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\begin{document}

\title{Increasing periphyton biomass and cyanobacteria in alpine streams with
retreating glaciers}



\author[1]{Georg Niedrist}%
\author[1]{Maria Chiara Vulcano}%
\author[2]{Martin Kainz}%
\author[1]{Leopold Füreder}%
\affil[1]{Universität Innsbruck}%
\affil[2]{WasserCluster Lunz}%


\vspace{-1em}



  
  \date{May 21, 2024}


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\selectlanguage{english}
\begin{abstract}
Deglaciation in mountain catchments changes water source contributions
and associated habitat conditions in usually cold and dynamic mountain
rivers. Although recent research has listed consequences of glacier
retreat for aquatic biodiversity, specific invertebrate and algal
groups, quantitative effects on the aquatic food web structure remain
poorly understood. In this study we assessed abiotic habitat conditions
together with the composition and the biomass of periphyton in 5
catchments in the Central European Alps over the snow-free period and
reveal significant links to declining glacier cover. We found that
reduced glaciation leads to decreasing sediment concentrations and also
to lower runoff, which in combination affects the stability of benthic
habitats. The reduced sediment load of the rivers dominates the
consequences of glacial retreat in subsequent river habitats more than
the decreasing nutrient supply. Periphyton biomass in glacier-fed rivers
is expected to increase with ongoing deglaciation and warming, which
subsequently favors particularly diatoms and cyanobacteria. This work
thus illustrates clear links between decreasing glacier cover and the
identity and relevance of aquatic producers in alpine river catchments,
and provides evidence for shifting resource base in terms of biomass
with potential consequences for its nutritional quality.%
\end{abstract}%



\sloppy


\section*{Introduction}

{\label{introduction}}

Accelerated glacier retreat in alpine regions and the European Alps is
reducing melt-water contributions to mountain rivers and is ultimately
changing the habitat conditions for aquatic life in these high-altitude
waters. The retreat of glaciers is linked to a reduced hydrological
variability, a decrease in sediment load, or altered water chemistry
(Milner \emph{et al.} , 2017), but also rising water temperatures
(Niedrist, 2023). Based on their preferences and traits, invertebrates
seem to be differently prepared to such climate change effects (Niedrist
\& F\selectlanguage{ngerman}üreder, 2023). While overall changes of specialized glacier-fed
river communities (i.e., their community structure) and effects of
glacier retreat on freshwater biodiversity are well studied
(Jacobsen\emph{et al.} , 2012; Füreder, 2012; Hotaling \emph{et al.} ,
2017; Cauvy-Fraunié \& Dangles, 2019), the quantitative response of
different biotic components is still poorly understood. In addition,
effects of habitat ameliorations on population structures, as observed
in other extreme ecosystems (Baum \emph{et al.} , 2005; Bærum \emph{et
al.} , 2021), have not been examined yet.

Periphyton, or benthic biofilm, is the dominant food source for
invertebrates in alpine streams (Niedrist \& Füreder, 2017) and
allochthonous input plays a minor role above the tree line
(Vannote\emph{et al.} , 1980). Given the dependency of first
invertebrate consumers upon sufficient basal production, the dietary
availability and quantity of periphyton may regulate grazing
invertebrate abundance with subsequent cascading effects on predatory
insects and cold-water fish populations in stream food webs. While the
occurrence of diatoms, or chrysophytes in mountain and alpine streams
(Hieber \emph{et al.} , 2001; Rott \emph{et al.} , 2006; Uehlinger
\emph{et al.} , 2010) and their response to deglaciation (Rott \emph{et
al.} , 2006; Fell \emph{et al.} , 2018), but also the detailed
composition of biofilms in glacier-fed streams (Brandani \emph{et al.} ,
2022), has been studied thoroughly, quantitative effects have not yet
been estimated and consequences of glacier retreat on periphyton
availability remain thus difficult to predict.

Different algal and bacterial groups that form periphyton (e.g.,
diatoms, green algae, brown algae, cyanobacteria) contain distinct
lipids and fatty acids (i.e., highly nutritious polyunsaturated fatty
acids) at different levels (Galloway \& Winder, 2015; Twining \emph{et
al.} , 2016) that are highly nutritional and required by invertebrate
grazers, so that the nutritional status of periphyton is largely linked
to its lipid composition. Since deglaciation leads to tremendous abiotic
habitat changes (Milner et al. 2017) and shifting niches for algal and
bacterial groups (Uehlinger \emph{et al.} , 2010), changes in periphyton
biomass and lipid composition, thus in the provision of dietary
nutrients are expected.

