Stable isotope analyses are increasingly employed to characterise population niche widths. The convex hull area (TA) in a δ13C–δ15N biplot has been used as a measure of isotopic niche width, but concerns exist over its dependence on sample size and associated difficulties in among-population comparisons. Recently a more robust method was proposed for estimating and comparing isotopic niche widths using standard ellipse areas (SEA), but this approach has yet to be tested with empirical stable isotope data. The two methods measure different kind of isotopic niche areas, but both are now widely used to characterise isotopic niche widths of populations. We used simulated data and an extensive empirical dataset from two fish populations to test the influence of sample size on the observed isotopic niche widths (TA and SEA). We resampled the original datasets to generate 5000 new samples for different numbers of observations from 5 to 80 to examine the statistical distributions of niche area estimates for increasing sample size. Our results illustrate how increasing sample size increased the observed TA; even sample sizes much higher than n = 30 did not improve the precision for the TA method. SEA was less sensitive to sample size, but the natural variation in our empirical fish δ13C and δ15N data still resulted in considerable uncertainty around the mean estimates of niche width, reducing the precision particularly with sample sizes n<30. These results confirm that the TA method is less appropriate for estimating population isotopic niche areas using small samples, especially when considerable population level isotope variation is expected. The results also indicate a need for caution when using SEA as a measure of trophic niche widths for consumers, particularly with low sample sizes and when the distribution and range for population isotope values are not known.

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1. We assessed spatial and temporal variation in carbon and nitrogen isotopic signatures in different compartments of a single lake ecosystem. Stable isotope analyses were made on samples of particulate organic matter (POM), zooplankton, periphyton, macrophytes, macroinvertebrates and fish collected from several locations throughout the ice-free period. 2. No spatial variation in d 13 C or d 15 N values was found for pelagic samples of POM and zooplankton. However, pelagic d 15 N signatures increased steadily through the summer resulting in an almost 6& average increase in POM and zooplankton. A concurrent decrease in epilimnetic nitrate concentrations suggested that the increase in d 15 N of POM and zooplankton could have resulted from a progressive 15 N-enrichment of the available inorganic nitrogen pool as the size of this pool was reduced. 3. Significant spatial variation in isotopic ratios was observed within littoral and profundal communities. Some spatial differences were likely related to lake-specific characteristics, such as a major inlet and a small harbour area and some were interconnected with temporal events. 4. Marked differences between spring and autumn d 15 N and d 13 C values of fish at one site probably reflected a spring spawning immigration from a larger downstream lake and also indicated limited dispersal of these immigrants. 5. Our results indicate that restricted sampling of ecosystem components from lakes may provide misleading single values for the isotope end members needed for quantitative uses of stable isotopes in mixing models and for estimating trophic position. Hence we strongly advise that studies of individual lakes, or multiple lake comparisons, that utilise stable isotope analyses should pay more attention to potential within lake spatial and temporal variability of isotope ratios.

We evaluated the potential utility of stable isotope analysis of tissues commonly archived by aquatic biologists. Previous studies with chemically preserved samples have shown contradictory results, which present an obstacle for the use of archived sample materials. We tested the effects of ethanol and formalin preservation on zooplankton and of ethanol on benthic macroinvertebrate d 13 C and d 15 N values. We found that neither formalin nor ethanol had a significant effect on d 13 C and d 15 N values of preserved zooplankton. Nor did ethanol significantly affect d 13 C or d 15 N values of macroinvertebrates. However, ethanol preservation slightly, but significantly decreased C:N ratios of both zooplankton and macroinvertebrates, probably reflecting some extraction of lipids. Overall, the effects of preservatives on d 13 C and d 15 N values that we observed were minor. We also compared d 13 C and d 15 N values analysed from roach scales and perch operculum bones with those analysed from muscle tissue. Decalcification of scales and operculum bones only slightly improved our comparison to muscle tissue d 13 C and d 15 N values. Decalcified scales had slightly higher d 13 C and lower d 15 N values. Similarly, decalcified operculum bones showed slightly increased d 13 C and decreased d 15 N values to those for fish muscle. Our results confirm that scales and operculum bones can provide a suitable proxy for fish muscle in isotope studies with minor correction. We conclude that various archived sample materials can indeed be used with confidence for historical reconstructions of freshwater food webs by stable isotope analysis.