Introduction
Diatoms are photoautotrophic unicellular organisms present in almost
every freshwater habitat (Zimmermann, Glöckner, Jahn, Enke, &
Gemeinholzer, 2015). They are one of the main components in the
nitrogen, phosphorus, silicon and carbon biochemical cycles, being
responsible of at least the 25% of the global carbon dioxide fixation
and the 20% of the global net primary production (Wilhelm et al., 2006;
Zimmermann, Jahn, & Gemeinholzer, 2011; Zimmermann et al., 2015). In
addition, benthic diatoms are very important ecological indicators as
they are highly sensitive to environmental conditions and have
relatively short generation times (Vasiljević et al., 2014; Visco,
Apothéloz-Perret-Gentil, Cordonier, Esling, Pillet, & Pawlowski, 2015;
Round, Crawford, & Mann, 1990). Consequently, analyzing their
communities is important to provide an overview of water quality and
allow the detection of environmental changes in freshwater assemblages
(Kermarrec et al., 2014).
Researches efforts have historically been focused on understanding how
ecological communities are assembled in space and time and how they vary
at local scale (Bishop, Robertson, van Rensburg, & Parr, 2015; Leibold
et al., 2004). In this vein, the metacommunity concept, first defined by
Leibold et al. (2004) as “a set of local communities linked by
dispersal of multiple potentially interacting species”, offers the
possibility to examine the complex ecological mechanisms, since it takes
into account the regional-scale processes in addition to local ones
(Leibold et al., 2004). From the very beginning (Brown, Sokol, Skelton,
& Tornwall, 2016), four not mutually exclusive paradigms -patch
dynamics, species sorting, mass effect and neutral perspective- have
been associated with metacommunity organization. Each of the four
paradigms is characterized by the amount of weight they place on a
combination of regional and local processes, disturbance, and the degree
to which species are equivalent in their functional biology (Brown et
al., 2016). More specifically, in neutral perspective, speciation,
extinction, migration and immigration processes participate structuring
ecological communities, while in patch dynamics there is a
colonization-competition trade off. In species sorting perspective,
environmental filtering and biotic interactions filter the occurrence of
the species, whereas in mass effect paradigm, high rates of dispersal in
addition to environmental factors are taking into account (Heino et al.,
2015; Göthe, Angeler, Gottschalk, Löfgren, & Sandin, 2013). Taking in
consideration a metacommunity perspective, the β-diversity concept (i.e.
variation in community composition and structure among sites in a
geographical area) is essential to understand ecosystem functioning,
since it provides important information about the patterns of diversity
and processes that modify the ecosystems (Bonecker et al., 2013;
Florencio, Díaz-Paniagua, Gómez-Rodríguez, & Serrano, 2014). As a
consequence, several studies examining diatoms β-diversity at various
spatial scales (Green et al, 2004; Smucker & Vis, 2011; Leboucher et
al., 2019; Rodríguez-Alcalá et al., 2019) have reported that spatial
structure is responsible of a significant proportion of the community
variance, which suggest that diatoms lack strict ubiquitous dispersal
and thereby exhibit clear biogeographical patterns (Soininen, 2007).
Morphological identification of diatoms species is subjected to a
bottleneck since the number of expert taxonomists is decreasing whereas
the number of identified taxa is increasing rapidly (Pečnikar and Buzan,
2013). Traditional taxonomic identification relies in morphological
traits and light or electron microscopy, so is time-consuming and
demands specialized knowledge (Blanco, 2020). In addition, the presence
of cryptic species and phenotypic plasticity found in some species may
hinder classical species identification (Hadi et al., 2016). In this
vein, several studies have recently revealed the presence of hidden
diversity on diatoms (Trobajo et al., 2010; Rovira, Trobajo, Sato,
Ibáñez, & Mann, 2015), suggesting that its diversity has like been
underestimated (Mann & Vanormelingen, 2013). As a consequence,
exclusive reliance on morphological traits may lead to ambiguous
identification of taxa (Zimmermann et al., 2011; Kowalska, Pniewski, &
Latała, 2019). To overcome the biases related with morphological
identifications, alternative techniques such as DNA barcoding have been
developed in recent years. The term DNA barcoding was first used by
Arnot, Roper, & Bayoumi (1993) to refer the possibility of
differentiate stocks and lineages of Plasmodium falciparum based
on targeting tandem repeats of circumsporozoite gene. DNA barcoding
relies in a short DNA fragment, which is sequenced and compared with a
reference library, allowing the identification of all taxa independently
of its life stage (Zimmermann et al., 2015; Rimet, Vasselon, Keszte, &
Bouchez, 2018; Hebert, Cywinska, Ball, & deWaard, 2003). In addition,
DNA metabarcoding combine DNA barcoding methods with high-throughput
techniques (HTS) allowing the sequencing of millions of DNA fragments
from many samples simultaneously (Rivera et al., 2017). Metabarcoding
has been applied regularly to inventory taxonomic diversity of
freshwater diatoms since the pioneering study of Kermarrec et al.
(2013). Since then, several studies have highlighted the usefulness of
metabarcoding approach in combination with morphological methods for
diatom biomonitoring (Mora et al., 2019; Zimmermann et al., 2015).
However, the incompleteness of reference database still remains as one
of the main biases constraining the accuracy of molecular-based
inventories.
Here, we used a comprehensive dataset of 22 ponds located in a
Mediterranean landscape to (i) assess if morphological and metabarcoding
approaches could be comparable methods in β-diversity studies of benthic
diatoms, and (ii) test whether environmental filtering and dispersal
limitation, predictably assemble diatom communities. Importantly, we
also assessed the influence of taxonomic resolution (genera and species
level) on the observed community-level patterns and processes. We
focused our study exclusively on incidence values, avoiding the use of
abundance values in order to prevent the biases related with the copy
number variation of the marker gene per cell (Vasselon, Domaizon, Rimet,
Kahlert, & Bouchez, 2017). Following recent works (Rimet et al., 2018;
Medlin, 2018; Kowalska et al., 2019), we hypothesized that morphological
methods based on light microscopy and metabarcoding approach provide
similar results when it comes to the β-diversity patterns of diatom
metacommunities (Hyphothesis 1 ). On the other hand, we expected
to find a relationship between environmental template and compositional
variation, since freshwater diatoms have usually been found to be
strongly related to environmental dissimilarity at regional extents
(Hyphotesis 2 ; Declerk, Coronel, Legendre, & Brendonck, 2011).