Discussion
We found that the ecotone, stretching from subalpine mountain birch
forest to treeless low-alpine vegetation, was the primary structuring
gradient shaping the soil biota. Both the fungal communities, as well as
the overall micro-eukaryotic communities, were structured primarily
along the ecotone. Variation in community composition between sites was
largely accounted for by regional variation in climate, as well as other
site-specific factors, such as slope, aspect and bedrock, the latter
affecting nutrient content.
We observed that the belowground community composition largely reflected
the aboveground shift in vegetation. However, for fungal communities,
the 18S and ITS2 markers showed substantial differences in abundance and
distribution of fungal groups across the ecotone. Most noteworthy,
Archaeorhizomycetes (Ascomycota) and Mucoromycota dominated in the 18S
dataset, and Chytridiomycota and Cryptomycota were also abundant across
the entire ecotone. In contrast, these groups were poorly represented in
the ITS2 dataset. On the contrary, the subphylum Pezizomycotina,
represented mainly by Leotiomycetes, Eurotiomycetes, Lecanoromycetes and
Dothideomycetes, was largely absent in the 18S dataset, but made up a
large proportion of the ITS2 dataset. We suggest that these contrasting
results can largely be explained by primer biases (Nilsson, Anslan, et
al., 2019; Rosling et al., 2011; Tedersoo et al., 2015). The employed
ITS primers have been shown to discriminate against e.g.
Archaeorhizomycetes (Ihrmark et al., 2012; Rosling et al., 2011;
Tedersoo et al., 2015) and they also seemingly amplify Mucoromycota,
Chytridiomycota and Cryptomycota poorly. The 18S primers we used in this
study, was expected to amplify all eukaryotes (Hadziavdic et al., 2014)
and provide a more general overview of the fungal community composition
compared to the ITS2 dataset. However, the low amount of Pezizomycotina
in the 18S data indicated that primer biases may be present. Indeed,
after a closer inspection, a mismatch in the reverse primer was observed
in numerous Pezizomycotina lineages. Irrespective of the biases, there
was a general trend of more Ascomycota above the forest line, most
notably Archaeorhizomycota (18S data), and Leotimycetes and
Eurotiomycetes (ITS2 data).
Several groups of Ascomycota in the ITS2 data represented plant
root-associated fungi. For example, the overall most common OTU showed
high sequence similarity to Pezoloma ericae , a common ErM fungus
(Smith & Read, 2008) . In the species ordination plot, most of the
root-associated ascomycetes, representing ErM and DSE fungi, clustered
in the alpine heath along the ecotone, where ericaceous plants,
especially Empetrum nigrum , dominated. The gradual increase of
Leotiomycetes and Eurotiomycetes towards the low alpine vegetation (ITS2
data) is in line with the pattern showed in Tedersoo et al.(Tedersoo et al., 2014), where these groups had highest relative
abundances in arctic tundra. ErM and DSE fungi living under stressful
conditions typically have melanized hyphae. Melanin is a recalcitrant
polymer of the hyphal cell wall that may protect fungi against
desiccation (Fernandez & Koide, 2013). Melanin induces slower
decomposition and the retention time of highly melanized fungi in the
soil may thus be longer, in comparison with non-melanized fungi
(Fernandez, Heckman, Kolka, & Kennedy, 2019; Fernandez & Koide, 2014).
Fungal necromass (i.e. dead biomass) is an important part of boreal soil
carbon pools (Clemmensen et al., 2015), and the longer retention time of
melanised mycelia may explain the strong positive correlation between
root-associated ascomycetes and percent C content in this study. We also
observed an increase of Archaeorhizomycetes towards the alpine heath.
Little is known about the ecology of the Archaeorhizomycetes (Rosling et
al., 2011), but is has been hypothesised that some Archaeorhizomycetes
may be root-associated mutualists (Menkis, Urbina, James, & Rosling,
2014). The high abundance of Archaeorhizomycetes in the low alpine
vegetation may indicate that they are linked to ericaceous plants, along
with ErM fungi and DSEs. Other studies confirm that Archaeorhizomycetes
are relatively abundant in stressful environments (Sterkenburg, Bahr,
Durling, Clemmensen, & Lindahl, 2015), e.g. in high altitude and
latitude ecosystems (Pinto-Figueroa et al., 2019; Schadt, Martin,
Lipson, & Schmidt, 2003).
In parallel to the root-associated ascomycetes, we observed that percent
soil C and ergosterol content had a strong positive correlation towards
the low-alpine vegetation, in line with our hypotheses 3. Clemmensenet al. (Clemmensen et al., 2015) showed a similar correlation
between ergosterol and total soil C stock, and argued that this
relationship is due to slower mycelial turnover rate leading to a
long-term build up and increased soil C sequestration (Clemmensen et
al., 2013; Clemmensen et al., 2015; Hagenbo et al., 2017; Kyaschenko,
Clemmensen, Karltun, & Lindahl, 2017). These processes may also account
for the high C and ergosterol content in the low-alpine vegetation.
