearson = 0.768 15 13 δ δ

5 -30

0 -35 0 5 10 15 20 -35 -30 -25 -20 -15 δ 15N (‰) δ 13C (‰) 20 ab-15

15 -20

y = 1.2001x - 0.4734 R² = 0.1948 Pearson: 0.441 10 -25 N (‰) C (‰) 15 13 δ δ

5 -30

0 -35 0 5 10 15 20 -35 -30 -25 -20 -15 δ 15N (‰) δ 13C (‰)

Fig. 4 Correlations between the isotopic signals (d15N and d13C) of samples from NF patches. Significant regression lines and 1:1 lines are the single components: soil host plant (a, b), host plant–aphid (c, given; slopes and Pearson’s correlation coefficients shown in Table 4 d) aphid–ant (e, f). Black points samples from F patches, light points:

123 K. Brewitt et al.

Table 4 Correlation relationship between the stable isotopes of components (hp = host plant of aphids) Components Isotope Treatment (n) Slope R2 Pearson P line regression

Aphid–ant d15N NF (6) 0.09 0.008 0.090 0.865 F (26) 0.06 0.006 0.079 0.702 Aphid–ant d13C NF (6) -0.69 0.333 -0.577 0.23 F (26) 0.08 0.005 0.068 0.741 hp-ant d15N NF (6) 0.37 0.311 0.558 0.329 F (26) -0.15 0.062 -0.249 0.210 hp-ant d13C NF (6) -1.42 0.640 -0.800 0.104 F (26) 0.24 0.059 0.243 0.222 hp-aphid d15N NF (16) 0.46 0.193 0.440 0.078 F (62) 0.84 0.657 0.811 <0.001 hp-aphid d13C NF (16) 0.278 0.109 0.330 0.195 F (26) 0.86 0.590 0.768 <0.001 Soil-hp d15N NF (16) 0.84 0.224 0.473 0.064 F (62) 1.20 0.195 0.441 <0.001 Soil-hp d13C NF (16) 0.32 0.356 0.597 0.015 F (62) 0.13 0.010 0.098 0.454 ‘‘Slope’’ and ‘‘R2’’ refer to the regression lines in Fig. 4

Fig. 5 Categorization of ant species into trophic functional groups by plotting Dd13C versus Dd15N between ants and attended aphids. Dotted line‘‘more trophobiotic species’’ (isotopic enrichment within expected range), broken line ‘‘less trophobiotic species’’ (Dd13C much higher than expected), Camponotus aethiops: nectarivore ant

d15N = 10.3 %; Mestre et al., 2013) and their potential food the question how much food is gained from the detritus- like hyphae from the fungi Agaricus campestris (d13C = based system, more research is needed. However, there may -21.4 %, d15N = 9.4 %) had enriched d13C-values similar be more reasons for the large differences in the d13C signal to several ant species. Field experiments and stable isotope between ants and aphids: (1) Ants could reflect an integrated analyses suggest Collembola to be an important food signal of their diet over their lifetime fixing a large part resource for ants in temperate grasslands (Reznikova and during larval development. Larvae could be fed with Panteleeva, 2001; Sanders and Platner, 2007). To answer prey (O‘Grady et al., 2010). Thus, d13C of ants

