1 INTRODUCTION
Role of forest ecosystems. Communities inhabiting tree canopies
are believed to contribute significantly to the maintenance of the
diversity, resiliency, and functioning of forest ecosystems (Thompsonet al. , 2009). On a global scale, there are more than 3 trillion
trees on Earth, of which 43% can be found in tropical and subtropical
regions and 22% in temperate biomes (Crowther et al. , 2015).
Forest ecosystems harbour a large proportion of global biodiversity,
contribute extensively to biogeochemical cycles, and provide countless
ecosystem services (Bonan, 2008). Moreover, tree canopies form the most
important interface between Earth’s terrestrial biomass and the
atmosphere (Ozanne et al. , 2003; Ellwood & Foster, 2004) and
contain a multitude of heterogeneous microhabitats conducive to the
evolution of epiphytic plants, animals and microorganisms (Nadkarni,
2001).
Environmental heterogeneity and biodiversity in tree canopies.One of the most general patterns in community ecology is the increase in
species richness with increasing habitat heterogeneity (MacArthur &
MacArthur, 1961; Williams, 1964). Accordingly, the presence of different
structurally complex microsites (microhabitats) within tree canopies was
shown to favour biodiversity of a variety of organisms, including
epiphytes (Lyons, Nadkarni, & North, 2000; Nadkarni, 2001), birds
(Goetz et al. , 2007), small mammals (Carey & Wilson, 2001) and
arthropods (Hijii, Umeda, & Mizutani, 2001; Ishii, Tanabe, & Hiura,
2004).
State of the art (microorganisms). In a similar way,
tree-colonizing microorganisms (i.e., Bacteria, Archaea and microfungi)
formed highly specific communities across broader microhabitat classes
(soil, stems, leaves) (Cregger et al. , 2018). In addition, it was
shown that different plant species harbour species specific
leaf-associated bacterial communities (Lambais et al. , 2006;
Vorholt, 2012) as well as highly specific bacterial communities inhabit
different cryptogamic epiphytes (bryophytes, macrolichens)
(Aschenbrenner et al. , 2017). Accordingly, it can be assumed that
unicellular eukaryotes (protists) show similar diversity patterns. So
far, molecular studies reported distinct protistan communities in mosses
(Mitchell, Bragazza, & Gerdol, 2004; Mieczan & Tarkowska-Kukuryk,
2014), lichens (Bates et al. , 2012; Mazei et al. , 2016),
phytothelmata (Carrias, Cussac, & Corbara, 2001; Dunthorn et
al. , 2012) as well as root associated communities (Turner et
al. , 2013; Dumack et al. , 2020). A recent study on protistan
diversity in tropical forest soils hypothesised that some soil protists
could be a subset of tree canopy communities that have rained down from
above (Mahé et al. , 2017). Nevertheless, a comprehensive
comparative assessment of the protistan communities across different
microhabitats from forest soils to the canopy region is still lacking.
Investigation of protistan niche-level composition across
trees. Accordingly, we hypothesised (1) to find microhabitat-specific
protistan communities in tree canopies, and (2) an increase of species
richness with habitat diversity. Following the terminology of Stein and
Kreft (2015), we define environmental heterogeneity as an
“umbrella term for all kinds of spatial heterogeneity, complexity,
diversity, structure, or variability in the environment”, while we are
focusing here in particular on the sub-categorical term habitat
diversity as a measurement of habitat richness, i.e. the number of
distinct (micro-)habitats and habitat types. To investigate our
hypotheses, we sampled numerous microhabitat compartments across a
vertical gradient, from forest soils to the canopy region of three
autochthonous tree species in a temperate floodplain forest. In a
metabarcoding approach, we focused on two exemplary prominent taxa of
protists which are commonly known to be plant associated (Ploch et
al. , 2016; Flues, Bass, & Bonkowski, 2017; Sapp et al. , 2018),
namely the Cercozoa (Rhizaria) and the Oomycota(Stramenopila). In order to improve coverage of investigated taxa,
taxon-specific primers were used to amplify protistan DNA. Considering
variation in taxonomic resolution of DNA barcodes, two different markers
were targeted in this study: the hypervariable V4 region of the 18S rRNA
gene and the Internal Transcribed Spacer 1 (ITS1) for barcoding
cercozoan and oomycete communities, respectively.
The aim of this study was to shed light on unicellular eukaryotic
diversity and community composition in forest soils and the canopy
region. Unveiling these protistan distribution patterns will contribute
to the understanding of environmental factors shaping cercozoan and
oomycete communities across different ecological compartments
represented by the structural complex ecosystem of tree canopies.