Taxonomic composition
Because different data sets in this study included ESVs, OTUs or
morphospecies, the direct comparisons of their taxonomic unity were
performed at the genus level. In this study, metabarcoding results from
10 g and 0.5 g of DNA extracts exhibited highly concurring taxonomic
composition, with only few mismatched taxa, which were represented by a
low relative abundance of reads. In accordance with the community
analyses, this indicates sample-size independent patterns when detecting
diatoms via metabarcoding from lake sediments. However, comparisons
between microscopy and metabarcoding data resulted in a higher number of
mismatched taxa (Fig. 4). Not completely matching identifications from
microscopy vs . metabarcoding have been reported in several
previous diatom-related studies (e.g. Visco et al. 2015; Riveraet al. 2018; Tapolczai et al. 2019), with the possible
reasons discussed within. One of the main reasons of such mismatches is
the incompleteness of the reference sequence databases, which consists
of a limited number of annotated taxa. For example, Sichuaniellalacustris , discovered only by morphological analyses, is the
unique representative of the genus Sichuaniella, which was
originally described from Sichuan Province on the southeastern Tibetan
Plateau (Li, Lange-Bertalot & Metzeltin 2013) and has no genetic
information in the public databases. Therefore, the identity of this
species in the metabarcoding data set cannot be confirmed. Additionally,
there are no reference sequences for genera such as Platessa ,Odontidium and Gomphosinica in the public databases.Gomphosinica has been separated from Gomphonema and
described as a new genus based only on their morphological differences
(Kociolek, You, Wang & Liu 2015). Thus, Gomphosinica in the
microscopy data set could potentially be represented asGomphonema in the metabarcoding data set.
The inter-investigator variation depending on changes in diatom taxonomy
and the use of synonym names could add additional layers for the
mismatches between microscopy and metabarcoding data. In this study, it
is difficult to consistently separate Staurosirella andPseudostaurosira (missing from metabarcoding data) fromStaurosira (present in metabarcoding data) under the light
microscope and even with support from SEM images. AlthoughPseudostaurosira was one of the most abundant genera in the
microscopy data, it was missing from the metabarcoding inventories,
whereas the relative abundance of Staurosira was high in latter
data sets (Table S3). Medlin, Yang and Sato (2012) have pointed out that
the molecular separation of Pseudostaurosira andStaurosirella from Staurosira is arguable. On the other
hand, in the few studies that have attempted to merge morphological- and
molecular-based phylogenies of the Fragilariaceae, the morphological
characterization is often poorly done (Morales et al. 2019). We
speculate that morphologically identified Pseudostaurosira(especially Pseudostaurosira brevistriata ) corresponds toStaurosira in the metabarcoding data, as their presence-absence
patterns in our sediment samples correlates well (Table S3).
Furthermore, identification of Pseudostaurosira in the
metabarcoding data sets was also limited due to a fact that almost all
originally named Pseudostaurosira were re-assigned toStaurosira in the curated R-Syst diatom database (Rimet et
al. 2016).
The majority of other missing genera from metabarcoding data sets were
represented in very low abundances in the microscopy data. Similarly,
Kermarrec et al. (2013) reported that morphologically identified
low abundance taxa (< 1% from 450 valve counts) were often
not detected in the DNA metabarcoding data set. These low abundance taxa
may indicate the transport of diatom valves with highly degraded DNA
from other locations (thus non-detectable with herein used primers). On
the other hand, environmental DNA could be carried along large distances
(Deiner & Altermatt 2014), which also could contribute to the observed
‘extra’ diatom taxa in metabarcoding data sets, which were not detected
via microscopy. Moreover, some of the diatom taxa with fragile and
weakly silicified valves, such as Cylindrotheca ,Entomoneis , Fistulifera , Reimeria ,Seminavis , that were detected only in the metabarcoding data
sets, might be sensitive to the chemical treatment (e.g. HCl and
H2O2) during sample preparation for
microscopy. Based on personal observations of water samples from Nam Co,
we have confirmed the presence of several Entomoneis andFistulifera species (data not shown), which further supports the
assumption that valves of fragile diatom species may be more prone to
dissolution and therefore undetectable in sediment samples. Thus,
incompleteness of the reference databases, together with the
continuously changing diatoms classification system and DNA
transportation characterizations contribute to at least some extent to
the issue of non-matching taxa between microscopy and metabarcoding
results.