123 Evaluating the importance of trophobiosis sampled feeding on aphids during the peak of aphid growth species Pheidole pallidula showed a high isotopic differ- might not reflect the trophic importance of honeydew, ence from tended aphids (Dd13C =?8.1 %; which could be important for maintaining the energy Dd15N =?4.0 %). As an opportunist, this species is metabolism of adult ants, without integration in tissues. (2) known to feed on remains of plant and animal origin Isotope fractionation changing d13C of the phloem sap could (Retana et al., 1992). Additional observations in the stud- occur in the metabolism of aphids (Sagers and Goggin, ied grove confirmed that Pheidole pallidula scavenge 2007). Phloem sap d13C-values are generally more dynamic upon other ant species, as nearly every nest entrance is than bulk leaf material and reflect seasonal changes in marked by remains of arthropod carcasses, mainly of the photosynthetic discrimination (Rascher et al., 2010). (3) dominant harvester ant Messor barbarus (Platner et al., Finally an ant-specific accumulation of d13C by endo- 2012; own obs.). The isotopic enrichment of the ant spe- symbionts could also add to the discrepancies (Fiedler et al., cies with the tiniest workers, Plagiolepis pygmaea, meets 2007; but for Camponotus see Feldhaar et al., 2010). the expectations for honeydew collecting ants. Despite the high numerical abundance of workers at the plantation at Trophic functional groups of ants all microclimatic conditions, it is very difficult to localize the nests and study their biology. Interestingly, we found The species-specific analysis of isotopic data confirmed the lowest d13C-values of the aphid attending ant com- our hypothesis that within the group of aphid attending munity for this species, contrasting to significantly higher ants different trophic levels between ant species can be d13C-values found in a previous study from Citrus trees identified. The highly abundant species Pheidole pallidula (Platner et al., 2012); while the trophic level (i.e., d15N) showed an unexpectedly high d13C enrichment in com- was similar in both studies. The omnivorous species parison to their attended aphids, which can be regarded as Formica subrufa shows a slight enrichment in the d15N an indication of other food sources, and thus of a different signal compared to their attended aphids trophic functional group (Fig. 5; Table 5). Seven of 23 ant (Dd15N =?1.36 %), combined with the high level of species of the plantation were observed to be active in enrichment in the d13C-signal (Dd13C =?3.08 %), which trophobiosis. Even of this obviously small subset of the suggests that Formica subrufa use honeydew to a great known local ant fauna, isotopic values of only four extent. Formica subrufa additionally feeds on arthropod species (Lasius grandis, Formica subrufa, F. carcasses, mainly of other ants, but also from other insect rufibarbis, and Plagiolepis pygmaea) match our expecta- groups. Seeds with elaiosomes, nectar, and sap are also tions for trophobiotic species (Fig. 5, dotted line). At least taken as food (Cavia, 1990). The enrichment of the two species (Pheidole pallidula and Pyramica omnivore Lasius grandis in relation to aphids was similar membranifera) with clearly higher isotope enrichment for d13C and d15N (Table 5). Early in the season, up to seems to form another functional group of less trophobi- 10 % of workers carry insect prey, mainly Psocoptera and otic species (Fig. 5, broken line). Peka´r and Mayntz aphids, other workers collect honeydew from diverse (2014) found typically a high amount of lipids for sources (Paris and Espadaler, 2010). Lasius grandis is Formicinae and lipids in tissues of most organisms are normally found throughout the year on the tree canopies known to be depleted in 13C compared to carbohydrates (Pin˜ol et al., 2012). The enrichment in d15N between and proteins (DeNiro and Epstein, 1978). In detail, the aphids and ants could be explained by predation of aphids and collection of honeydew. Formica rufibarbis and La- sius grandis are well known as aphid mutualists, but they Table 5 Mean shifts in isotope values d13C und d15N between aphid could also be predacious to meet their nitrogen needs, and ants, with SE, sorted by size of d13C difference, n in parentheses, especially during larvae development (O‘Grady et al., not differentiated by fertilized/non-fertilized (Fig. 5) 2010; Seifert, 2007; Pontin, 1978). For more ‘‘typical Difference SE Difference SE Mediterranean’’ ant species, like Pheidole pallidula, the d13C ant– d 13 d15N ant– d 15 enrichment in d13C seems to be too large for a mainly aphid C aphid N trophobiotic feeding behavior, indicating utilization of Formica subrufa (12) 3.08 0.25 1.36 0.61 other C-sources. Further research should, therefore, focus Lasius grandis (14) 3.14 0.28 3.05 0.78 on species-specific potential food sources. The trophobi- Plagiolepis pygmaea (2) 3.25 0.36 -1.53 2.27 otic species in this study separated clearly from the group Formica rufibarbis (1) 4.44 4.44 of phytophagous ants, Camponotus spp., and Messor spp. 15 Camponotus aethiops (1) 6.37 -1.76 with significantly lower d N-values. This is in contrast to Pyramica membranifera (1) 7.33 4.17 studies on ants in the tropics, where trophobiotic and Pheidole pallidula (10) 8.12 0.50 4.04 0.74 phytophagous species differ less in their isotopic signals (Davidson et al., 2003; Blu¨thgen et al., 2003). 123 K. Brewitt et al.

