Discussions of the factors regulating nutrient recycling by consumers have focused on predictions from Ecological Stoichiometry (ES) and the Metabolic Theory of Ecology (MTE). ES posits that imbalances between the composition of an animal’s body tissues and its diet should determine its nutrient excretion rates, whereas the MTE predicts that excretion should directly reflect metabolic activity arising from body size and temperature. Each framework has been supported by data, but they are rarely tested together. In this study, we measured excretion rates of nitrogen (NH4), phosphorus (SRP) and N:P excretion ratio, body N:P stoichiometry, body size, and temperature for 12 species of fish from an Atlantic rainforest stream in Brazil. We fitted 8 competing models reflecting different combinations of ES (body N:P, armor classification, diet group) and MTE (body size, temperature) variables. For both N and P excretion, as well as excreted N:P ratio, only body size was included in the best model, and interspecific differences in size-scaling were greater for N than for P. Fitted size scaling coefficients were lower than the MTE prediction of 0.75 for both N (0.59, 95% CI = 0.45, 0.73) and P (0.56, 95% CI = 0.40, 0.77). There was only weak evidence that body armor in 3 of 12 species led to more retention of P, and there was no discernable effect of diet group, body N:P, or water temperature. We conclude that differences in nutrient excretion among species within a shared environment primarily reflect contrasts in metabolic rates arising from body size, rather than disparities between consumer and resource stoichiometry. Our findings align with those from other ecosystems and synthesis across aquatic taxa, expanding support for the MTE as the primary framework for predicting nutrient excretion rates. Key words: ecological stoichiometry, metabolic ecology, animals, nitrogen, phosphorus, freshwater.

DNA metabarcoding is used to enumerate and identify taxa in both environmental samples and tissue mixtures. The composition and resolution of metabarcoding data depend on the primer(s) used. Markers that amplify different genes can mitigate biases in primer affinity, amplification efficiency, and reference database resolution, but few empirical studies have evaluated markers for complementary performance. Here, we assess the individual and joint performance of 22 markers for detecting species in a DNA pool of >100 species of primarily marine and freshwater fishes, but also including representatives of elasmobranchs, cephalopods, and crustaceans. Marker performance includes the integrated effect of primer specificity and reference availability. We find that a portfolio of four markers targeting 12S, 16S, and multiple regions of COI identifies 100% of reference taxa to family and nearly 60% to species. We then use the four markers in this portfolio to evaluate metabarcoding of heterogeneous tissue mixtures, using experimental fishmeal to test: 1) the tissue input threshold to ensure detection; 2) how read depth scales with tissue abundance; and 3) the effect of non-target material in the mixture on recovery of target taxa. We consistently detect taxa that make up >1% of fishmeal mixtures and can detect taxa at the lowest input level of 0.01%, but rare taxa (<1%) were detected inconsistently across markers and replicates. Read counts showed weak correlation with tissue input, suggesting they are not a valid proxy for relative abundance. Despite this limitation, our results demonstrate the value of a primer portfolio approach—tailored to the taxa of interest—for detecting and identifying both rare and abundant species in heterogeneous tissue mixtures.