The utility of ASVs for community ecology
All the above conclusions about the predominant processes shaping
community composition of soil microarthropods across the Troodos forests
were very similar when based on OTUs or ASVs, contributing to the
broader discussion about whether OTUs should be replaced by ASVs in
metabarcoding studies (Callahan et al., 2017; Porter & Hajibabaei,
2020). As recent read-filtering methods have overcome the need of
clustering to account for amplification and sequencing errors, the
user-defined OTUs (traditionally used as proxies of species-level
entities) could be redundant. However, the exclusive use of ASVs could
affect the biological conclusions drawn from biodiversity analyses, as
patterns of haplotypic diversity can reflect demographic attributes of
populations, and do not always coincide with diversity patterns at the
species level (Martin et al., 2021). In our system, while β diversity
estimates at ASV and OTU levels were correlated and likely shaped by the
same ecological processes as described above, we observed distinct α
diversity (richness) patterns between them, which were explained by
statistically significant differences in geographic or topoclimatic
predictors. OTU richness per site was primarily explained by
topoclimatic conditions, with assemblages hosting fewer OTUs as
elevation and precipitation increased and temperature decreased (Table
1; Figure S3), thus following the general rule of declining species
richness with increasing elevation (Rahbek, 1995), commonly interpreted
as an outcome of environmental filtering driven by temperature or
productivity gradients (Graham et al., 2014; Peters et al. 2016). In
contrast to OTUs, ASV richness varied significantly along a longitudinal
axis with local communities harbouring more haplotypes westwards (Table
1; Figure S3), which might be interpreted as a signature of higher on
average intraspecific genetic diversity in the Western part of the
mountain range, which has historically been less affected by
anthropogenic disturbance (Delipetrou et al., 2008). This finding is in
accordance with population genetic studies of forest trees that observed
high genetic diversity in the western populations of Pinus brutia (Eliades, Aravanopoulos, & Christou, 2018) and Cedrus brevifolia (Eliades, Gailing, Lenemann, Fady, & Finkeldey, 2011). This is
potentially a consequence of the local topography facilitating the
maintenance of higher effective population sizes in this region during
the Pleistocene climatic oscillations, and/or of less intensive
historical human impact (livestock grazing and logging) than in Eastern
Troodos (Eliades et al., 2018). The decline of haplotypic diversity in
soil microarthropod assemblages eastwards may therefore indicate
incipient biodiversity loss, as genetic variation tends to be eroded
more quickly than species diversity under scenarios of global change
(Balint et al., 2011). However, such differences between species and
haplotype diversity were not reflected in patterns of community
uniqueness, as our ASV- and OTU-based estimates of LCBD (local
contribution to β diversity) were correlated and similarly explained by
topoclimatic variation (Table 1; Figure S3), without any clear signature
of historical contingencies, as those likely affecting longitudinal
haplotypic richness patterns across Troodos. Our results therefore
highlight the complementarity of OTUs and ASVs for community
metabarcoding, as such side-by-side comparisons can help to detect
processes that produce uncoupled patterns between the two levels of
diversity (e.g., Reisch & Schmid, 2019).
Our ASV-level analyses were facilitated by the application of the
metamate tool that utilised local and public reference sequence
databases to discard non-authentic ASVs and retain only true biological
sequence variants (Andújar et al., 2021). Although applying the most
stringent filtering in metamate might have caused the removal
of valuable rare biological haplotypes, appreciable intraspecific
genetic variation (on average 2.35 ASVs per OTU) was still retrieved and
produced reasonable haplotype diversity patterns as explained above.
Based on our results, we advocate stringent ASV filtering, as it can
provide informative datasets without compromising the required
reliability for haplotype-level metabarcoding. Even if we cannot be
fully confident that all erroneous haplotypes were filtered out, as the
performance of the approach depends on the completeness of the reference
sequence catalogue (Andújar et al., 2021), the future incorporation of
intraspecific genetic data in local reference databases will provide
further confidence and the opportunities for intra-OTU analyses of ASV
variation (Elbrecht et al., 2018; Zizka, Weiss, & Leese, 2020).