Conclusions University of Bielefeld. http://nbn-resolving.de/urn:nbn:de:hbz: 361-26546725 Cavia V. 1990. Re´gimen alimenticio de la hormiga Formica subrufa The individual trophic levels in trophobiotic subsystems are (Hymenoptera: Formicidae). Ses. Entom. ICHN-SCL 6: 97-107 13 15 supported by the analysis of d C and d N isotope ratios. Cerda´ X., Retana J. and Cros S. 1997. Thermal disruption of transitive The unexpected high d13C enrichment from aphids and hierarchies in Mediterranean ant communities. J. Anim. Ecol. 66: plants to some ant species and the remarkable d13C differ- 363-374 Cerda´ X., Retana J. and Cros S. 1998. Critical thermal limits in ences between ant species indicate the importance of other Mediterranean ant species: trade-off between mortality risk and carbon sources in addition to trophobiosis. For the whole ant foraging performance. Funct. Ecol. 12: 45-55 community in the studied Mediterranean ecosystem, Collingwood C.A. 1978. A provisional list of Iberian Formicidae with trophobiosis is of less importance than expected from pre- a key to the worker caste (Hym, Aculeata). EOS 52: 65-95 Davidson D.W., Cook S.C., Snelling R.R. and Chuta T.H. 2003. vious studies in tropical and temperate systems. Explaining the abundance of ants in lowland tropical rainforest Connectedness of the samples, guaranteed by direct obser- canopies. Science 300: 969-972 vation, allows disentangling the food web, starting from the Delabie J.H.C. 2001. Trophobiosis between Formicidae and Hemiptera soil over plants and aphids up to the ants posing the highest ( and Auchenorrhyncha): an Overview. Neotrop. Entomol. 30: 501-516 trophic level in this study. A close relationship between host DeNiro M.J. and Epstein S. 1978. Influence of diet on the distribution plants and their related aphids was clearly demonstrated by of carbon isotopes in animals. Geochim. Cosmochim. Acta 42: tracing isotopic signals from fertilizer up to aphids. Ant 495-506 species showed distinct isotopic signatures directly related DeNiro M.J. and Epstein S. 1981. Influence of diet on the distribution 15 of nitrogen isotopes in animals. Geochim. Cosmochim. Acta 45: to their trophic functional group. Using the fertilizer N 341-351 label confirmed the importance to establish an isotopic Dormann C.F., Gruber B. and Fru¨nd J. 2008. Introducing the bipartite baseline in food web studies. Thus, combining isotopic Package: Analysing Ecological Networks. R News 8: 8-11 analyses with direct field observations provides an efficient Feldhaar H., Gebauer G. and Blu¨thgen N. 2010. Stable isotopes: past and future in exposing secrets of ant nutrition (Hymenoptera: method to disentangle trophic interactions from isotopic Formicidae). Myrmecol. News 13: 3-13 values. Fiedler K., Kuhlmann F., Schlick-Steiner B.C., Steiner F.M. and Gebauer G. 2007. 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Electronic supplemental material (ESM)

Evaluating the importance of trophobiosis in a Mediterranean ant community: a stable isotope analysis

Katrin Brewitt1, Josep Piñol2, 3, Christiane Werner1,4, Wolfram Beyschlag1, Xavier Espadaler2, 3, Nicolás Pérez Hidalgo5,

Christian Platner1

1Department of Experimental and Systems Ecology, University of Bielefeld, 33615 Bielefeld, Germany

2CREAF, 08193 Cerdanyola del Vallès, Spain

3Univ Autònoma Barcelona, Campus Bellaterra, 08193 Cerdanyola del Vallès, Spain

4AgroEcoSystem Research, University of Bayreuth, 95447 Bayreuth, Germany

5 Department of Biodiversity and Environmental Management, University of León, 24071 León, Spain

e-mail: [email protected]

Table S1

Table S2

Table S3

Figure S1

Table S1 Number of observed contacts between aphids (sc=scale insect), their host plants and ants in June 2010: The number of ant species interacting with a single aphid species

(total with ant), the number of aphid contacts per ants species, the number of different host plants for a single aphid species, the number of different aphid species per host plant species and the total number of aphid colonies found on a single host plant species (total with aphids) are given.

Formicidae (n=7) Host plants (n=18)

um

different

pycnocephalus avellana

parviflora clementina

europaea Lasius grandis Formica subrufa Pheidole pallidula Plagiolepis pygmaea Pyramica membranifera Camponotus aethiops Formica rufibarbis No ant Total with ant Sonchus tenerrimus Citrus Malva Pyrus communis Urtica urens Chaerophyllum sp. Avena barbata Carduus Prunus dulcis Corylus Plantago sp. Asteraceae Prunus persica Punica granat Olea Hordeum murinum Rumex sp. Hirschfeldia incana Number of host plants per aphid species 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 Aphis spiraecola 1 2 10 1 6 13 1 9 1 2 3 3 1 7 Aphis gossypii 2 1 6 2 7 9 1 6 1 2 1 5 Baizongia pistaciae 3 3 1 2 6 6 1 Geoica urtricularia 4 3 1 2 6 6 1 Dysaphis plantaginea 5 5 2 5 7 1 Aphis sp. 6 1 2 1 1 1 5 2 3 2 Aphis umbrella 7 4 1 4 5 1 Brachycaudus amygdalinus 8 1 3 1 4 1 Aphididae Gen. sp. 1 9 2 2 2 1 Brachycaudus cardui 10 1 1 1 2 3 1 Sitobion avenae 11 1 5 1 5 1 2 Brachycaudus persicae 12 4 4 4 1 Brevicoryne brassicae 13 1 0 1 1

