Abstract
An exhaustive assessment of biodiversity is a major challenge of
ecological research, and molecular approaches such as the metabarcoding
of environmental DNA are boosting our ability to perform biodiversity
inventories. Are we actually able to assess the whole community, to
unravel the intricate interactions between organisms and the impacts of
global changes on the different trophic levels? The majority of
metabarcoding papers published in the last years used just one or two
markers and analyzed a limited number of taxonomic groups. Nevertheless,
approaches are emerging that might allow ”all-taxa biological
inventories”. Exhaustive biodiversity assessments can be attempted by
combining a large number of specific primers, by exploiting the power of
universal primers, or by combining specific and universal primers to
obtain good information on key taxa while limiting the overlooked
biodiversity. Multiplexes of primers and shotgun sequencing may provide
a better coverage of biodiversity compared to standard metabarcoding,
but still require major methodological advances. We identify the
strengths and limitations of different approaches, and suggest new
development lines that might improve broad scale biodiversity analyses
in the near future.
Key words:
Environmental DNA; freshwater and marine biodiversity; multi-trophic
analyses; primer cocktails; shotgun sequencing; soil biodiversity
1. INTRODUCTION
An exhaustive assessment of biodiversity has always been a major
challenge for ecologists. In principle, all the organisms living in an
ecosystem can interact with each other: some insects and mammals feed on
plants, plants interact with soil fungi, protists can feed on bacteria
or parasitize other eukaryotes, and of course many other interactions
occur. Ideally, we should assess the occurrence (and perhaps the
abundance) of all the organisms, shall we want to unravel the impact of
environmental changes on biodiversity, eventually taking into account
the potential biotic interactions. Unfortunately, we all know that this
is only rarely possible. If we want to use traditional approaches (e.g.
morphological identification of species), thousands systematists should
work together for weeks to produce an ”all-taxa biological inventory” of
just a hectare of tropical forest (Lawton et al., 1998). The emergence
of molecular approaches (starting with DNA barcoding) has certainly
revolutionized biodiversity inventories, as it allows a much faster and
cheaper assessment of present species, particularly for taxonomic groups
including many difficult to identify, cryptic or undescribed taxa
(Floyd, Abebe, Papert, & Blaxter, 2002; Hebert, Cywinska, Ball, &
DeWaard, 2003; Hebert, Penton, Burns, Janzen, & Hallwachs, 2004). DNA
metabarcoding now allows the contemporary assessment of a huge number of
species, starting from both environmental samples and from tissues (as
nicely shown by many papers in this special issue). Does this mean that
we are finally able to assess the whole community, and to unravel the
intricate interactions between organisms?
So far, beside rare exceptions, this does not seem the case. Out of 70
papers using DNA metabarcoding to study biodiversity variation published
in target journals during the last two years (see supplementary
materials for details on methods), the majority used just one or two
primer pairs and focused on just one (e.g. arthropods, fish, fungi,
plants…) or two taxa (e.g. plants + mammals; bacteria +
micro-eukaryotes; Fig. 1a; Supplementary Table S1). Several studies had
a broad taxonomic scope and used generalist primers (particularly
targeting COI and 18S) to amplify very broad groups (e.g. all the
eukaryotes, all the animals…), while very few attempted an
exhaustive biodiversity analysis using multiple primer pairs each of
which targets a different taxon (Fig. 1).
Nevertheless, in principle several strategies may be adopted to obtain
detailed information over a broad spectrum of taxa, and attempt a
nearly-complete reconstruction of communities on the basis of DNA
metabarcoding related approach that might disclose new avenues to
biodiversity and ecological research. In this short contribution, we
describe some of these approaches, we discuss their strengths and
limitations (Table 1), and suggest new development lines that might
improve broad scale biodiversity analyses in the near future.
2. POTENTIAL STRATEGIES FOR ALL-INCLUSIVE BIODIVERSITY ANALYSIS USING
MOLECULAR APPROACHES
2.1 Using a large number of metabarcodes in the same study
A very large number of primers has been developed and tested for
metabarcoding studies. For instance, Taberlet, Bonin, Zinger, and
Coissac (2018) proposed 62 distinct primers pairs for DNA metabarcoding,
some of which were extremely generalist and amplified very broad taxa
(e.g. all the bacteria and archaea; all the eukaryotes…) and
others being much more specific, focusing on well-defined taxa (e.g.
turtles, the plant family Asteraceae…). In principle, we can
amplify the eDNA extracted from one single environmental sample using
multiple primers, and then combine the results to attempt an overall
reconstruction of biodiversity (Jurburg, Keil, Singh, & Chase, 2021).