In this study we quantify and characterize benthic periphyton in
mountain streams that drain glaciated catchments in the eastern European
Alps. These high-altitude catchments vary in the extent of glaciation
and form a gradient of catchment ice cover that served as a proxy for
different stages of glacier retreat for this study.

The aim of this field study was to; a) quantify habitat-specific
environmental changes relevant for the growth of primary producers
(i.e., turbidity, water temperature, nutrients) that are linked to
differences in catchments' glacier coverage; b) quantify the periphyton
biomass along the gradient of glaciated catchment cover, and; c)
identify group-specific responses to changing habitat conditions linked
to glacier retreat. This study tested the hypothesis that reduced
glaciation in mountain stream catchments is negatively linked with the
harshness of physical habitat conditions for primary producers (i.e.,
water temperature, sediment load), resulting in higher periphyton
biomass availability. Further, we hypothesized that the amelioration of
physical habitat conditions might also provide opportunities for
previously non-dominant primary producers to thrive and increase their
abundance. By testing these assumptions, this research provides insight
into the effects of glacier retreat on habitat conditions and the
biomass and composition of primary producers in high-altitude rivers,
thus contributing to a deeper understanding of the impacts of climate
change-driven glacier retreat on primary producers in glacier-fed
streams.

\section*{Material and Methods}

{\label{material-and-methods}}

\subsection*{Sampling design}

{\label{sampling-design}}

This field study was conducted at five alpine streams in the Central
Eastern Alps in the summer of 2022 (sampling in July, August, and
September). The study design included sites at similar elevations (2127
m -- 2363 m a.s.l.), with comparable topography and similar distance
from the water source, but in catchments with differing glacier areas
(Fig. 1) and different stages of glacier retreat.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image1/image1}

\end{center}
\end{figure}

Fig. 1. Study sites and catchments in the European Alps with indicated
glacier cover (right; CORINE land coverage 2018).

\subsection*{Characterization of habitat conditions and
catchments}

{\label{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 Whatman\textsuperscript{TM} 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 (NH\textsubscript{3}-N), dissolved nitrogen,
and dissolved and total phosphorous concentrations. The concentration of
suspended sediment (mg L\textsuperscript{-1}) was assessed by combusting
filters at 450\selectlanguage{ngerman}°C for 4 hours. We modelled the sediment load (mg
s\textsuperscript{-1}) by combining discharge (L s\textsuperscript{-1})
and sediment concentrations (mg L\textsuperscript{-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.

\subsection*{Periphyton screening}

{\label{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 \emph{in
vivo}fluorescence 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
\emph{a}concentrations and relative contributions of the different
periphyton groups across a river. We analyzed 10-15 cobble surfaces
(diameter \textgreater{}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.

\subsection*{Periphyton sampling and biomass
quantifications}

{\label{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 \selectlanguage{greek}µ\selectlanguage{english}m;
Whatman\textsuperscript{TM}) and one was kept frozen until fatty acid
analyses. Ash-free dry mass (AFDM, expressed in mg
cm\textsuperscript{-2}) was quantified by drying filters for 24 hours at
60\selectlanguage{ngerman}°C followed by ashing at 450°C for 4 hours (loss on ignition (Heiri,
Lotter \& Lemcke, 2001)). The chlorophyll \emph{a} content (\selectlanguage{greek}µ\selectlanguage{english}g
cm\textsuperscript{-2}) was quantified using a U-3900H spectrophotometer
(Hitachi, Tokyo, Japan) following the extraction protocol as described
elsewhere (Lorenzen, 1967).

\subsection*{Fatty acid analysis}

{\label{fatty-acid-analysis}}

Lipids and their FA were analyzed as described by (Guo \emph{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
(\textasciitilde{}5 mg) were dissolved in ice-cold chloroform (2 mL) and
stored under N\textsubscript{2} atmosphere over night at -80\selectlanguage{ngerman}°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
H\textsubscript{2}SO\textsubscript{4} methanol solution for 16 hours at
50°C. All FAME were stored at -80°C until being separated using gas
chromatography (THERMO\textsuperscript{TM} Trace GC) and detected using
flame ionization detection (FID). FAME were separated by a Supelco\selectlanguage{english}
SP-2560 column (100 m, 25 mm i.d., 0.2 \selectlanguage{greek}µ\selectlanguage{english}m 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).

