Fig. 1. Study sites and catchments in the European Alps with indicated
glacier cover (right; CORINE land coverage 2018).
Characterization of habitat conditions and
catchments
At each site, river water temperature was recorded continuously (hourly
measurements) with digital loggers (Onset Tidbit2, MA, U.S.A.) and water
was filtered through pre-combusted WhatmanTM GF/F
filters (0.7 um) to quantify sediment concentrations (3x 0.5 L). Water
discharge was estimated using depth/velocity transects and the Ott
discharge probe (Ott MF Pro, Kempten, Germany), and water samples were
taken to quantify nitrate (NH3-N), dissolved nitrogen,
and dissolved and total phosphorous concentrations. The concentration of
suspended sediment (mg L-1) was assessed by combusting
filters at 450°C for 4 hours. We modelled the sediment load (mg
s-1) by combining discharge (L s-1)
and sediment concentrations (mg L-1). Each catchment
was delineated using QGIS (QGIS Development Team, 2009) using highly
resolved digital elevation data retrieved from the Austrian data
repository (www.data.gv.at). The ratios of
glacial land-cover were calculated based on the freely available CORINE
Land Cover data
(https://land.copernicus.eu/pan-european/corine-land-cover) from
2018.
Periphyton screening
We used the BenthoTorch² (bbe Moldaenke, Germany) to differentiate among
the main periphyton groups that estimates the composition of the
following larger periphyton groups based on their in vivofluorescence excitation spectra: cyanobacteria (pigment-group A),
diatoms and chrysophytes (pigment-group B), and green algae
(pigment-group III). The fast non-invasive measurements allowed
spatially highly resolved assessments of total chlorophyll aconcentrations and relative contributions of the different periphyton
groups across a river. We analyzed 10-15 cobble surfaces (diameter
>10 cm) in each of the 3 transects across each study stream
on three occasions during summer 2022 (July, August, September),
resulting in up to 45 sampled cobble surfaces per study river and month.
Periphyton sampling and biomass
quantifications
Periphyton was sampled over the same three transects across each study
reach with up to 7 sampled cobble surfaces per transect. All scraped
surfaces per transect were pooled and divided into 50 mL fractions (3):
two were transferred onto GF/C filters (1.2 µm;
WhatmanTM) and one was kept frozen until fatty acid
analyses. Ash-free dry mass (AFDM, expressed in mg
cm-2) was quantified by drying filters for 24 hours at
60°C followed by ashing at 450°C for 4 hours (loss on ignition (Heiri,
Lotter & Lemcke, 2001)). The chlorophyll a content (µg
cm-2) was quantified using a U-3900H spectrophotometer
(Hitachi, Tokyo, Japan) following the extraction protocol as described
elsewhere (Lorenzen, 1967).
Fatty acid analysis
Lipids and their FA were analyzed as described by (Guo et al. ,
2015). Briefly, total lipids from freeze-dried (i.e., all lipids and FA
were reported as dry weight; DW) periphyton retained on filters
(~5 mg) were dissolved in ice-cold chloroform (2 mL) and
stored under N2 atmosphere over night at −80°C to
improve lipid extraction efficiency. Samples were then further extracted
in chloroform-methanol (2:1) and NaCl (0.8 mL; salt wash), vortexed and
sonicated, and subsequently analyzed gravimetrically in pre-weighed tin
capsules (total lipid content determination). Fatty acids from total
lipid extracts were derivatized to fatty acid methyl esters (FAME) in a
H2SO4 methanol solution for 16 hours at
50°C. All FAME were stored at -80°C until being separated using gas
chromatography (THERMOTM Trace GC) and detected using
flame ionization detection (FID). FAME were separated by a Supelco™
SP-2560 column (100 m, 25 mm i.d., 0.2 µm film thickness), identified by
comparison to the retention times of known standards (37-component FAME
Mix, Supelco 47885-U; Bacterial Acid Methyl Ester Mix, Supelco 47080-U).
The FAME concentrations were quantified using calibration curves based
on known standard concentrations. All FAME analyses were replicated
within the study design (e.g., 3 periphyton samples per sampling at each
transect).
Data analysis
Habitat conditions crucial for primary producers in alpine rivers (water
temperature indices, sediment concentration and sediment load,
concentrations of nutrients such as nitrate and dissolved phosphorous)
were linked to glacier cover in the catchment (GCC, %) using general
linear models (GLMs) or generalized additive models (GAMs), depending on
the type of the relationship. The percentage of explained deviance (R²)
was used to evaluate model performance. As different water temperature
indices we calculated the average of daily means, daily ranges, and
daily maximum and minimum for two weeks after sampling (July 2022). For
comparisons of sediment and nutrient concentrations, we also considered
data from the previous year, 2021. This extended dataset allowed us to
assess the variability for these parameters.
The relationships between glacier cover (and associated habitat
conditions) and the biomass of periphyton groups were tested using
GLMs/GAMs. Data manipulation and testing was done using R v.4.2.2
(R Core Team, 2021), GAMs were built using the mgcv package
(Wood, 2011), graphics were produced using ggplot2 (Wickham,
2016, 2) and ggtern (Hamilton & Ferry, 2018). Biotic data were
assessed in the summer months (July, August, and September) in the year
2022 only.