4 DISCUSSION
Why did the protists not obey the rule of increased species
richness with increasing habitat diversity? Our findings show how
discrete microhabitat niches lead to compositional heterogeneity of
microbial communities within tree canopies, and ultimately within whole
ecosystems (Peay, Kennedy, & Talbot, 2016; Cregger et al. ,
2018). Application of taxon-specific primers ensured an exhaustive
coverage of the investigated protistan taxa, a crucial precondition to
minimise the number of taxa absent due to undersampling (Supplementary
Figure 2). Furthermore, a study comparing the outputs of general and
group specific primers has shown that structuring effects of the
environment on protistan communities could only be seen in the data sets
generated with specific primers (Lentendu et al ., 2014). With
data obtained this way, we could report a further increase in the
diversity of investigated protistan taxa, as it was already demonstrated
by Fiore-Donno et al. (2018). Thus, the majority of detected OTUs
account for a large amount of undescribed diversity (Figure 1) and may
represent so far uncharacterised lineages, especially in the taxon of
oomycetes, as only 34% of the OTUs were 97%–100% similar to any
known sequence.
A world‐wide survey on forest litter to investigate patterns of
diversity in a group of testate amoebae observed that this common group
of terrestrial protists behave like macroscopic organisms. Documented
community structures were strongly correlated with climatic and
physicochemical parameters but also with geographical barriers (Laraet al. , 2015). We observed Canopy communities to be strikingly
different in terms of beta diversity compared to litter and soil
communities on the ground, and as expected, different microhabitats
within tree canopies were further colonized by distinct protistan
communities, especially by Cercozoa (Figure 3A). However, the
hypothesised rule of increased species richness with increasing habitat
diversity could not be confirmed due to the almost ubiquitous
distribution of protistan taxa (Figure 5). Our data exemplify that
microorganisms do not always obey ecological rules assigned to
multicellular organisms. Moreover, our findings are in contrast to
patterns observed for bacteria (Lundberg et al. , 2012; Ottesenet al. , 2013; Wagner et al. , 2016), epifoliar fungi
(Gilbert, Reynolds, & Bethancourt, 2007) or lichens (Boch et
al. , 2013; Marmor et al. , 2013). Most of these groups are
directly dependent on specific environmental conditions or the
availability of resources offered by the habitat (Vandenkoornhuyseet al. , 2015; Sasse, Martinoia, & Northen, 2018). However, high
turnover rates and the ability of protists to form cysts as resting
stages imposes less constraints on their distribution over large
distances due to a reduced likelihood of isolation and high diversity of
source pools for local community assembly (Fenchel & Finlay, 2004;
Bahram et al. , 2016). Accordingly, habitat diversity strongly
favoured certain protistan taxa in terms of relative abundance, but the
effect of OTU richness on community composition was negligible due to
their almost ubiquitous distribution.
Mahé et al. (2017) hypothesised that protists in soils may be a
subset from the canopy that have rained down from above; a pattern
confirmed for leaf endophytic fungi in one-year old beech litter of
temperate forests (Guerreiro et al. , 2018). On the other hand,
there is growing evidence of cercozoan species particularly adapted to
life in the phyllosphere (Dumack et al. , 2017; Flues, Bass, &
Bonkowski, 2017, Sapp et al. , 2018). Cercozoan phyllosphere
communities, isolated from fresh canopy leaves, were overall indeed
surprisingly similar to leaf litter communities on the ground (Figure
3A). However, this cannot be confirmed for protists in general, as
differences in oomycete communities between phyllosphere and ground
litter showed strikingly different patterns. While fresh canopy leaves
and ground litter had highest OTU richness of Cercozoa (Figure 5A),
ground litter contained a significantly depleted diversity of oomycetes
(Figure 2B). Interesting were the resembling patterns of beta diversity
between phyllosphere and deadwood Cercozoa and oomycetes which deserve
further attention in future studies. The small, but significant
differences of oomycete communities between tree species (Figure 4B,
Supplementary Table 5) might be explained by differences in host
specificity, since oomycetes are well known to contain specific
pathogens infecting leaves, stems and roots of forest trees (e.g. Rizzo
& Garbelotto, 2003; Lehtijärvi et al. , 2017). Apparently, tree
species do not shape their associated protistan communities to the same
degree as bacteria, where even different genotypes of the same tree
species can show distinct spatial patterns in their colonizing bacterial
communities (Redford et al. , 2010; Leff et al. , 2015;
Cregger et al. , 2018). Heterotrophic (or organotrophic) bacteria
experience a direct selection pressure by differences in nutritional
resource composition between plant microhabitats (Thapa et al. ,
2017), while the proportion of bacterivorous Cercozoa at the next
trophic level lack this direct dependency on the host-tree species
(Figure 4A).
Compared to the highly specific bacterial communities of tree bark,
mosses and lichens (Aschenbrenner et al. , 2017), canopy protists
appear to rather depend on microhabitat characteristics. This is best
exemplified within the cryptogamic epiphytes. Lichen and the two moss
taxa harboured quite similar protistan communities (Figure 3). These
epiphytes are characterised by rapidly changing conditions with rapid
swelling and storage of moisture from morning dew and after rainfall to
severe dryness at sunshine (e.g. Jonsson et al. , 2014; Benítezet al. , 2018) and to a certain degree may act as environmental
filters selecting specific protistan communities.
Protistan communities of arboreal soil samples showed high variability,
spanning from moss-like communities to soil-like communities (Figure 3).
Importantly, this indicates that protistan communities resembling those
of mineral soil are not restricted to the forest floor. Community
variability in arboreal soil might be due to the varying degree of decay
of the sampled material and its distinct physico-chemical properties
(Nadkarni et al. , 2002), and further strengthens our hypothesis
that increasing habitat richness may result in increasing compositional
heterogeneity of protistan communities.
Conclusions
Beta diversity of protists was solely driven by differences in the
relative abundance of OTUs, because almost all taxa were ubiquitously
distributed among tree crowns and soil of the floodplain forest.
Accordingly, species richness did not increase with habitat diversity as
hypothesized, although strong differences in beta diversity between
protistan communities of the forest floor and tree crowns, and among
microhabitats within tree crowns, demonstrate strong differences in
relative abundance. Different tree species had a surprisingly low
influence on protistan community assembly; even the mostly
plant-parasitic oomycetes did not show a high degree of
host-specificity. However, a high number of OTUs from canopy communities
could not be assigned to any known sequence, giving evidence that
protistan communities of tree canopies are largely understudied. Both
strata show unique protistan communities, indicating no top-down
relationship of investigated protistan taxa in trees. Thus, our findings
illustrate that the diversity of soil protists is solely shaped by the
habitat itself, from which no conclusions regarding the total diversity
of the canopy can be drawn. The occurrence of only a few specialist OTUs
does not imply functional homogenization at the community level across
microhabitats, but rather indicates increasing functional diversity to a
greater extent than increasing OTU richness with increasing habitat
diversity (Ofek-Lalzar et al. , 2014). Future studies will be
needed to further address this hypothesis, supplementing functional
traits to the taxonomical assignment of the investigated protistan OTUs.