This dataset includes stable isotopes δ¹⁵N and δ¹³C to explore the effects of body size on fish community trophic structure and niches in one of Australia’s largest river basins — the Murray-Darling. First, we test whether Trophic Position (TP) and δ¹³C scale with body mass within and among species and functional guilds (predator; micro-carnivore; omnivore; algivore-detritivore). Secondly, we test whether isotopic niche breadth scales with body size within and among species or community mass-classes ranging from < 1 g to > 8192 g.

The importance of within species, size-based, trophic structure in our study contrasts with previous evidence suggesting that river food webs are not size-structured.  Food web models and tests of theory which have assumed a single, species-level, TP or δ¹³C are unlikely to fully capture the complex intraspecific size-based trophic dynamics of river fish communities. 

Between March and May 2018, fish tissues and basal carbon sources from primary producers were collected from five large rivers of the MDB. Rivers sampled were the Edward-Wakool, Goulburn, Lachlan, Lower Murray, and Murrumbidgee (Supplementary Fig. 1).  Primary producers were sampled from the Gwydir River, but fish tissue samples were not available from this system. Fin-clips from eight additional Murray cod ranging from 600 mm to 1100 mm TL sampled from the Mid-Murray River, and 36 juvenile Golden perch (Macquaria ambigua) from the Tala Creek in the Murrumbidgee River system were also included to fill gaps in small and large individuals. The fish species sampled make-up the bulk of fish community abundance and biomass in lowland river-floodplain ecosystems of the MDB (Davies et al., 2010), but did not include several rare small-bodied species, predominantly inhabitants of upland or coastal river reaches. Fish were sampled using standardised boat-electrofishing and fyke netting. In each river, fish were sampled at 10 sites, spanning lengths of 20-100 km. Each site was sampled once using 10 fyke nets and 2880 s of electrofishing ‘on time’. Fish were identified and the length of the first 50 individuals of each species per site was measured (mm).  Body mass of individuals was estimated from length using previously published length-weight relationships (Llewellyn, 2011).

Whole fish or a caudal fin-clip of up to 30 individuals from each fish species per river, spanning the sampled body size range, were collected for stable isotope analysis.  A caudal fin-clip (30-50 mm2) from the upper lobe was collected from fish larger than 150 mm, while fish smaller than 150 mm were euthanised to ensure enough tissue for stable isotope analyses and to minimise post-release animal welfare concerns.  Fin-clips were stored in 1.5 mL non-stick centrifuge tubes and whole fish in plastic zip-lock bags on ice and then frozen before preparation for stable isotope analyses.

Basal carbon sources

To provide baseline δ15N for trophic position estimates, and to estimate δ13C of basal resources, four basal resources were sampled during the same period as fish community sampling activities were being undertaken. These included two replicates from each of three reaches within each river located between 10-120 km apart (Figure S1). At each site, the basal resources sampled were: 1) periphyton (benthic algae, filamentous algae and associated biofilm); 2) seston (phytoplankton and suspended fine particulate matter); 3) macrophytes; and 4) terrestrial riparian plants. Periphyton was scraped from woody debris, rocks and macrophytes using five, 2 cm diameter, circular dish scouring pads and placed in zip-lock bags. Seston was collected from epilimnetic water samples in 1 L bottles prefiltered through 250‐μm mesh and then filtered onto precombusted Whatman GF/F filters (pore size 0.7 um). Macrophytes, grasses and plant leaves, seeds and stems were collected from a mixed variety of species visually assessed within a 500 m reach at each site, cut into pieces, placed into plastic zip-lock bags and frozen.

Stable isotope and laboratory analyses

Frozen basal resources, fin-clips and whole small-bodied fish were thawed and rinsed (excluding seston filter papers) with reverse osmosis purified water and dried in glass vials or petri dishes in an oven at 60°C for 48 h (Arrington and Winemiller, 2002). Where possible, fin-clips from a total of at least 20 individuals of each species, representing the full body size range sampled and each river, were processed for bulk stable isotope ratios of nitrogen (15N/14N) and carbon (13C/12C) at the University of Western Australia.  Samples were analysed for δ15N and δ13C, using a continuous flow system consisting of a Delta V Plus mass spectrometer connected with a Thermo Flush 1112 via Conflo IV (Thermo-Finnigan/Germany). The isotope values δ15N and δ13C are reported in relation to [‰, Air] and [‰, VPDB] respectively according to international standards (Skrzypek, 2013).

Trophic guilds and trophic position

Fishes were classified into four trophic guilds based on a diet analysis of adult Australian freshwater fishes by Stoffels (2013). The trophic guilds included: 1) Predator/Piscivore (P): fish that primarily consume fish and in some cases decapod crustaceans and other macroinvertebrates; 2) Omnivore (O): trophic generalists that consume a range of phytoplankton, benthic algae and aquatic or terrestrial invertebrates; 3) Algivore-Detritivore (AD): fish that consume detritus or algae; 4) Micro-Carnivore (MC): fish with diets dominated by insects, aquatic microcrustaceans or other invertebrates. We combined the guilds ‘microcrustacivores’ and ‘aquatic insectivores’  (Stoffels, 2013) into MC since some species could not be clearly distinguished between the two guilds. 

The trophic position (TP) of fishes was calculated according to the equation (Post, 2002, Vander Zanden and Rasmussen, 1999): TP = 1+ (δ15Nconsumer - δ15Nbasal source) / TDF, where δ15Nconsumer is the signature of individual fish, and δ15Nbasal source was the mean δ15N of the basal resources within respective rivers where fish were sampled including (δ15N, mean ± SE): EW (2.85 ± 1.28); GB (2.90 ± 1.30 SE); LC (4.44 ± 1.98); LM (2.54 ± 1.14); and MB (3.59 ± 1.61).  The trophic discrimination factor (TDF) represents the shift in nitrogen between its ingestion by a consumer and its assimilation into the consumers’ tissue. Firstly, we used a constant TDF of 3.4 which is a common baseline used in freshwater food web studies (Post, 2002). To evaluate the sensitivity of our results to uncertainties associated with the TDF, we compared TPs of models based on the constant TDF (TPconstant) with scaled (TPscaled) based on a global meta-analysis (Hussey et al., 2014) and TDFs estimated for fish predators (5.7), microcarnivores (3.4), omnivores (4.3) and herbivores (3.9) in Australian rivers (TPAus.riv.) (Bunn et al., 2013). TPscaled (Hussey et al., 2014) was calculated according to the equation: TPscaled =1+ (δ15Nconsumer - δ15Nbasal source) / 2.94 + 0.22 × (δ15Nbasal source - 3.4).  The TP values shown in the dataset represent TPscaled values.