We demonstrate isotopic evidence for substantive incorporation of salmon-derived nitrogen into multiple trophic levels of terrestrial litter-based invertebrates from two salmon bearing watersheds. Enrichment in δ15N in terrestrial invertebrates occurs through two possible pathways: 1) direct consumption of salmon tissue and/or predation off of direct salmon consumers such as larval blowflies; or 2) indirect enrichment through δ15N enriched soil and vegetation N pools. Here, the use of the dual isotope method provides insight into the mechanism of salmon nitrogen utilization by terrestrial invertebrates. Direct consumption of salmon, with approximate δ15N and δ13C values of +11.2‰  and -21‰  respectively, would lead to enriched signatures of δ15N and δ13C in animal tissues. For example, consumption of salmon carcasses by larval blowflies (Calliphoridae) has been documented through the dual isotope method . However, terrestrially derived carbon through C3 photosynthesis dominates δ13C pools in coniferous forest soils and salmon-derived carbon is assumed to contribute little to total carbon in litter and soil. The process of indirect utilization of salmon-derived nitrogen by animals has been observed previously in small mammals , whereby individuals were enriched in δ15N but not δ13C. Because we found little differences in δ13C in all trophic groups collected above versus below the waterfalls, this suggests that the primary mechanism of δ15N enrichment is by indirect processes through salmon-derived nitrogen subsidies to soil and vegetation N pools.
δ15N / δ14N ratios of forest nitrogen pools are influenced by the isotopic values of nitrogen inputs and outputs and fractionation that occurs during nitrogen transformations within ecosystems . Nitrogen inputs to typical Pacific coast forest ecosystems include atmospheric deposition and biological nitrogen fixation. In the case of forests adjacent to salmon streams there is substantial evidence that marine-derived nitrogen from salmon is transferred to forest ecosystems through predator activity [11, 12, 14–16], flooding events  and hyporheic zone transfer , and is incorporated into soil N pools through uptake by vegetation [6, 7, 11–13].
Vegetation δ15N values tend to parallel those in the soil and litter across multiple sites and are typically slightly depleted in δ15N relative to the soil source [22, 23]. Recent estimates for the contribution of marine-derived nitrogen from salmon in riparian ecosystems to total plant nitrogen have ranged from 15.5–24% [6, 12, 13]. These values may be conservative as they are based on the assumption of no plant fractionation from the original source nitrogen. In the case of high nitrogen inputs from salmon, vegetation may preferentially assimilate isotopically light nitrogen (even though it is also originally from salmon). However, in nutrient rich habitats fractionation from the source is potentially not as marked compared with nutrient poor soils [23, 24], making %MDN estimates challenging. %MDN estimates from hemlock (Mathewson & Reimchen unpublished data), possibly constituting a large percentage of litter biomass, vary from 23–34% on the Clatse River and 49–66% on the Neekas River depending on degree of fractionation from the source. These estimates are higher than previously reported, yet remain the baseline for comparison with %MDN estimates in our litter-based invertebrate community.
Ponsard and Arditi  observed substantial site variation in litter and soil δ15N due to variations in soil processes and nitrogen sources across small scales (< 1 km). Soil and litter δ15N and δ13C values are not yet available for our sites. However, δ15N values in litter-based terrestrial invertebrates are known to parallel the δ15N values in the litter and soil [25, 26]. We suspect that because vegetation and all invertebrates collected below the waterfall barrier to salmon migration are enriched in δ15N, that soil and litter δ15N are also enriched at these sites. Our data demonstrates that terrestrial invertebrates exhibit a substantial shift in δ15N over a sharp ecological discontinuity (ca. 250 m) in the source of nitrogen to the forest community, as a consequence of a distinct salmon-derived nitrogen subsidy to litter, soil and vegetation N pools. We estimate that %MDN to multiple trophic levels of litter-based invertebrates ranges from 19–71% on the Clatse River and 34–70% on the Neekas River depending on trophic grouping, and on the extent of fractionation from the original source nitrogen. These values are similar to %MDN estimates of hemlock and indicate that salmon-derived nitrogen is cycled from primary producers through multiple trophic levels of litter-based terrestrial invertebrates.
Grouping all invertebrate samples over the entire 100 m riparian zone may have reduced the extent of statistical differences for δ15N in our comparisons above and below falls. This occurs because of a potential isotopic gradient of decreasing δ15N from salmon in terrestrial vegetation with increasing distance from the stream over a relatively small scale (< 100 meters) [11–13]. Nevertheless, our %MDN estimates are higher than any other study investigating salmon nutrient transfer into terrestrial ecosystems and emphasizes the magnitude of the discontinuity that occurs across the waterfall barrier to salmon migration in these watersheds.
These %MDN estimates assume salmon tissue δ15N as the marine end-member in the model. However, there are other factors that can influence these estimates. Vertebrate urine, particularly from bears (Ursus spp.) , faeces and guano deposition may contribute highly to nitrogen inputs during the salmon spawning season. Despite the fact that these inputs are ultimately from salmon tissue consumption, high fractionation during multiple transformation steps prior to nitrogen availability, such as ammonia volatilization , may lead to unknown shifts in the δ15N levels of the source nitrogen. This may increase the microspatial variability in δ15N in litter, soil, and vegetation, and subsequently invertebrates, along the salmon spawning channel.
Variation in δ15N in carabid beetles and spiders collected below the waterfall barrier was substantially greater than above the falls. It was only marginally higher (non-significant) in root feeding weevils and millipede detritivores, possibly due to low sample sizes. This may indicate higher microspatial variability in δ15N in soil, litter and vegetation N pools, increased range of prey resources below the falls, and/ or invertebrate dispersal from other habitats into the zone of substantial salmon transfer.