For example, we might study soil biodiversity by analyzing markers
specific for bacteria, fungi, earthworms, insects, springtails…,
while freshwater diversity can be assessed by combining primers that
amplify bacteria, protists, insects, fishes, amphibians…
(Guerrieri et al., 2022; Li, Qin, Wang, Zhang, & Yang, 2023).
Combining multiple markers allows a good resolution for the selected
focal taxa, particularly if each marker has a well-defined and limited
taxonomic scope. The integration of results of different primers can
allow assessing the response of multiple taxa to environmental
gradients, and even attempting the reconstruction of interaction
networks (Li et al., 2023).
Unfortunately, targeting multiple taxa increases the cost and labor
associated with the laboratory and sequencing, as using many markers
means running many PCR reactions, sequencing lanes and so on.
Furthermore, even if unlimited resources were available (and this is
rarely the case), the amount of eDNA available for amplification remains
limited. Imagine you have extracted 100 μL of eDNA from water, each PCR
reaction requires 2 μL of template DNA, and you want to run eight
replicated PCRs per sample to detect rare species with a limited rate of
false negatives (Ficetola et al., 2015). In this case, the template DNA
is only enough for a maximum of six primers, thus some key taxon will
always be missed. For instance, if we analyze water biodiversity using
primers amplifying bacteria, diatoms, mollusks, insects, fish and
amphibians we will miss key taxa such as crustaceans and most of
micro-eukaryotes.
2.2 Using very generalist or degenerated primers
In principle, we might choose a few very generalist, universal primers,
such as the ones amplifying all the eukaryotes or most of the animals
(e.g. 18S rDNA or COI-based primers). Several studies have adopted this
approach (Fig. 1b) as it has clear advantages, including relatively
cheap cost, and relatively easy implementation (see Jurburg et al., 2021
for additional discussions on limitations and recommendations). In
principle, with 2 / 3 primer pairs (e.g. one eukaryote and one
prokaryote marker) we might try amplifying the whole tree of life (e.g.
Holman et al., 2021; Martinez-Almoyna et al., 2019). Unfortunately, the
search for perfect, truly universal primers has been compared to the
search for the Holy Grail (Rubinoff, Cameron, & Will, 2006). On the one
hand, some ”universal” primers have limited resolution, or have
heterogeneous resolution across the three of life. For instance, some
primer pairs focusing on 18S (e.g. the Euka02 primer pair, Guardiola et
al., 2015) amplify most eukaryotes and have a reasonable resolution for
some taxonomic groups (e.g. nematodes), but a very poor resolution for
other taxa (e.g. plants), with complex consequences for data analyses
(Jurburg et al., 2021). On the other hand, generalist primers such as
those amplifying COI have heterogeneous amplification rate among the
target species. The taxa with less mismatches will be amplified
preferentially, and this can reduce the success over other taxa. Highly
degenerated primers have additional issues such as frequent
amplification of non-target regions, and the amplification of non-target
taxa (e.g. bacterial DNA amplified with COI primers) (Hintikka,
Carlsson, & Carlsson, 2022).
Recently, long-read metabarcoding has been proposed to overcome the
limited resolution of many generalist primers (Jamy et al., 2022). With
this approach, a very long (e.g. 4500 bp) DNA fragment is amplified with
universal primers and then processed through technologies that allow the
sequencing of long reads (Jamy et al., 2022). The long-read
metabarcoding provides unprecedented taxonomic resolution compared to
traditional generalist primers, still poses major technical issues (e.g.
chimaera formation) and is much more expensive than short-read
metabarcoding. Furthermore, long-read metabarcoding is still rarely
applied, and several aspects of this approach will deserve future
adjustments and analyses, including the actual universality of primers.
2.3 Combining very generalist and more specific primers
In order to overcome the limitations of strategies 2.1 and 2.2, it is
possible to analyze the same environmental DNA using both specific
primers targeting taxa with particular ecological role (e.g. high
taxonomic diversity, keystone functions…), and generalist
primers. For instance, for the analysis of soil biodiversity we might
complement primers amplifying insects, springtails, earthworms and
fungi, with a primer that amplifies all the eukaryotes and can give an
idea of the diversity for groups not amplified with the previous ones
(micro-eukaryotes, nematodes, rotifers…) (Bloor, Si-Moussi,
Taberlet, Carrère, & Hedde, 2021; Calderón-Sanou et al., 2022;
Guerrieri et al., 2022). This approach has the advantage of providing a
reasonable representation of biodiversity, with good information on
selected key taxa and few taxa completely missing, and might thus allow
exploring complex relationships between multiple taxonomic groups (Bloor
et al., 2021; Calderón-Sanou et al., 2022). Nevertheless, similarly to
approach 2.1, it remains costly and labor-intensive.
