4. Discussion
DNA metabarcoding has become an essential tool for species inventory and
monitoring. However, its use in identifying ichthyoplankton is still
incipient in the Neotropics, with several methodological challenges and
biases still needing to be tackled (Carvalho, 2023). Considering the
crescent demand for innovative techniques to unravel the complex
reproductive dynamics of fish communities for both research and
practical applications, there is an underlying need for the continuous
refinement of this methodology. The COI gene has been commonly used as
the marker of choice (Mariac et al., 2018; Nobile et al., 2019) because
of its well-established primers and complete reference sequences
libraries encouraged by the global initiative Fish Barcode of Life.
Here, each molecular marker recovers a distinct community structure when
considering both quantitative and qualitative analysis.
Using a marker of choice still raises concerns since using several
markers is still expensive when using HTS and because each marker has
distinct amplification biases and taxonomic resolution (Deagle et al.,
2014). The high interspecific variability of the COI gene, when compared
to other mitochondrial genes (Hebert et al., 2003), can help
differentiate closely related species. However, the same high
variability creates the need to use universal degenerate primers with
lower specificity than those designed for more conserved genes. Also, it
hinders the design of internal minibarcodes for COI (Deagle et al.,
2014). In the present study, while COI presented 93.33% sequencing
success and some of its sequences remained unassigned or were assigned
to Bacteria, both 12S markers were successfully sequenced for all
samples, and all their sequences were assigned to fish taxa.
Additionally, the technology used for sequencing limits the total
fragment size to 600bp, precluding the merging of both COI strands from
forming the full-sized barcode, therefore, each strand was analyzed
independently. The loss of resolution power caused by this could explain
why COI detected fewer species, genera, and families than the 12S
markers and why two of the three exclusive species level identifications
were assigned to nonnative fishes closely related to species from São
Francisco.
Minibarcode markers for the 12S gene have been developed and applied to
environmental DNA metabarcoding studies (Milan et al., 2020; Miya et
al., 2020; Sales et al., 2021) and, more recently, to ichthyoplankton
studies as well (Jiang et al., 2022; Van Nynatten et al., 2023). One of
the main concerns when using these markers is the conserved nature of
the gene, which can impact their ability to differentiate closely
related species, especially in diverse regions. However, the current
study shows that both MiFish and NeoFish were able to successfully
identify and distinguish multiple congeneric species, such asLeporinus piau and L . taeniatus ,Megaleporinus elongatus and M . reinhardti ,Pimelodus fur , P . maculatus and P .pohli , and Prochilodus argenteus and P .costatus . Moreover, the 12S markers have a higher species
detection sensibility than COI, considering that the exclusive fishes
they retrieved were underrepresented, with low RRA. This could result
from low-efficiency primer binding by COI, which can lead to a lack of
amplification (Zhang et al., 2020).
Database completeness is another variable that directly impacts species
detection, as a lack of reference sequences for a given species may
hamper accurate taxonomic assignment (Collins et al., 2019). This aspect
has affected both COI and 12S markers in this study. For instance, whilePachyurus squamipennis is not represented by any COI reference
sequence in the public databases and was exclusively detected by 12S,
the only native species retrieved solely by COI, Bergiaria
westermanni , does not have any 12S representative sequence in neither
the public nor our custom library. These limitations highlight the
importance of continuous sequencing efforts to broaden reference
sequence databases, especially for megadiverse regions.
Considering that each marker has advantages and limitations, some
studies suggest combining multiple primer sets to increase taxonomic
coverage (Liu and Zhang 2021; Zhang et al., 2020). In a metabarcoding
study using multiplexed markers to identify zooplankton mock
communities, Zhang et al. (2018) demonstrated that a multi-maker
approach can improve species detection and allow the cross-validation of
taxa detected by each marker. Our results support this conclusion, as
using the three markers combined increased the genera detection by up to
87.5% and species detection by up to 61.54%. Therefore, employing
multiple markers, whenever feasible, reduces the likelihood of
overlooking species or incorrectly classifying them due to the absence
or mislabeling of sequences in the reference database (Locatelli et al.,
2020).
Discrepancies between markers were observed in the quantitative analysis
using the RRA estimative. Although some studies with mock samples of
eggs (Duke and Burton, 2020) and larvae (Nobile et al., 2019) yielded a
positive correlation between input organisms and output reads for each
species, the results from this study support the idea that amplification
bias is one of the main pitfalls for quantitative metabarcoding
analyses, as already reported (Carvalho, 2023; Fonseca, 2018). While
MiFish and COI presented RRA resemblance for samples in which both
detected similar taxa, low-efficiency primer binding to Siluriformes and
especially Pimelodidae sequences resulted in completely different
abundance patterns for NeoFish.
In conclusion, using multiple markers from two distinct genes and
lengths allowed an increased taxonomic coverage and robust taxonomic
classification of complex neotropical ichthyoplankton communities.
Finally, precaution is still needed when inferring species abundance
based on DNA metabarcoding data when using PCR-dependent protocols since
it is marker dependent. Nonetheless, ichthyoplankton metabarcoding
offers superior resolution and feasible scalability compared to
traditional techniques, and provides qualitative information, which is
paramount for characterizing reproducing species and definition of
conservation strategies.