\subsection*{Data analysis}

{\label{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 (R\selectlanguage{ngerman}²)
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 \emph{R} v.4.2.2
(R Core Team, 2021), GAMs were built using the \emph{mgcv} package
(Wood, 2011), graphics were produced using \emph{ggplot2} (Wickham,
2016, 2) and \emph{ggtern} (Hamilton \& Ferry, 2018). Biotic data were
assessed in the summer months (July, August, and September) in the year
2022 only.

\section*{Results}

{\label{results}}

\subsection*{Habitat conditions}

{\label{habitat-conditions}}

Water turbidity (i.e., sediment concentration) was mostly differing
among sites and positively linked to glacier cover in the catchment (\%
GCC, Fig. 2A). Along the gradient of decreasing glaciation, sediment
concentration and sediment load (product of concentration and runoff, in
g s\textsuperscript{-1}) decreased exponentially from 30\% to 0\%
glaciation (with an average rate of -12\% and -21\% per 1\%-decrease in
GCC, for sediment concentration and sediment load, respectively) (Fig.
2).

The concentration of nitrate, dissolved nitrogen and dissolved/total
phosphorus was not related with the glacier cover in both years
(p\textgreater{}0.05). Similarly, averaged water temperature metrics for
July 2022 (daily means, daily ranges, daily maxima and minima) were not
related to the glacier cover of the catchments (linear models,
p\textgreater{}0.05, Fig. S1).\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image2/image2}

\end{center}
\end{figure}

Fig. 2. Relationship between the suspended sediment concentration (left)
and the sediment load (right) with the relative glacier cover in the
catchment (\% GCC) in the study streams during July 2021 and 2022 with
indications of relationships (polynomial regressions in orange, linear
model as grey text). Dashed lines indicate 95\% confidence intervals,
vertical dotted line indicates the breakpoint of the relationship.
Y-axis has a log-scale.

\subsection*{Periphyton biomass in differently glaciated
catchments}

{\label{periphyton-biomass-in-differently-glaciated-catchments}}

The mean dry mass of biofilm in the study streams ranged from 1710 to
5740 mg m\textsuperscript{-2}, whereas the mean organic fraction
(ash-free dry mass, AFDM) was significantly lower (ranging from 130 to
693 mg m\textsuperscript{-2}; mean values). Single highest AFDM
densities reached 1181 mg m\textsuperscript{-2} in the least glaciated
catchments. The periphyton dry mass (g m\textsuperscript{-2}) was not
related to the degree of glaciation in the studied catchments (no
significant linear relationship, p=0.78, R\selectlanguage{ngerman}²=0.01, Fig. 3 A). Instead,
AFDM was significantly negatively linked to glaciation in the catchment
(F\textsubscript{1,13}=28, p\textless{}0.001, R²=0.68, Fig. 3 B) in all
sampled months. In streams with larger glacial influence, the AFDM
reached 300 mg m\textsuperscript{-2}, whereas the AFDM of riverbeds with
little (10\%) or no glacial input reached mean values of 500-600 mg
m\textsuperscript{-2}. Compared to the potential link between nutrients
(i.e., NO\textsubscript{3}-N) and AFDM (no significant relationship,
p=0.062), the effect size of the relative glacier cover in the catchment
exceeded the one of nitrate concentration (R²\textsubscript{partial}=
0.45 vs. 0.10).

The biofilm dry mass per study stream varied among transects, with large
deviations found for the total dry mass (estimates within same streams
deviate between 29-48\%, Fig. S2) and smaller variability for the AFDM
(organic fraction; 14 -- 35\% (95\% CI) (Fig. S2). Chlorophyll \emph{a}

contents (\selectlanguage{greek}µ\selectlanguage{english}g cm\textsuperscript{-2}) were negatively related to
glaciation (polynomial model, R\selectlanguage{ngerman}²=0.63, p=0.002, Fig. 3).\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image3/image3}

\end{center}
\end{figure}

Fig. 3. Periphyton mass dynamics along glacier-fed rivers with different
degrees of glaciated catchment (\% GCC) with dry mass (top), ash-free
dry mass (AFDM, center), and chlorophyll a concentration (bottom).

