Study area and site selection
The study area is located in the Northwest of the Chubut Province, Patagonia, Argentina, in the ecotone between the Subantarctic forest and the Patagonian steppe, and exhibits a marked altitudinal gradient (2,000 to 600 m above sea level (m.a.s.l.)). Perennial (Austrocedrus chilensis, Nothofagus dombeyi, and Maitenus boaria) and deciduous (Nothofagus pumilio and Nothofagus anctarctica) tree species constitute the Subantarctic forest. Rivers at the study area have a pluvionival regime; therefore, discharge pattern is bimodal, with one peak associated to winter rains (June to July) and a second one originated by snowmelt in spring (September to October) (Coronato and del Valle 1988). The watercourses selected for this study were Nant y Fall (NyF) and Cabeza de Vaca (CVA) streams (2nd order streams). The sampled site at NyF (43° 13′ 24″ S, 71° 25′ 17″ W) is located 3.7 km downstream of Rosario lake (19 km2) in the Futaleufú-Yelcho basin, at an altitude of 690 m.a.s.l. NyF's sub-basin has 15% of the area dedicated to pastures, 24.8% correspond to lakes, and 60.2% to the timberland (H. Claveri unpublished information). Land-use adjacent to the lake and the stream is mainly pasture, with agricultural and extensive livestock grazing activity.
The sampled site selected at CVA (740 m.a.s.l.) (43° 30′ 02″ S, 71° 20′ 49″ W) is located 25.8 km from the headwater and belongs to the Carrenleufú basin. CVA's sub-basin is covered by pastures (29.4%) and timberland (65.4%), and 5.2% of the area corresponds to lakes (H. Claveri unpublished information). Land-use around the stream is primarily wood extraction.
Environmental characterization and sampling procedure
Sampling sites were visited in early autumn (May), late winter (September), and late spring (December) of 2005 and during late summer (March) of 2006, under stable environmental conditions. At each site, substrate size composition was estimated as percentages of boulders, cobbles, gravel, pebbles, and sand in the reach, using a 1-m2 grid (n = 3). Digital pictures of the grid were obtained and processed in the laboratory. Water velocity (ms−1) was measured in mid-channel (thalweg) on three occasions by timing a float (average of three trials) as it moved over a distance of 10 m (Gordon et al. 1994). Average depth (cm) was estimated from five measurements along one transversal profile across the channel with a calibrated stick. At each site (run-riffle areas), water temperature (°C) was measured with a mercury thermometer.
During each sampling, specific conductance (μS20 cm−1), pH, and dissolved oxygen (DO, mg O2l−1) were measured with a multiparameter probe (Hach sensION 156, Hach Instruments, Lovedale, CO, USA). For nutrient analyses, water samples were collected below the water surface and kept at 4°C prior to analysis and transported to the laboratory. Total suspended solids (TSS), nitrate + nitrite nitrogen (NO3 + NO2), ammonia (NH4), and soluble reactive phosphate (SRP) were analyzed following standard methods (APHA 1994).
Biological data collection
Fish and macroinvertebrates were sampled seasonally at the four mentioned dates. Fish were sampled along reaches of 100 m long employing a portable backpack electrofishing gear (Coffelt Mark-10 CPS, output 350 V). The width of the sampling area was coincident with the stream width. In situ, the individuals caught were preserved in cooled containers. At laboratory, fish were counted, weighted (g), and measured in total length (cm) and the stomachs were separated and fixed with 90% alcohol for posterior diet analysis.
Macroinvertebrate samples were obtained using a Surber sampler (0.09 m2; 250-mm mesh size). Three samples from riffles (n = 3) and three from pools (n = 3) were taken at each reach. Samples were fixed in situ with 4% formaldehyde for posterior analysis.
At the laboratory, all the invertebrates represented in the samples, both in fish stomach contents and in benthos samples, were sorted (using a binocular microscope at × 5 magnification), identified to the lowest taxonomic level possible using available keys (Domínguez and Fernández 2009) and counted. Individuals from stomach and benthos samples were identified to the same taxonomic level. Terrestrial invertebrates (including those adults of aquatic insects with aerial phases) in the stomach contents were grouped into a single category (terrestrial items). Algae and vegetal fragments were grouped into a single category (vegetal items). Inorganic material (i.e., little stones) was classified as inorganic items. Total body length of each individual, excluding antennae and terminal cerci, was measured to the nearest 0.1 mm using an ocular micrometer (Zeiss stereomicroscope Stemi DV4, Zeiss, Jena, Germany).
Data analysis
In order to calculate the contribution of a food item to the diet, the dietary coefficient (Q) (Hureau 1970) was employed. This method reduces the biases associated to the use of numeric or weight methods, because it is the product of the percentage by number (%F) and the percentage by mass (%M) of each prey type (Q = %F × %M). According to this index, the prey items were separated into the following categories: main preys, Q > 200; secondary preys, 200 > Q > 20; and occasional preys, Q < 20.
To estimate diet width, the Levins (1968) index was calculated with 95% confidence limits as follows: B = 1/Σp
2
i
, i = 1…n, where p
i
is the proportion of each prey type i in the diet and equals Ai expressed as fraction rather than percentage.
Diet diversity was assessed using the Shannon-Wiener index of diversity (Krebs 1989) according to:
$$ H=-{\displaystyle \sum_{i=l}^s\left({p}_i \ln \kern0.5em {p}_i\right)} $$
where p
i
is the fraction of items in benthos sample that are of category i.
Comparisons between proportions of preys in the rainbow trout stomach contents and in benthos samples were carried out using Ivlev's index. Ivlev (1961) electivity index (Ei) was selected because it handles the situation where a prey item is represented in the diet but absent from field samples or where an item occurs in the field but is absent from the stomach contents. The electivity index ranges between 1 (the prey is represented in the stomachs but absent from benthos samples) and −1 (the prey is absent from the stomach but present in benthos samples) as it is estimated according to the following equation:
$$ \mathbf{I}=\frac{Ei- Bi}{Ei+ Bi} $$
where Ei is the percentage by number of taxon i in the stomach contents and Bi is the percentage by number of taxon i in benthos samples. Additionally, to identify if trouts select preys according to their size, the Ivlev's electivity index was calculated for three size ranges of each prey species (small, medium, and large size).
To identify differences in prey availability and density between streams, habitats, and seasons, we employed one-way analysis of variance. Similarly, to identify seasonal changes in the size of macroinvertebrates from the benthos, we used ANOVA and the post hoc Newman-Keuls test (Sokal and Rohlf 1995). Prior to analysis data were tested for normality and homogeneity of variances using Kolmogorov-Smirnov and Levene's tests, respectively, and log (x + 1)-transformed when appropriate (Gotelli and Ellison 2005). Mann–Whitney test was used to verify whether the length distributions between individuals represented in stomach contents and in benthos samples were different (Sokal and Rohlf 1995).
To compare the size of rainbow trouts between streams, a Kruskal-Wallis test was applied (Sokal and Rohlf 1995).
In order to summarize the variation among seasons in the distribution of fish based on their diets, we performed a Non-Metric Multidimensional Scaling (N-MDS) ordination technique based on the Bray-Curtis similarity coefficient using the software Statistica (version 6.0) (Ludwing and Reynolds 1988; Marshall and Elliott 1997; Clarke and Warwick 1994). Pearson correlation matrices based on quantitative data of trout stomach contents were built for each stream (NyF and CVA). The proximity of points in a resulting 2-D plot graphic indicates a higher degree of similarity, whereas more dissimilar points are positioned further apart.