3 RESULTS
3.1 Sequencing and bioinformatic
pipeline
We obtained 550 OTUs from 1.381.839 sequences (Cercozoa) and 331 OTUs
from 1.610.374 sequences (Oomycota). The average number of cercozoan
OTUs was 516 ± 15 and 546 ± 3 per microhabitat and tree species,
respectively, while the average number of oomycete OTUs was 236 ± 25 and
304 ± 4 per microhabitat and tree species, respectively. Tree canopies
contained a substantially unknown diversity of oomycetes, with 57
oomycete OTUs with less than 70% percent identity to any reference
sequence, while this was the case for only three cercozoan OTUs (Figure
1). While most of the reads and OTUs showed a similarity of 97-100% to
any known reference sequence, oomycete reads revealed two additional
peaks at about 76% and 87%, indicating that a small number of highly
abundant OTUs in oomycetes is still not taxonomically recorded (see
Supplementary Figure 1 and Supplementary Tables 1 and 2 for taxonomic
composition and annotation).
3.2 Alpha diversity
The used taxon-specific primers recovered the majority of OTU richness
from canopy and ground samples (soil and litter) as indicated by
rarefaction curves (Supplementary Figure 2). The extrapolation revealed
that doubling the sequencing depth would have yielded no more cercozoan
and oomycete OTUs, respectively, for all samples combined. All sampled
microhabitats showed high alpha diversity (Figure 2), except for
oomycetes in the ground leaf litter (ANOVA F value = 10.79, p
<0.001; Figure 2B).
3.3 Beta diversity
Most variation in protistan beta diversity was explained by microhabitat
differences (permANOVA, Cercozoa: R 2 0.45, p =
0.001; Oomycota: R 2 0.30, p = 0.001). Another
significant proportion of beta diversity was explained by the
differences between the two strata ground and canopy (permANOVA;
Cercozoa: R 2 0.13, p = 0.001; Oomycota:R 2 0.14, p = 0.001). Only the beta diversity of
the oomycete communities, but not that of the cercozoan, differed
between tree species, yet with only a small amount of explained variance
(permANOVA; Cercozoa: R 2 0.04, p = 0.1;
Oomycota: R 2 0.06, p = 0.01) (Supplementary
Table 5). Non-metric multidimensional scaling of protistan community
profiles revealed a strong separation of canopy and ground strata
(Figure 3). In Cercozoa, communities inhabiting fresh leaves in the
canopy were most distinct to those in mineral soil on the ground (Figure
3A). Leaf litter communities on the ground scaled closer to fresh canopy
leaves than to the underlying soil communities. A clear transition in
beta diversity occurred from fresh canopy leaves to deadwood, bark and
lichen, and finally the moss communities (Hypnum sp. andOrthotrichum sp.). Communities of arboreal soil were highly
variable, ranging from samples with high similarity to mosses to samples
closely resembling the mineral soil communities underneath the litter
layer. Also in oomycetes (Figure 3B), canopy and ground communities were
most distinct. Again, the two ground samples, mineral soil and leaf
litter, showed no overlap, with leaf litter having a low alpha
diversity. In contrast to Cercozoa, oomycete communities of canopy
microhabitats showed a high overlap, and communities of fresh canopy
leaves were clearly distinct from litter on the ground. The only common
pattern between cercozoan and oomycete communities in the canopy was
their high variability in the arboreal soil.
A db-RDA showed the same pattern, but revealed a bit clearer the
similarity of communities of fresh canopy leaves to deadwood in both
protistan taxa (Figure 4). For the Cercozoa (Figure 4A), both axes
explained approximately the same amount of variance. Bark and epiphyte
communities were again more similar to each other; thus these factors
explained the same kind of variance in the community compositions. The
soil communities were clearly different, while leaf litter clustered
together with fresh leaves and deadwood communities. Little variance was
found between tree species and arboreal soil. In the Oomycota (Figure
4B) a similar pattern could be observed. Bark and epiphytes communities
were similar, and soil - together with leaf litter communities -
appeared as separated from the other microhabitats. Tree species
communities appeared further away along the second RDA axe, which
however only explained approx. 5% of the variance.
3.4 Shared OTUs
Despite high differences in beta diversity, the majority of OTUs were
shared between all microhabitats irrespective of the protistan phylum
(Figure 5). Only few combinations yielded more than ten unique OTUs
shared between distinct microhabitats, which is negligible given the
high OTU richness per sampled microhabitat, which varied between 498
(deadwood) and 537 OTUs (fresh leaves) for the Cercozoa (Figure 5A) and
between 189 (leaf litter) and 270 (Orthotrichum moss) for the
Oomycota, respectively (Figure 5B). Because almost all OTUs were shared
between all microhabitats with the species accumulation curve showing
only a flat increase, communities revealed no patterns of nestedness
(Supplementary Figure 3, Supplementary Figure 4).