\subsection*{Periphyton community
composition}

{\label{periphyton-community-composition}}

The analysis of the community composition (relative abundance of the
three groups) revealed the dominance of pigment-group B (diatoms and
chrysophytes) over the other two groups, cyanobacteria and green algae
(Fig. 4). While we found individual transects being dominated by green
algae or cyanobacteria, all stream reaches and most sampled transects
(87 \%) were dominated by pigment-group B (diatoms and chrysophytes).
Across all samples (n=45), diatoms made up between 62 \% and 74 \% (95\%
CI) of the producer communities.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image4/image4}

\end{center}
\end{figure}

Fig. 4. Ternary plot of periphyton compositions illustrating that most
sites and transects are dominated by diatoms and/or chrysophytes. Colors
highlight the dominance in all subsamples (transects). Each circle
corresponds to a composite transect sample (each consisting of 7 single
measurements). Shades of grey illustrate sample density in the
respective corner(s), most samples are dominated by
diatoms/chrysophytes.

The biomass of diatoms and chrysophytes was significantly negatively
linked with glaciation (2\textsuperscript{nd} order polynomial model,
R\selectlanguage{ngerman}²=42, p\textless{}0.001, Fig. 5 B), with biomass doubling when
glacier-cover lowers from 30\% to 0.5\%. Pigment-group A was also
negatively related to glacier-cover (R²=0.25, p\textless{}0.001), but
biomass remained low (\textless{}3.2 mg cm\textsuperscript{-2}; Fig. 5
A). In contrast, the biomass of pigment-group C was positively related
to the glacier-cover in a catchment, but remained \textless{}2.5 mg
cm\textsuperscript{-2} (=8 to 22\% of total periphyton biomass, 95\%-CI)
across all study streams. The composition and the contribution of
different groups did not differ significantly among different summer
months (Fig. 5).

The estimated diatom biomass was related to the palmitoleic acid
(16:1n-7) content, a diatom-related fatty acid (F=8.1, p=0.01, R²=0.4,
Fig. 6).\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image5/image5}

\end{center}
\end{figure}

Fig. 5. Biomass of periphyton pigment groups along the gradient of
glaciated catchment cover. Points represent means (n=3) and deviations
limit standard errors of each transect. Lines are polynomial models
(2\textsuperscript{nd} order) best describing the relationships of mean
biomass per stream (n=9). The colors indicate different sampling months
(07 = July, 08 = August, 09 = September).\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image6/image6}

\end{center}
\end{figure}

Fig. 6. Relationship of diatom biomass with the diatom-specific
palmitoleic acid (16:1n-7). Points illustrate mean concentrations per
measured transect in the different study sites. The solid orange line is
a simple linear regression with 95\% confidence interval (brown area),
the fill color of points corresponds to the \%glaciation in the
catchment (with higher values indicated by brighter colors).

\section*{Discussion}

{\label{discussion}}

Glacier-retreat in Alpine catchments is progressing fast with
accelerating rates (Hugonnet \emph{et al.} , 2021), altering the
geomorphological and hydrological characteristics of downstream rivers
(Milner \emph{et al.} , 2017). While consequences for invertebrates
(esp. cold-water specialists) have been extensively studied (e.g.,
Jacobsen\emph{et al.} , 2012; Cauvy-Frauni\selectlanguage{ngerman}é \& Dangles, 2019) and are
well predicted (Wilkes \emph{et al.} , 2023), impacts on primary
producers remain poorly understood (but see Fell \emph{et al.} (2018) on
expected decrease of diatom richness as glaciers retreat). In this study
we quantified how glacial contribution regulates periphyton biomass in
glacier-fed mountain streams, which expands our understanding of glacier
loss effects on primary production in these fast transforming headwaters
(Sudlow, Tremblay \& Vinebrooke, 2023). Abiotic shifts appear to
ameliorate habitat conditions that favor periphyton growth, as evidenced
by higher biomass in less glaciated catchments. Also, a reduced glacial
influence seems to promote the colonization and establishment of less
nutritious basal resources, such as cyanobacteria, demonstrating that
the provision of dietary primary producers to stream consumers is
fundamentally linked with flow regimes of glacial streams.

