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.