Aphididae (n=21) Myzus persicae 14 1 0 1 1 Corylobium avellanae 15 2 0 2 1 Hyalopterus amygdali 16 2 0 2 1 Myzocallis coryli 17 2 0 2 1 Uroleucon sp. 18 3 0 1 2 2 Uroleucon sonchi 19 4 0 4 1 Aphis fabae 20 8 0 1 1 3 1 2 5 Hyperomyzus lactucae 21 17 0 17 1 sc Ceroplastes sp. 22 (1) (2) Number of different contacts 7 3 5 5 3 1 1 5 3 3 2 4 2 3 3 1 2 1 3 3 1 2 1 1 1 Total with aphids 24 15 8 8 8 6 17 5 4 4 3 5 7 2 0 1 1 1

Evaluating the importance of trophobiosis in a Mediterranean ant community: a stable isotope analysis

Katrin Brewitt, Josep Piñol, Christiane Werner, Wolfram Beyschlag, Xavier Espadaler, Nicolás Pérez Hidalgo, Christian Platner (2015)

Insectes Sociaux

Table S2 Metrics of a tripartite network analysis of a trophobiotic mutualist network

Number of Mean links Connectance Nestedness Weighted H′2 Robustness Generality combinations per species temperature nestedness (higher level (links) (NODF) species) 1.Level (Host-plant- 40 1 0.101 17.5 2.057 0.72 0.655 2.231 Hemiptera) 2. Level 25 1.3 0.286 19.8 9.596 0.53 0.627 4.244 (Hemiptera-ants)

Connectance ranges from 0 to 1 and equals the proportion of realized interactions in the network. Nestedness measures departure from systematic arrangement of species by niche width and ranges from 0 for fully nested (“cold”) to 100 for non-nested, chaotic (“hot”) networks. The weighted index of nestedness WNODF expresses better how the nested pattern resembles abundance gradients among species and ranges from 0 for non-nested to 100 for fully nested networks (Almeida-Neto M., Ulrich W. 2011. A straightforward computational approach for measuring nestedness using quantitative matrices. Environ. Modell. Software 26:173–178). The network-level measure of specialization H′2 ranges from 0 to 1 and quantifies the standardized two-dimensional entropy across the matrix (Blüthgen et al. 2006). Robustness R ranges from 0 to 1 and quantifies the secondary extinction risks of aphids following random extinctions of plant species and accordingly the robustness of ant species against random extinctions of aphid species. Generality is the effective mean number of host plant species per aphid species and of the visited aphid species per ant species, respectively.

Table S3 Mean shifts in stable isotope 13C und 15N contents between host-plant and aphid, with standard error of the mean (S.E.), sorted by Δδ13C, number of analyzed samples in parentheses.

difference aphid host 13 difference aphid host 15 13 S.E. δ C 15 S.E. δ N plant δ C plant δ N

Corylobium avellanae (1) -3,54 2,45 Ceroplastes sp (2) -1,12 0,68 3,50 0,81 Aphis umbrella (4) -0,92 1,12 3,83 1,04 Baizongia pistaciae (2) -0,71 0,19 2,59 1,59 Brachycaudus amygdalinus (2) -0,56 0,57 -1,53 1,32 Brachycaudus persicae (1) -0,51 2,03 Aphis spiraecola (8) -0,48 0,38 0,41 1,22 Hyperomyzus lactucae (16) -0,43 0,24 -3,78 0,81 Aphis sp (3) -0,37 0,57 -3,69 2,01 Aphis fabae (2) -0,28 0,96 0,49 0,10 Sitobion avenae (5) 0,22 0,02 1,57 0,75 Uroleucon sonchi (5) 0,28 0,32 0,50 0,86 Brachycaudus cardui (2) 0,45 0,14 4,28 4,16 Hyalopterus amygdali (1) 0,49 -3,59 Dysaphis plantaginea (3) 0,55 0,79 3,31 1,10 Aphis gosypii (11) 1,03 0,47 -2,38 0,66 Brevicoryne brassicae (1) 1,28 -6,97 Myzus persicae (1) 1,84 0,25

3

Evaluating the importance of trophobiosis in a Mediterranean ant community: a stable isotope analysis

Katrin Brewitt, Josep Piñol, Christiane Werner, Wolfram Beyschlag, Xavier Espadaler, Nicolás Pérez Hidalgo, Christian Platner (2015)

Insectes Sociaux

Pheidole pallidula (10)

Pyramica membranifera (1)

Camponotus aethiops (1)

Formica rufibarbis (1)

Plagiolepis pygmaea (2)

Lasius grandis (14)

Formica subrufa (12)

-6 -4 -2 0 2 4 6 8 10

difference 15N ant-aphid difference 13C ant-aphid

Fig. S1 Mean difference in δ13C- and δ15N-values between ants and attended aphid colonies, with standard error of the mean (error bars), sorted by Δδ13C, number of analyzed samples in parentheses (n differs from those in

Table S1, because more contacts were observed than samples were analyzed).

4