\subsection*{Ameliorating habitat
conditions}

{\label{ameliorating-habitat-conditions}}

Contrary to the general assumption that stream water temperatures
increase in catchments with lower glacial cover (Füreder \& Niedrist,
2020), our findings revealed that waters from catchments with larger
glaciers exhibited higher summer temperatures compared to those fed by
smaller glaciers. Sites with higher glacial coverage also had the
highest daily minimum temperatures. These temperature patterns suggest
that glacier-fed river temperatures are not simply driven by the
quantity of ice in the catchment, but indicate to a more complex
relationship with glacier cover, discharge, and river morphology
(Williamson, Entwistle \& Collins, 2019). Flat and broad floodplains
might lead to larger river surface areas and allow water to warm faster
(Williamson \emph{et al.} , 2019; O'Sullivan, Devito \& Curry, 2019), as
likely the case in this study. Since periphyton growth is generally
enhanced by increasing temperatures through its direct influence on
metabolic rates and nutrient uptake (Demars \emph{et al.} , 2011),
considering such antagonistic temperature dynamics is important to
predict periphyton shifts as glaciers keep retreating. During summer,
however, water temperature and nutrients may not be the limiting factor
for periphyton growth in glacier-fed streams, but physical habitat
conditions are. In particular, this study confirmed that the effects of
physical limitation outcompetes a potential effect of nutrients on the
periphyton growth in glacier-fed rivers (as hypothesized by
(Sudlow\emph{et al.} , 2023).

This field study across catchments with varying glacier coverage
revealed an exponential decrease in meltwater turbidity and sediment
load as glaciers recede. This milder physical habitat condition enables
periphyton to thrive even during high flow conditions. The reduced
sediment concentrations in the waters reduce drag forces, facilitating
the colonization of habitats by species with outward growth forms
(Rott\emph{et al.} , 2006), rather than solely early colonizers with
small cells (Biggs, Goring \& Nikora, 1998). Furthermore, the study
indicates that the consequences of glacier retreat for sediment load and
water turbidity are significant. These effects can be partially
predicted based on the degree of glaciation in the catchment, with
sediment concentration and load decreasing exponentially when the degree
of glaciation drops \textless{}30\%. On average, sediment concentration
and load decreased by 12\% and 21\% per 1\% decrease in relative glacier
cover, indicating a disproportionally higher decrease of sedimented
substrates in glacial streams with respect to discharge (-8.9\% per 1\%
decrease in relative glacier cover). However, it is important to note
that sediment concentration in glacier-fed rivers can vary considerably
and may be influenced by factors such as glacial activity, river slope,
and flow velocity (Mao \emph{et al.} , 2019).

Reduced sediment concentrations in less glaciated catchments generally
improve the underwater photosynthetically active radiation (PAR) and can
promote periphyton growth to certain extend (Boix Canadell \emph{et al.}
, 2021). In return, sediment transport can, depending on the flow
velocity, abrade and scour periphyton biomass. Despite the potential
photoinhibition by high UV-radiation in clear waters (Martyniuk,
Modenutti \& Balseiro, 2014; Jacobsen \& Dangles, 2017; Elser \emph{et
al.} , 2020), we detected highest periphyton biomasses in rivers with no
or little turbidity and less in waters with higher glacial
contributions. We thus suggest that the light regime and the UV-stress
in the studied sites was of minor importance for primary productivity
than the physical abrasion by high discharge or transported sediment
(Rinke, Robinson \& Uehlinger, 2001; Hoyle \emph{et al.} , 2017;
Sudlow\emph{et al.} , 2023).

Glacial water typically carries higher nutrient concentrations,
particularly nitrogen and phosphorous, which can influence periphyton
growth, as summarized by Sudlow \emph{et al.} (2023). The nitrogen
delivered by glacier melt originates from atmospheric deposition
(Saros\emph{et al.} , 2010). Unlike non-glaciated catchments, glacial
streams still exhibit higher nitrogen concentrations (Hood \& Scott,
2008; Slemmons \emph{et al.} , 2013; Sudlow \emph{et al.} , 2023), but
with glacier retreat, a general decline in nutrient levels is
anticipated to occur across the river network level. However, these
assumptions were only partly confirmed in this study (i.e., we observed
a weak negative link of nutrients with \%glaciation in the catchments),
assuming area-specific (and geology-based) differences. This potential
interplay of factors during glacier retreat underscores the importance
of understanding glacial stream nutrient dynamics to evaluate the
(future) productivity of these ecosystems.

\subsection*{Increasing and diversifying periphyton biomass with
declining glacial
influence}

{\label{increasing-and-diversifying-periphyton-biomass-with-declining-glacial-influence}}

Decreasing glacier-cover in the catchment has been linked to increasing
biomass of benthic biofilms in glacial streams. Thus, warmer, slower,
and less turbid waters seem to promote periphyton growth as suggested by
(Sudlow \emph{et al.} , 2023), suggesting that the suppression by
physical disturbance in glacial streams appears to be greater than the
inhibition by UV radiation in clear mountain waters (Hoyle \emph{et al.}
, 2017). Depending on how periphyton is quantified (dry weight vs.
ash-free dry mass), our analysis reveals different biomass patterns
across the rivers with different glacial influence. We attribute such
difference to the accumulation of glacial flour in the biofilm because
silt accumulation in epilithon that has been reported previously
(Graham, 1990). Thus, this underscores the importance of quantifying
periphyton biomass using ash-free dry mass, also in slightly turbid
rivers.

