Insect. Soc. 54 (2007) 393 – 402 0020-1812/07/040393-10 DOI 10.1007/s00040-007-0959-0 Birkhuser Verlag, Basel, 2007
Insectes Sociaux
Research article
Stable N-isotope signatures of central European ants – assessing positions in a trophic gradient K. Fiedler 1, F. Kuhlmann 2, B.C. Schlick-Steiner 3,4,5, F.M. Steiner 3,4,5 and G. Gebauer 6 1
2 3
4
5 6
Department of Population Ecology, Faculty of Life Sciences, University of Vienna, Althanstr. 14, A-1090 Vienna, Austria, e-mail:
[email protected] Department of Animal Ecology I, University of Bayreuth, D-95440 Bayreuth, Germany Institute of Zoology, Department of Integrative Biology and Biodiversity Research, Boku, University of Natural Resources and Applied Life Sciences, Gregor-Mendel-Str. 33, A-1180 Vienna, Austria Institute of Forest Entomology, Forest Pathology and Forest Protection, Department of Forest and Soil Sciences, Boku, University of Natural Resources and Applied Life Sciences, Hasenauerstr. 38, A-1190 Vienna, Austria School of Marine and Tropical Biology, DB23, James Cook University, Townsville, Queensland 4811, Australia Laboratory of Isotope Biogeochemistry, Bayreuth Center of Ecology and Environmental Research (BayCEER), University of Bayreuth, D-95440 Bayreuth, Germany
Received 20 March 2007; revised 17 June and 24 August; accepted 11 September 2007. Published Online First 24 September 2007
Abstract. Studies employing stable isotope technology have greatly contributed to understanding trophic relationships of tropical ants, but temperate-zone ants remain under-explored. We studied d15N values of 43 ant species from three subfamilies sampled across central Europe. After statistically accounting for the effects of elevation and geographical location of habitats, which alter the isotopic composition of nitrogen in ecosystems, significant patterns in N-isotope signatures were detected. These signatures hint at differences across ants in the contribution of plant-derived nitrogen obtained via trophobiosis and nectarivory relative to nitrogen obtained via predation and scavenging. In general, Myrmicinae had higher d15N values than Formicinae, in line with a greater relative importance of trophobiosis in the latter. The genus Myrmica scored especially high, indicating predominantly predacious nitrogen sources. Remarkably, also the granivore Messor cf. structor had high d15N values. This suggests that, despite the major portion of food uptake being made up by plant seeds, this ant could derive substantial fractions of its nitrogen budget from feeding on arthropod corpses or vertebrate faeces. Moreover, this highlights that deductions from observed quantities of ingested food on its relative contribution to ants matter balance should be accompanied by isotope analyses. At the other end of the spectrum, Camponotus and Plagiolepis had low d15N values. In line with multiple field observations, this suggests a contribution of trophobiosis not only to their energy, but also to their nitrogen budget. Formica and Lasius had intermediate 15N values,
which is in agreement with the current view that these ants have mixed diets with a balance between trophobiosis and predation. A possible influence of endosymbiotic bacteria on the isotope signatures of several genera is discussed. This study provides a first application of stable isotope technology to estimate the role of plant-derived nutrients to the nitrogen budget of a larger range of central European ants. Furthermore, it shows that Nisotope analysis is applicable across extended ecological and geographical gradients. Future studies along this line are promising to complement our current understanding of the nutritional ecology of temperate-zone ants. Keywords: Ants, Europe, N-isotope signatures, macroecology, trophic relationships.