We detected variation in δ15N at different stream reaches, most likely as a function of abundance and species of spawning salmon. On the Clatse River, δ15N values decreased with increasing distance upstream. Potentially, this might result from a gradient in marine subsidies other than salmon as a function of distance from the estuary . However, this trend was not observed on the Neekas River where δ15N values remain high, even at 2 km upstream. The difference between these two watersheds in the distribution of marine-derived nitrogen appears to be due to topography and the species and distribution spawning salmon. Clatse River is pink salmon dominated, with the majority of spawning, and subsequent predator activity, occurring in the lower 500 meters of the spawning channel  (personal observations). Above 600 meters the stream narrows and the riparian profile becomes increasingly steep on both sides. The Neekas River has high density chum spawning to the base of the falls with high salmon nutrient transfer and predator activity occurring in this region  (personal observations). Chum salmon contain twice the biomass of nitrogen than pink salmon, and this may partly explain the higher %MDN estimates obtained on the Neekas River compared to the Clatse. The distribution of δ15N in these terrestrial invertebrate groups thus appears to be directly correlated to salmon spawning density and biomass, and subsequent predator activity, a pattern that has been observed for δ15N in ground beetles (Carabidae) occurring between watersheds on Vancouver Island .
Differences in the variance of isotopic signatures within a population provide insight as to the range of diet available to the individual. For example, this has been found in stable isotope studies of marine mammals and chimpanzees [29, 30]. In the case of carabid beetles and spiders, high variability in δ15N along the salmon-spawning channel compared to above the falls, may indicate higher prey variability in this region. Variance in isotopic signatures can also indicate mobility between habitats [31, 32]. Carabid beetles, particularly on the Neekas River, exhibited high variance in signatures. The carabid beetle species collected, although brachypterous, can move freely between habitats , and captured individuals may not have obtained their nutrition along the salmon spawning channel for their entire life history.
Correlations between δ15N and δ13C values provide further resolution into individual niche variability. We observed a significant positive correlation between δ15N and δ13C values in carabid beetles and spiders below waterfalls, with access to salmon nutrients, but not above falls. Both groups feed on a diverse array of prey including primary and secondary consumers, and in the case of the ground beetles, vegetative matter as well. Individuals within each group that fed at a higher average trophic level would be expected to exhibit more enrichment for δ15N and δ13C [34, 35]. Alternatively, individuals that fed on salmon directly or on prey that fed on salmon would also demonstrate isotopic enrichment in both isotopes [3–7]. Positive relationships in δ15N and δ13C below the falls and the absence of that relationship above the falls hints that direct consumption of salmon or salmon consumers below the falls may be a factor for some individuals of these species. However, increased range of food resources below the falls would also be consistent with this finding. Furthermore, smaller sample sizes above the falls may have reduced our ability to detect relationships. For the majority of the spiders and ground beetles, direct uptake of the marine isotopes most likely contributes only a minor component to yearly protein intake, as uptake of marine-derived nitrogen occurs by indirect means. The use of dual isotope model becomes most relevant when investigating terrestrial organisms that use salmon protein as a major contributor to diet. This is the case for several terrestrial necrophages including flies (Diptera: Calliphoridae, Scathophagidae, Anthomyiidae), and beetles (Coleoptera: Silphidae, Leiodidae, Staphylinidae)  (Hocking unpublished data).
Animals are isotopically enriched in δ15N and δ13C relative to their dietary intake as a consequence of preferential excretion of the lighter isotope in metabolism , and this allows insight into relative trophic position within a community. Isotopic enrichment varies widely by body tissue, but there is an approximate stepwise enrichment of 3.4 ± 1.1‰ for δ15N [35, 37] and 0.4 ± 1.4‰ for δ13C [34, 38] for each sequential trophic level. Ponsard & Arditi  suggest that there are on average two trophic levels within litter-based invertebrate communities. We also find general evidence for two general trophic levels within the litter-based community at Clatse and Neekas Rivers usually consisting of: 1) root feeders and detritivores (weevils and millipedes) as primary consumers of plant material, and 2) predators (carabid beetles and spiders) that feed on these and other presumed plant feeders within the litter community. Our data, however, provides substantial evidence for a gradient in trophic level among our litter-based invertebrates rather than two distinct trophic groupings, a finding that coincides with that of Scheu & Faica . Millipedes, for instance, were often found to be enriched in δ15N compared to root feeders, a finding that suggests that either weevils (Curculionidae) feed on roots that are somewhat depleted in δ15N compared to litter, or that millipede detritivores utilize some δ15N enriched protein food sources such as bacteria in their guts, or both . Spiders were enriched in δ15N in all cases over those in carabid beetles, and below the falls on the Neekas this constituted a mean difference greater than a single trophic level. Evidence for omnivory is emerging in the carabid beetles [33, 39–42] and the observed discrepancy between spiders and carabid beetles is most likely a result of the purely predaceous versus omnivorous life histories of these groups. Spiders also demonstrated trophic enrichment in δ13C over carabid beetles at all sites. However, spiders were not consistently enriched over root feeders at each site and carabid beetles exhibited the lowest δ13C values. We conclude that, in general, carbon is a poor trophic level indicator . Overall, this suggests that increased trophic and individual niche resolution in stable isotope studies will more likely extend from a detailed taxonomic separation rather than with guild analyses .