Furthermore, with this approach, the resolution of markers can be
extremely heterogeneous among taxa amplified by specific and generalist
primers. For instance, the above-cited combination of primers would
result in an excellent taxonomic resolution for earthworms and
springtails, but a very coarse one for other taxa (e.g. rotifers).
Combining taxonomic tables with very different resolution in ecological
analyses can be extremely complex, and comparing the biodiversity (e.g.
taxonomic richness) of taxonomic groups amplified with different markers
is certainly problematic. Even if some analytical strategies can help
combining information from disparate groups (Jurburg et al., 2021),
understanding the consequences of analyzing altogether taxa with very
different taxonomic resolution remains a major methodological challenge
associated with this approach.
2.4 Multiplex of primers
An alternative approach is combining multiple metabarcoding primers in
the same PCR mix, to simultaneously amplify and sequence multiple
taxonomic groups. So far, primer cocktails have been rarely used, but
might provide extremely comprehensive information on biodiversity. For
instance, Govender, Singh, Groeneveld, Pillay, and Willows-Munro (2022)
used six primer cocktails, each amplifying a different fragment of the
COI-5P gene region, to explore the diversity of marine zooplankton. By
combining primers optimized for different phyla, they were able to
characterize at high resolution the diversity of the major taxonomic
groups, including crustaceans, fish, echinoderms, mollusks, cnidarians
and more. Govender et al. (2022) included up to four different reverse
primers within the same PCR reaction, all targeting the same DNA
fragment. However, in principle an even larger number of primers could
be combined, to maximize the number of taxa that are amplified at high
resolution, and the multiplex might include primers targeting different
genomic regions, if they have comparable performance (see below). Such
multiplexes including a large number of markers might boost the number
of taxa amplified at high resolution, efficiently exploiting the
available template DNA while limiting costs.
Nevertheless, this approach still needs major methodological
developments. Primers often show strong variation in amplification
efficiency, and the eDNA of different taxa normally is found at
different concentrations. In standard PCRs, this is taken into account
by tuning key parameters (e.g. number of cycles), but in a multiplex all
the primers undergo the same number of cycles, therefore the mix should
ideally include primers with comparable amplification performance, and
targeting taxa with similar DNA concentration. Preliminary analyses can
assess the similarity of primers, for instance checking via qPCR if they
show analogous amplification patterns under the same conditions.
Alternatively, multiplexes including markers with different efficiency
and / or abundance of template DNA can be optimized by increasing the
concentration of the primers with lower performance. Furthermore,
designing a multiplex requires the identification of primers with
similar annealing temperatures, but amplifying complementary groups.
Specific bioinformatics tools have boosted our ability to identify the
most appropriate metabarcoding primers (e.g. Riaz et al., 2011), but
designing a multiplex will certainly need further developments for both
bioinformatics and wet lab. Finally, current popular bioinformatics
pipelines are optimized to process one marker at a time, and specific
developments can be required to retrieve information from multiple
metabarcodes from the same study (Porter & Hajibabaei, 2022).
2.5 Shotgun sequencing
Shotgun sequencing and other metagenomics approaches can extract a huge
amount of information from the environmental DNA, and potentially allow
the reconstruction of the whole community, without targeting a specific
group (Gusareva et al., 2019; Parducci et al., 2019; Pedersen et al.,
2016; Wang et al., 2021). In principle, this approach should bypass the
DNA barcode amplification bias, might allow use the whole DNA available
in the environment, providing information on all the trophic layers, and
can even help to obtain information on the relative abundance of present
taxa (Garrido-Sanz, Senar, & Piñol, 2022; Parducci et al., 2017), thus
overcoming many of the limitations associated to standard DNA
metabarcoding.
