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 spatial and 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, the possibility to detect a present lotic
macroinvertebrate community can be strongly affected by the selected
sampling position in the water. Similar to stratified aquatic
environments where non-mixed layers need to be considered in the
sampling design (Jeunen et al., 2019; Lawson et al., 2019), eDNA
molecule distribution may also differ between different positions in
lotic environments, such as the water surface versus the riverbed.
Accordingly, small scale differences in sampling position both
vertically and horizontally may recover different lotic communities
(Berger et al., 2020; Macher & Leese, 2017; Thalinger et al., 2021),
producing different perspectives of biodiversity change depending on
where sampling is conducted. 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). Second, 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 .
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 spatial or temporal scales
often suffer from an insufficient resolution (Jackson & Fuereder, 2006;
Pilotto et al., 2020), studies using high resolution spatiotemporal data
at smaller scales are still scarce. Especially for macroinvertebrates,
eDNA metabarcoding data has the potential to assess small temporal and
spatial 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 the
sampling position (i.e., different positions in the river’s cross
section), discharge and temperature, and sampling season on stream
macroinvertebrate community composition determined from eDNA. The time
series comprises 102 total 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: (i) the river surface; (ii) the river bottom; and
(iii) the river bank. Community composition was determined using
high-throughput mitochondrial cytochrome c oxidase subunit I (COI) gene
metabarcoding. We tested three hypotheses:
- Change in community composition will be driven by seasonality but,
because of turbulent flow and mixing, not or less the sampling
position. Differences in seasons will follow a cyclic pattern
throughout the year (‘seasonal clock’) irrespective of the sampling
position.
- Differences in community composition will reflect the diverging life
cycles of different mero- and hololimnic taxa. For example, the
species numbers of merolimnic taxa like Ephemeroptera, Plecoptera and
Trichoptera (EPT) will decline in summer after emergence, while
differences in species numbers 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 community composition, although we had no a prioriexpectations of what these effects would be.