1. Introduction
Biodiversity in streams and rivers is being impacted by multiple
anthropogenic stressors (Jackson et al., 2016). To understand these
impacts, their functional consequences, and management effectiveness
taxonomically highly resolved information with high temporal resolution
is important. However, such information is difficult to obtain through
traditional morphological assessments as many invertebrate species are
small or present only in juvenile stages that are difficult to identify.
Molecular taxonomic approaches, in particular metabarcoding of
environmental DNA (eDNA) collected from water, offer a fast and
cost-effective way to assess biodiversity and are routinely used in
aquatic bioassessments around the world (Deiner et al., 2017). eDNA
metabarcoding is based on extracted DNA shed by organisms via sloughed
cells, feces, gametes or other particles into the water and is thus a
non-invasive method to assess community composition because assessment
is based on water rather than organismal bulk samples (Taberlet et al.,
2012; Valentini et al., 2016). DNA metabarcoding uses high-throughput
sequencing methods to generate comprehensive taxa lists (Brantschen et
al., 2022; Leese, Sander et al., 2021). However, since the reference
databases used to assign taxonomic names to the obtained sequences are
still incomplete (Weigand et al., 2019), not all sequences can be
assigned to species level. Therefore, molecular Operational Taxonomic
Units (OTUs) that are generated according to genetic distance-based
similarity thresholds can be used as surrogates for species. Using OTUs
in addition to species can reveal further insights into ecological
processes (e.g. Beermann et al., 2018).
Despite the obvious advantages, several factors hinder the direct
interpretation of eDNA data (Barnes & Turner, 2016; Harrison et al.,
2019). First, several abiotic factors can influence DNA transport and
detectability and may thus distort the inferred community (Barnes &
Turner, 2016; Harrison et al., 2019), such as discharge and water
temperature. Discharge is an important factor influencing eDNA
detectability in streams because high discharge could lead to more
species being detected by eDNA signals from transported DNA or whole
organisms being swept downstream (Fremier et al., 2019; Shogren et al.,
2017; Carraro et al., 2018). In contrast, high discharge can also dilute
the eDNA signal thus making it more difficult to detect all present
species (e.g., Thalinger et al., 2021), which may particularly impede
the detection of rare species that are already at low abundance. In
addition to discharge, temperature also affects eDNA detectability
(Strickler et al., 2015), either negatively if higher temperatures
reduce eDNA persistence due to increased enzymatic activity or
positively if higher temperatures increase DNA shedding rates (Jo et
al., 2019; Kasai et al., 2020; Strickler et al., 2015). These
potentially contrasting effects of discharge and temperature make it
difficult to predict how they will affect estimates of community
composition.
Second, as a consequence of the phenology of organisms, eDNA
detectability in streams might also be influenced by sampling season.
Depending on the stream type and region, characteristic abundance
patterns can be found for different macroinvertebrate orders, genera and
species throughout the year and across years (Cowell et al., 2004,
Wagner et al., 2011; Wagner, 2004). In addition, the biology of the
different macroinvertebrate taxa has a strong effect on seasonal
community composition. One important factor are differences in
organismal life cycles. While hololimnic species (species with a fully
aquatic life cycle) are presumed to be present in the water the whole
year, merolimnic species (species with aquatic larvae and aerial adults)
leave the water after hatching and have distinct emergence periods,
often lasting up to few months, which can lead to a sudden decline in
sampled benthic communities (Füreder et al., 2005; Jackson & Füreder,
2006). Besides life cycle based community composition changes, streams
also differ systematically with respect to their functional feeding
groups (FFG) in both space and time (Vannote al., 1980). For example,
shredders are typically more abundant in autumn, when the amount of
allochthonous material in streams is highest (Cummins et al., 1989) and
grazers in spring and summer, due to sun exposure supporting the growth
of large biofilms. The functional composition of macroinvertebrate
communities in the form of different FFG affects ecosystem functioning
and is therefore also included in bioassessment approaches (Šporka et
al., 2006). Detecting these community dynamics patteres is important in
aquatic ecology. There is ample evidence that these types of seasonal
differences are reflected in eDNA metabarcoding data (Bista et al.,
2017; Dunn et al., 2017; Zizka et al., 2020) and that season or even
month of sampling lead to different biological assessment results with
eDNA metabarcoding (Jensen et al., 2021; Zizka et al., 2020).
While biodiversity studies addressing larger temporal scales often
suffer from an insufficient resolution (Jackson & Fuereder, 2006;
Pilotto et al., 2020), studies using high resolution temporal data at
smaller scales are still scarce. Especially for macroinvertebrates, eDNA
metabarcoding data has the potential to assess small temporal changes in
community composition time- and cost-effectively to complement future
long-term bioassessment of streams.
Using time series data from a Long-Term Ecological Research (LTER; Mirtl
et al., 2018) site, the aim of this study was to test the effect of
discharge and temperature, and sampling season on stream
macroinvertebrate community composition and species richness determined
from eDNA. Further we included three biological replicates to test for
small scale differences between sampling positions in the river’s cross
section. Small scale differences in sampling position may recover
different lotic communities (Macher & Leese, 2017) but alternatively,
sampling position may have no effect given that eDNA can be transported
over long distances of >12 km in streams (Deiner &
Altermatt, 2014), which may homogenize eDNA community signals across
sampling positions (Macher et al., 2021).
The time series comprises in total 102 eDNA samples taken every two
weeks for 15 months (from 24.05.2017 to 29.08.2018) at three sampling
positions at the same location which served as replicates: (i) the river
surface; (ii) the river bottom; and (iii) the river bank. Community
composition and species richness was determined using high-throughput
mitochondrial cytochrome c oxidase subunit I (COI) gene metabarcoding.
We tested three hypotheses:
- Change in species richness and community composition will be driven by
seasonality and differences in seasons will follow a cyclic pattern
throughout the year.
- Differences in species richness will reflect the diverging life cycles
of different mero- and hololimnic taxa. For example, the biomass of
merolimnic taxa like Ephemeroptera, Plecoptera and Trichoptera (EPT)
will decline in summer after emergence leading to lower species
richness detectable with eDNA metabarcoding, while differences will be
less pronounced for hololimnic taxa like Annelida and Coleoptera.
- eDNA metabarcoding will detect seasonal differences in community
composition for different functional feeding groups (FFG). In
particular, more grazer species will be present in spring and summer,
whereas more shredder species will be found in autumn and winter based
on seasonal food availability. For parasite species that are dependant
on other organisms, overall species occurrence will not follow any
seasonal pattern, as it is linked to the presence of different host
taxa.
Moreover, we also tested the effects of discharge and water temperature
on species richness, although we had no a priori expectations of
what these effects would be.