Introduction Stable isotope analysis has evolved into a highly sensitive standard method to disentangle food webs and to assess the trophic position of organisms (reviewed by Fry, 2006). The basic principle of such studies is the process of fractionation: many metabolic processes in organisms discriminate between the lighter and heavier non-radioactive isotopes of, e.g., carbon or nitrogen. This leads to characteristic shifts in isotope signatures from the inorganic environment across primary producers to consumers of biomass at higher trophic levels. Typically,
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organisms at higher positions in a food web are characterized by relative enrichment of the heavier stable isotopes. Many important discoveries have been made with this technique, e.g., about the extent of intra-guild predation among predacious arthropods (Rickers et al., 2006) or nutrient fluxes in decomposer food-webs (Hendrix et al., 1999). These results frequently contradict expectations based on direct, yet anecdotal observations of nutritional links in food webs, or deduced from taxonomic affiliation. Stable isotope studies have also been applied from early onwards to and become increasingly popular with social insects, including termites (e.g., Boutton et al., 1983; Hyodo et al., 2006) and ants (e.g., Rico-Gray and Sternberg, 1991; Blthgen et al., 2003; Davidson et al., 2003; Tillberg and Breed, 2004). Ants are generally viewed as important predators, especially of other arthropods (Carroll and Janzen, 1973; Stradling, 1978; Hçlldobler and Wilson, 1990). Due to physiological constraints as genuine predators, most ants lack a direct trophic link to plant biomass. Exceptions to this rule include the seed harvesters (which specialize on protein- and lipid-enriched food that strongly differs from vegetative plant tissues in structure and nutrient content; Hçlldobler and Wilson, 1990), and the fungus-growing leaf cutter ants (which feed on both plant sap and fungal products, with quantitative contributions differing across species and developmental stages: Wirth et al., 2003). Seed harvesters and leaf cutters are restricted in their occurrence to a few ant clades. In contrast, an alternative mode of gaining direct access to plant-derived nutrients is extremely widely distributed in the ant world: the collection of liquid nutrient sources such as plant nectar, the excretions of plant sap-sucking insects (honeydew), or the nectar-like secretions of certain specialist herbivorous insects (Daniels et al., 2005). Blthgen et al. (2003) and Davidson et al. (2003) showed that ants exhibiting high fidelity to nectar or honeydew differ strikingly in their N-isotope signatures from omnivorous or entirely predacious ants. Thus, these nectarivores not only obtain energy in terms of fuel for adult metabolism from their plant-derived diet but also plant-derived nitrogen that plays a significant role in their matter budget during growth and development. In extreme cases, nectarivorous ants may have d15N values as low as those of true herbivores. These studies have contributed to solving a long-standing puzzle (Tobin, 1991; Davidson, 1997) of how putatively predacious organisms like ants can be so hyper-abundant in tropical rainforest canopies despite the apparent scarcity of potential prey (but see Discussion for a potential influence of endosymbiotic bacteria as explanation for low d15N values). While quite a number of stable isotope studies have been published for tropical ants (e.g., Fisher et al., 1990; Sagers et al., 2000; Fischer et al., 2002; Blthgen et al., 2003; Davidson et al., 2003; Tillberg and Breed, 2004; Blthgen et al., 2006), there is a surprising paucity of such investigations in the temperate zones (but see Fischer et al., 2005; Mooney and Tillberg, 2005; Platner, 2006;
Stable N-isotopes of central European ants
Tillberg et al., 2006). Moreover, based on in-depth analysis of intracolonial and intraspecific variation of N and C isotopes in temperate-zone ants, notes of caution have recently been sounded regarding the interpretation of stable isotope data in social insects in general (Tillberg et al., 2006). Besides methodological aspects (preservation of samples, body part analysed) and the need for coverage of intraspecific variation, these authors highlighted properties unique to social insects, such as a potential nutritional worker-brood divide which should be considered. The present study aims at providing a first insight into stable isotope patterns in central European ants. We accounted for the methodological caveats made by Blthgen et al. (2003) and Tillberg et al. (2006), also by confining the analysis to N-isotope signatures which are considered robust to ethanol preservation of samples and were shown to be more indicative of trophic levels than were C-isotope signatures. We included samples from a wide range of central European ant species, with respect to diets and systematic affiliation, from a variety of habitats to a degree hitherto unparalleled in isotope studies on ants, and aimed at including samples from as diverse localities as possible. Moreover, we collated ant individuals on the level of sampled colonies, trusting that for this baseline study, exploring variation across species, rather than within colonies, would be of relevance. Specifically, we address the question whether it is possible to infer the relative importance of nitrogen derived from plant nectar and / or trophobiosis for different ant species. While qualitative observations (e.g., Seifert, 2007) indicate that trophobiosis or harvesting of extrafloral plant nectar occurs widely in the central European ant fauna, few studies have thus far aimed at quantifying this impact and are largely confined to Formica red wood ants (e.g., Horstmann, 1970). Yet, since these studies were based on observational evidence, they could not answer the question as to whether the large amounts of honeydew and nectar that wood ant colonies collect are only used for the energy requirement to maintain the ants, or also contribute to the build-up of new ant biomass. One would expect highest d15N values in ants that are exclusively predators and scavengers, and lowest d15N values in honeydew consumers and nectarivores (e.g., Blthgen et al., 2003; Davidson et al., 2003).