Percent C content correlated strongly with the community composition in
the ordination diagrams, but with respect to decomposition and cycling
processes, C content is not regarded as a driver, rather a consequence
of the fungal community composition (Clemmensen et al., 2015). However,
it must be emphasised that in this study we only have data on percent C
content and not overall C stock measurements. Hence, to conclude about
overall C stock dynamics, additional data must be collected.
In accordance with hypothesis 2, the distribution of EcM fungi across
the ecotone reflected their host plants distributions, with higher
abundances of EcM fungi below the mountain birch forest line. Our
observations are in line with what observed by Vašutová et al .
(Vasutova, Edwards-Jonasova, Baldrian, Cermak, & Cudlin, 2017), where
EcM fungi declined with altitude. Many EcM fungi are associated with
relatively fast soil organic matter turnover (Bodeker et al., 2014;
Clemmensen et al., 2013; Lindahl & Tunlid, 2015) and may, together with
the saprotrophic fungi, account for the lower amount of soil C observed
below the forest line in our study.
In general, we observed a higher proportion of saprotrophic fungi in the
alpine birch forests compared to the heath, which may account for the
lower fraction of soil C observed here, due to faster decomposition.
Based on the 18S dataset, Mucoromycetes dominated the fungal community
below the forest line. These fungi are foremost known as saprotrophs,
and there is likely a higher abundance of available litter in the forest
compared to the alpine heath. However, it has also been observed that
Mucoromycotina, as fine root endophytes, are important symbionts with
cryptogams (Field et al., 2015; Hoysted et al., 2019), receiving plant C
in exchange of soil N. Hence, Mucoromycetes may play different
functional roles, which may explain the high relative abundance of
Mucoromycota across the entire ecotone.
Although our study was mainly focused on fungi, we found that,Metazoa
was about equally abundant as fungi in the soil measured as sequence
reads. As pinpointed earlier, some fungi Ascomycota might have been
poorly amplified by the 18S marker, so it remains unclear whether the
proportions reflect an unbiased picture of the belowground biota. The
two most abundant Metazoa groups, Annelida and Nematoda, were common
across the entire ecotone, whereas Arthropoda and Rotifera were
relatively more abundant above the forest line. Other studies have shown
that annelids and nematodes typically are more abundant at high
latitudes and altitudes (Phillips et al., 2019; Procter, 1990). This is
in line with the result of this study, where annelids and nematodes are
the dominant groups. Rotifera has also been shown to be abundant in
soils at high latitudes and altitudes, and are often present in mosses
and lichens (Bielańska-Grajner, Ejsmont-Karabin, & Yakovenko, 2011;
Fontaneto & Ricci, 2006), which corresponds well with their relatively
higher abundance above the forest line. Studies have shown that soil
moisture strongly influence protist communities (Bates et al., 2013; de
Araujo et al., 2018; Stefana, Cornelia, Jorg, & Michael, 2014), while
others regard soil pH as more important (Heger, Derungs, Theurillat, &
Mitchell, 2016; Shen et al., 2014). Heger et al. (Heger et al.,
2016) also showed a correlation between protist communities and
altitude. In general, Cercozoa is commonly observed in various soil
types, whereas Ciliophora and Apicomplexa have been shown to be
relatively more abundant in humid soils (Bates et al., 2013). Due to the
parasitic lifestyle of Apicomplexa, their distribution is likely largely
dependent on the presence of host species (Arthropodes) (Mahe et al.,
2017; Seppey et al., 2020). However, at kingdom and phylum level, none
of the protists showed any clear distribution pattern across the
ecotone.
The ecotone, stretching from subalpine mountain birch forest to treeless
low-alpine vegetation, represents a corresponding shift in belowground
fungal communities, from soils dominated by EcM and saprotrophic fungi
to soils dominated by root-associated ascomycetes, respectively. This
study has shown there is a parallel shift in belowground ergosterol
content across the ecotone, which is strongly associated with the
abundance of root-associated ascomycetes in the low-alpine vegetation.
Our findings corroborate the view that despite higher aboveground
productivity, shrubification and raise in forest line in northern
ecosystems may lead to soil C loss because of higher soil respiration
and C cycling (Parker et al., 2015; Sjogersten & Wookey, 2009; Sorensen
et al., 2018), through corresponding shifts in fungal communities.
However, C stock data is needed to conclude on this matter. On a
technical note, our study also underlines that in future community
studies of fungi, a wider set of DNA-markers and primers should be
considered to obtain a comprehensive picture of soil fungal communities.