Application of nitrogen and carbon stable isotopes (δ¹⁵N and δ¹³C) to quantify food chain length and trophic structure

Increasingly, stable isotope ratios of nitrogen (δ¹⁵N) and carbon (δ¹³C) are used to quantify trophic structure, though relatively few studies have tested accuracy of isotopic structural measures. For laboratory-raised and wild-collected plantinvertebrate food chains spanning four trophic levels we estimated nitrogen range (NR) using δ ¹⁵N, and carbon range (CR) using δ¹³C, which are used to quantify food chain length and breadth of trophic resources respectively. Across a range of known food chain lengths we examined how NR and CR changed within and between food chains. Our isotopic estimates of structure are robust because they were calculated using resampling procedures that propagate variance in sample means through to quantified uncertainty in final estimates. To identify origins of uncertainty in estimates of NR and CR, we additionally examined variation in discrimination (which is change in δ¹⁵N or δ¹³C from source to consumer) between trophic levels and among food chains. δ¹⁵N discrimination showed significant enrichment, while variation in enrichment was species and system specific, ranged broadly (1.4% to 3.3%), and importantly, propagated variation to subsequent estimates of NR. However, NR proved robust to such variation and distinguished food chain length well, though some overlap between longer food chains infers a need for awareness of such limitations. δ¹³C discrimination was inconsistent; generally no change or small significant enrichment was observed. Consequently, estimates of CR changed little with increasing food chain length, showing the potential utility of δ¹³C as a tracer of energy pathways. This study serves as a robust test of isotopic quantification of food chain structure, and given global estimates of aquatic food chains approximate four trophic levels while many food chains include invertebrates, our use of four trophic level plant-invertebrate food chains makes our findings relevant for a majority of ecological systems.