Material and methods Ant collection From nests in the field, ants were hand-collected at 52 sampling sites, and stored immediately in 70 % ethanol, then oven-dried at 908C for 48 h, and kept dry until analysis. Samples were identified at species level according to Seifert (2007), and voucher specimens were stored in the personal collection of BCS & FMS. Earlier tests had not indicated any effect of alcohol storage on N-isotope ratios (Blthgen et al., 2003; Tillberg et al., 2006). From each ant, the gaster was removed at the petiole before drying. This was important to eliminate the effect of
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undigested food in the ants crop on isotope measurements (Blthgen et al., 2003; Tillberg et al., 2006), though perhaps with some effect due to production of substantial quantities of nitrogenous defensive secretions by some taxa and not others. Our material included 166 samples from 43 ant species from 18 genera and the three subfamilies Formicinae, Myrmicinae, and Dolichoderinae (Appendix). Each sample contained typically 10 ants from the same colony, but often fewer or more, depending on availability and size (between 1 and 30). Intracolonial variation of isotope composition was ignored in the present study. Samples came from Germany (northern Bavaria, Saxony, Sachsen-Anhalt, and Brandenburg), eastern Austria, and Switzerland (Appendix) and spanned an altitudinal range of sites from 40 – 1440 m a.s.l. We aimed at covering a broad range of central European ant species with regard to systematic affiliation and nutritional ecology. All ant species covered in our analyses are qualitatively known, or at least suspected, to be omnivorous in the sense that they do feed on arthropods as well as nectar-like fluids (Seifert, 2007). Available information suggests that the degree of nectarivory varies strongly across species, although quantitative data to underpin this notion are largely absent. Isotopic analysis Ant samples were homogenized in a mortar, weighed (0.5 – 1.3 mg dry weight) on an electronic balance (Sartorius M25D, Gçttingen, Germany), and placed in tin capsules. Isotopic N composition of each sample was measured using an elemental analyser – isotope ratio mass spectrometer (EA-IRMS) coupling (EA type 1108, Carlo Erba, Milano, Italy; ConFlo III interface and gas-IRMS delta S, both Finnigan MAT, Bremen, Germany). The deviation of the sample from the international standard in per mil (%) is expressed as: d15 N ¼ ½ðRsample Rstandard Þ 1 103 where Rsample denotes the ratio between the heavy isotope and its lighter counterpart (Rsample = 15N/14N) for the sample, and Rstandard the ratio for the international standard (N2 in the air). N2 from lecture bottles calibrated against the reference substances N1 and N2 for the N isotopes was used as laboratory standard (Gebauer and Schulze, 1991). Reference substances were provided by the International Atomic Energy Agency, Vienna. Reproducibility of isotope measurements for N2 based on the above-described equipment is typically 0.15% or better (Gebauer and Schulze, 1991). Reproducibility was routinely controlled by measurement of acetanilide (Merck, Germany). Acetanilide was furthermore used to calibrate N concentration measurements (Gebauer and Schulze, 1991). Data analysis We first explored whether N-isotope signatures of ants reveal geographical patterns. This is expected since the sources of nitrogen in habitats, and hence the isotopic signature of this element, strongly depend on climatic variations as well as human activities such as fertilization and fuelling. Highest d15N values are expected in habitats which have a strong N input from fertilization or atmospheric deposition, and lowest d15N values at near-natural sites (e.g., Gebauer et al., 1994; Bauer et al., 2000; Stewart et al., 2002; Bragazza et al., 2005; Kellmann, 2005; Choi et al., 2006). Anthropogenic N fertilization of ecosystems inevitably causes an enrichment in the heavier 15N isotope due to saturation effects that open up the naturally more closed N-cycle. Losses of N-compounds from ecosystems (into the ground water or atmosphere) occur primarily with substances that are depauperate in 15N. The consequence is a higher 15N fraction of the nitrogen that remains in the ecosystem and then enters into food webs. We used a multiple regression model with the geographical location of the sampling site, i.e., latitude, longitude and altitude, as predictors, and d15N as response variable. This analysis revealed a clear effect of sampling location on ant d15N values (Table 1). Altitude had by far the largest influence, followed by latitude and, just marginally significant,
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Table 1. Results of a multiple regression of ant d15N values on latitude, longitude, and altitude of the habitats of the ant colonies. Given are standardized regression coefficients b, their standard errors, the partial correlation coefficients r*, the t-statistics, and respective P-values. The regression model was highly significant (R=0.376, F3;162=8.868, P
