1. There is growing recognition of the importance of food quality over quantity for aquatic consumers. In streams and rivers, most previous studies considered this primarily in terms of the quality of terrestrial leaf litter and importance of microbial conditioning. However, many recent studies suggest that algae are a more nutritional food source for riverine consumers than leaf litter. To date, few studies have quantified longitudinal shifts in the nutritional quality of basal food resources in river ecosystems and how these may affect consumers.

2. We conducted a field investigation in a subalpine river ecosystem in Austria to investigate longitudinal variations in diet quality of basal food sources (submerged leaves and periphyton) and diet source dependence of stream consumers (invertebrate grazers, shredders, filterers and predators, and fish). Fatty acid (FA) profiles of basal food sources and their consumers were measured.

3. Our results indicate systematic differences between the FA profiles of terrestrial leaves and aquatic biota, i.e., periphyton, invertebrates and fish. Submerged leaves contained very low proportions of long-chain polyunsaturated fatty acids (LC-PUFA), which were conversely rich in aquatic biota. While the FA composition of submerged leaves remained similar among sites, the LC-PUFA of periphyton increased longitudinally, which was associated with increasing nutrients from upstream to downstream.

4. Longitudinal variations in periphyton LC-PUFA were reflected in the LC-PUFA of invertebrate grazers and shredders, and further tracked by invertebrate predators and fish. However, brown trout (Salmo trutta) contained a large proportion of docosahexaenoic acid (DHA, 22:6ω3), a LC-PUFA almost entirely missing in basal sources and invertebrates. The fish accumulated eicosapentaenoic acid (EPA, 20:5ω3) from invertebrate prey and may use this FA to synthesize DHA.

5. Our results provide a nutritional perspective for river food web studies, emphasizing the importance of algal resources to consumer somatic growth and the need to account for the longitudinal shifts in the quality of these basal resources.

This study was carried out in the subalpine River Ybbs catchment, Austria (47°45'N, 15°12'E). The area has a temperate climate with evenly distributed precipitation over the year. The primary land use in the catchment is forestry, with alpine meadows and agriculture constituting only a small area in the lower catchment. All study streams were selected in the upper catchment, Weiße Ois, which had little or no human disturbances in the study reaches or their upstream catchments. The substrata of study streams were cobbles, and the underlying geology was primarily limestone. Nine study streams were chosen, with consideration of different levels of riparian canopy cover (shade vs open), altitude (associated with temperature) and nutrient concentrations (dissolved inorganic nitrogen (DIN) and soluble reactive phosphorus (SRP)), including 2 headwater streams with shaded canopy, 2 headwater streams with open canopy, 2 midstream sites with open canopy and 3 downstream sites with open canopy.

Basal food sources, i.e., submerged leaves and periphyton, and their consumers, i.e., macroinvertebrates and fish were collected from 9 streams in October 2016. All samples were collected along a 20-m reach from each stream. Submerged leaves were picked from water by hand. Periphyton and macroinvertebrates were sampled using a 1.5 m x 1.5 m quadrat. Three replicated samples of periphyton were collected from three quadrats, respectively, and each periphyton sample was collected from five different cobbles by scraping them with brushes. Macroinvertebrates clinging to those cobbles within each quadrat were washed into a white tray, identified to Genus and assigned to FFG (Cummins & Klug, 1979). Macroinvertebrate grazers in the study streams included Ecdyonurus sp., and Baetis sp.; shredders were Nemoura sp., Leuctra sp., Allogamus sp., and Potamophylax sp.; The only filterer collected was Hydropsyche sp.; predators included Rhyacophila sp., Plectrocnemia sp., Perla sp., Perlodes sp., and Isoperla sp.. A separate invertebrate sample was preserved for further taxonomic identification. Fish samples were collected by electrofishing, anesthetized and killed in situ according to the Federal Act on the Protection of Animals, Austria (http://www.ris.bka.gv.at). Total body length (mm) of each collected fish was measured.  Three fish species were collected in the study catchment, including Brown trout (Salmo trutta), Rainbow trout (Oncorhynchus mykiss) and European Bullhead (Cottus gobio). However, only juvenile brown trout were abundant and occurred in most sampling sites, and therefore this species was used for FA analyses. Their body lengths ranged from 55 to 218 mm.

All samples were placed in zip-lock plastic bags, stored on ice and kept in the dark in a portable freezer in the field. The dorsal muscle tissue of fish samples was extracted immediately for FA analyses when back in lab. All FA samples were placed in a -80 °C freezer until further processing, and the separate invertebrate sample was preserved in 75% ethanol for invertebrate identification.

All FA samples of fish, macroinvertebrates, periphyton and leaves were freeze-dried (Virtis Genesis Freeze Dryer). After freeze-drying, each sample was homogenized with a glass rod and/ or a food processor. A dry mass of each invertebrate sample (5-7 mg), each fish sample (5-7 mg), each periphyton sample (10 mg) and each leaf sample (50 mg) was used for lipid extraction and FA methylation according to the methods described in Guo et al. (2016). Fatty acid methyl esters (FAME) were analysed using a gas chromatograph (Thermo Trace) equipped with a temperature-programmable injector and an autosampler. FAME were separated by a Supelco^TM SP-2560 column (100 m, 25 mm i.d., 0.2 um film thickness), identified by comparison of their retention times with known standards (37-component FAME mix, Supelco 47885-U; Bacterial Acid Methyl Ester Mix, Supelco 47080-U) and quantified with reference to seven-point calibration curves based on known standard concentrations. FA compositions were expressed as percentage values relative to total FA (FA%).