Nevertheless, several issues continue to limit the broad-scale
application of shotgun sequencing compared to the more standard
metabarcoding. First, shotgun sequencing is much more expensive than
PCR-based metabarcoding, and the associated bioinformatics pipelines
remain complex. Furthermore, taxonomic identification relies on the
existence of complete genomic databases. Unfortunately, so far genomic
information outside the barcode regions is mostly limited to
vertebrates, some plants (Alsos et al., 2020), and commercially
important species. As a consequence, evidences of the advantage of
shotgun sequencing over PCR-based metabarcoding for broad-scale
environmental analyses remain mixed, so far (Bell et al., 2021; Parducci
et al., 2019; Paula et al., 2022). Despite these issues, the continuing
advances of sequencing and bioinformatics technologies suggest that
shotgun will play an increasingly important role in the analysis of
community-level variation, particularly for topical study systems such
as ancient eDNA (Pedersen et al., 2016; Wang et al., 2021).
3. CONCLUSION: CHALLENGES AND OPPORTUNITIES FOR AN ALL-INCLUSIVE
COMMUNITY ECOLOGY USING METABARCODING
One decade of advances on eDNA metabarcoding has fostered our ability to
obtain biodiversity data, filling long-standing gaps on many components
of both terrestrial and aquatic environments. Nevertheless, just a few
studies have taken the challenge of attempting analyses covering
multiple taxonomic groups, and trying to identify the complex
multi-trophic interactions between them (but see Bloor et al., 2021;
Calderón-Sanou et al., 2021; Calderón-Sanou et al., 2022;
Martinez-Almoyna et al., 2019). Several approaches can now allow an
all-inclusive community ecology, potentially allowing unprecedented
understanding of patterns and processes underlying biodiversity
variation, but both technical and conceptual developments will be
required for a more widespread application of the all-inclusive ecology,
and some challenges are shared by most approaches. So far, strong
efforts have been devoted to the development of massive databases for
standard barcodes, but just one or a few barcodes are unlikely to be
enough to enable the characterization of the whole community. New
reference databases can be generated using high-throughput sequencing
approaches (e.g. genome skimming) that would allow covering broad
sections of the genome (i.e. organelle(s) and nuclear ribosomal DNA),
and might even serve as starting point for the identification of new
markers (Coissac, Hollingsworth, Lavergne, & Taberlet, 2016).
Furthermore, analyses of biotic interactions involving a large number of
taxa remain extremely challenging. Novel frameworks have been proposed
during the last years for the multi-trophic and multi-taxa analysis of
communities, but a lot of work remains to be done to assess their power,
strengths and limitations (e.g. Burian et al., 2021; D’Amen, Mod,
Gotelli, & Guisan, 2018).
It is now clear that ongoing global changes determine very intricate
effects on the organisms. For instance, species responses to climate
change often alter the existing biotic interactions, and predicting a
specie’s response while ignoring interactions with its predators,
foodsource or pathogen can lead to highly biased results (Sirén,
Sutherland, Karmalkar, Duveneck, & Morelli, 2022; Urban et al., 2016).
Yet, accounting for species interactions requires well-resolved
information that is often missing, and that cover a large subset of
existing biodiversity (Gilman, Urban, Tewksbury, Gilchrist, & Holt,
2010; Urban et al., 2016). DNA metabarcoding can heavily contribute to
such endeavors of biodiversity studies, and we hope that methodological
and conceptual advances, allowing an all-inclusive community ecology,
will remain an active research area in the near future.
ACKNOWLEDGMENTS
GFF is funded by the European Research Council under the European
Community’s Horizon 2020 Programme, Grant Agreement no. 772284
(IceCommunities).
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DATA ACCESSIBILITY
All the relevant data are provided as supplementary material (Table S1).
AUTHOR CONTRIBUTIONS
The two authors jointly designed the study. GFF drafted the first draft
of the manuscript, with sustantial contribution of PT.
SUPPORTING INFORMATION
Additional supporting information may be found online in the Supporting
Information section
ORCID
Gentile Francesco Ficetola: https://orcid.org/0000-0003-3414-5155
Pierre Taberlet:https://orcid.org/0000-0002-3554-5954
Figure legends
Figure 1. Number and typologies of markers analyzed in 70 papers
published in 2021-22 in seven representative scientific journals. We
considered papers extracted from the Web of Science using the search
term “DNA metabarcoding” and analyzing biodiversity variation.
“Generalist markers” are markers that amplify multiple distantly
related phyla and / or an entire domain of life, while studies focusing
on “specific taxa” focus on a given taxonomic group (phylum,
super-phylum or finer). Note that some studies focused on one specific
taxon (e.g. fish), but used more than one marker to improve coverage.
Additional details are provided in the Supplementary Methods and in
Supplementary Table S1.
Table 1. Summary of approaches for all-inclusive community ecology, with
examples of their strengths and limitations.