This dataset contains data of fatty acid profiles described in the paper: “Guo et al. (2021) The dark side of rocks: an underestimated high-quality food resource in river ecosystems. Journal of Ecology, https://doi.org/10.1111/1365-2745.13647”.

This study was a field investigation conducted along a subalpine river continuum in Austria and aimed to identify the nutritional role of a “hidden” food resource for aquatic consumers; the biofilms growing on the underside of rocks (dark biofilms). Dark and light (i.e., upper surface of rocks) biofilms, and invertebrates were collected, and their fatty acid (FA) composition was analyzed.

Main results were: (1) compared with light biofilms, dark biofilms contained greater proportions of bacterial FA, long-chain saturated FA (biomarkers of terrestrial plants), and oleic acid (18:1ω9; a fungal biomarker), but a lower proportion of algal FA, especially omega-3 polyunsaturated FA (ω3 PUFA). (2) The ω3 PUFA composition in dark biofilms was strongly correlated with that in light biofilms. (3) the overall FA profiles of dark biofilms were significantly associated with invertebrate FA profiles. (4) Strong correlations were also observed between invertebrates and dark biofilms for bacterial FA and the ω3 PUFA eicosapentaenoic acid (EPA, 20:5ω3).

Sample collection

Biofilms and macroinvertebrates were sampled at 8 riffles from upstream to downstream in the subalpine River Ybbs catchment, Austria,  across three seasons, i.e., summer (July), fall (October) and winter (November) in 2017. Three paired samples of light and dark biofilms were collected for FA analyses along a 20-m reach from each riffle. Each paired sample was collected using a quadrat (1.5 m * 1.5 m), and five cobbles (diameters ranging from 8 to 16 cm) within the quadrat were randomly picked. Light and dark biofilms were scraped from the substrate with a toothbrush and kept separate.

Macroinvertebrates clinging to the above cobbles within each quadrat were washed into white trays. Ecdyonurus sp. (Heptageniidae, Ephemeroptera), which is the most abundant algal grazer in the study streams (Kühmayer et al., 2020), was selected for analysis. Although there are four Ecdyonurus species from our study streams, including E. helveticus (Eaton, 1885), E. venosus (Fabricius, 1775), E. dispar (Curtis, 1834) and E. picteti (Meyer-Dur, 1864), more than 95% of the larvae were late instars of E. helveticus (Moog, 2002). All four species have identical feeding habits and are assigned to the same functional feeding groups: 50% grazer and 50% detritivore/gatherer/collector (Moog, 2002). One replicate with at least 10 individuals of Ecdyonurus sp was collected from each riffle. All samples were immediately placed in zip-lock plastic bags, stored on ice and kept in the dark in a portable freezer. Samples were brought to the laboratory within 4 hours and placed in a -80°C freezer until further processing.

Due to the heavy snow in early November, there was no access to one of the upstream sites (WO), where biofilms and invertebrates were not collected. There was limited access to the sites FB, TB and WO during winter and invertebrates were not collected.

Sample processing

All biofilm and macroinvertebrate samples were freeze-dried (Virtis Genesis Freeze Dryer). Dry mass from each biofilm sample (~10 mg) and from each invertebrate sample (5-7 mg) was used for the lipid extractions. Lipids were extracted and methylated according to the methods reported in Guo et al (2016c), with nonadecanoic acid (19:0) used as an internal standard. Fatty acid methyl esters (FAME) were analysed using a gas chromatograph (THERMO Trace; FID 260°C, Carrier gas: He: 1 ml/min, Detector gases: H₂: 40 ml/min, N₂: 45 ml/min, air: 450 ml/min, temperature ramp: 140°C (5 min)– 4°C/min–240°C (20 min) = 50 min) 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 mm film thickness), and FA peaks were identified by comparison of their retention times with known standards, i.e., 37-component FAME mix (Supelco 47885-U) and 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 percentages relative to the total FA (FA%).