Generally, the screened periphyton in all study streams was dominated by
the pigment-group B (diatoms and chrysophytes). Although cyanobacteria
and green algae were present at most sites, diatoms and chrysophytes
contributed between 62 and 74\% to the total chlorophyll a content,
similar to the reported dominance of species in such ecosystems
(Rott\emph{et al.} , 2006; Fell \emph{et al.} , 2018) or previous
pigment screenings (Niedrist, Cantonati \& Füreder, 2018). The
quantifications of this pigment group correlated with the content of the
diatom-specific palmitoleic acid in the same samples, which generally
verified the quantifications based on fluorescence measurements. Along
the gradient of glaciation, we found increases of diatoms and
chrysophyte densities. We attribute this to the more benign habitat
conditions in less glaciated catchments (i.e., decreased turbidity,
calmed discharge, elevated temperature) that generally promotes and
allows growth of present periphyton groups (Cauvy-Fraunié \emph{et al.}
, 2016; Sudlow\emph{et al.} , 2023). The more favorable habitat
conditions also support other periphyton groups, such as cyanobacteria.
We observed an increasing cyanobacteria pigment content in the
periphyton as glaciation decreased. Although densities remain below
those of diatoms or chrysophytes even in unglaciated catchments, this
confirms the general advance of cyanobacteria also in oligotrophic
waters (e.g., Reinl\emph{et al.} , 2023) and the known positive growth
response of the cells to increasing temperatures (Lürling \emph{et al.}
, 2013). However, the share of immigrating species in this increase of
biomass remains unknown. To estimate and compare the many species
quantities at taxonomically higher level, methods such as qPCR will be
necessary.

\section*{Conclusion}

{\label{conclusion}}

The rapid retreat of glaciers in mountain catchments (Sommer \emph{et
al.} , 2020; Hugonnet \emph{et al.} , 2021) is causing significant
changes in the hydrological and geomorphological characteristics of
downstream rivers (Milner \emph{et al.} , 2017). Besides the
well-studied effects on invertebrates, especially cold-water
specialists, the impacts on primary producers are only partly understood
(e.g., Niedrist \emph{et al.} , 2018; Fell \emph{et al.} , 2018). This
study focused on quantifying the regulation of periphyton biomass and
the composition by glacial contribution, expanding our understanding of
glacier loss on primary production in these rapidly transforming
headwaters. We revealed that reduced glacial influence leads to
ameliorated habitat conditions favoring periphyton growth, with higher
biomass content observed in less glaciated catchments. Thereby,
reductions in sediment load have been identified most beneficial for
periphyton growth, whereas summer water temperature alone may not be the
limiting factor for periphyton growth in these systems. Given the
regulatory role of sediment for biota in glacier-fed rivers, predictions
of sediment dynamics are crucial for understanding future habitat
developments in mountain streams. However, developing such predictive
models requires data from diverse glacial streams with varying
topographical characteristics. Therefore, we recommend a collaborative
approach that utilizes existing data from observed glacial streams to
improve the accuracy of predictions.

Additionally, the colonization and establishment of groups previously
underrepresented in highly glaciated catchments, such as cyanobacteria,
are promoted by the reduced glacial influence. This study also
highlights the importance of quantifying periphyton biomass using
ash-free dry mass, even in slightly turbid rivers, since sediments might
blur the estimations using dry mass only. Overall, this research
contributes to our understanding of the complex interactions between
glacier retreat, periphyton communities, and their habitats in mountain
rivers, emphasizing the need for collaborative efforts and further
investigation to predict future habitat developments in the face of
ongoing glacier loss across the globe.

\section*{Acknowledgements}

{\label{acknowledgements}}

This research was funded by the Austrian Science Fund (FWF): P 34310.

\section*{Competing interests}

{\label{competing-interests}}

There are no competing interests to declare that are relevant to the
content of this article.

\section*{Data availability}

{\label{data-availability}}

The data used for this study is published in the KNB repository and can
be accessed using this DOI: 10.5063/F1154FH3.

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