Revealing the potential and limitation of eDNA metabarcoding
inventories
Fishes are the most diverse group of vertebrates on Earth with varying
body sizes, environmental niches and diets. Monitoring fish assemblages
in marine biodiversity hotspots like the Coral Triangle is a great
challenge particularly for small, rare, cryptobenthic or elusive
species. Here we show that the percentage of sequenced species is highly
variable among families preventing any robust estimation of species
richness. Instead Operational Taxonomic Units have the potential to
reveal the presence of a broad range of fish species, i.e. from
different lineages and with contrasted life-history traits. For
instance, cryptobenthic families have been poorly documented and are
often ignored in traditional visual censuses ((7) Brandl et al 2018)
while they strongly influence ecosystem functioning ((13) Brandl et al
2019). Similarly, traditional visual censuses often miss highly mobile
and elusive species such as sharks ((9) Boussarie et al 2018).
Among the 310 assigned fish species, we detected the presence of small
cryptobenthic species such as Gobiodon histrio orOstorhinchus selas , a goby and a cardinalfish with a maximum
length below 40 mm, respectively. We also detected large pelagic fish
such as the dogtooth tuna (Gymnosarda unicolor ) or the thresher
shark (Alopias pelagicus ) reaching over 2 m and 4 m long,
respectively. Flagship species for conservation were also present in our
DNA samples such as the over-exploited Napoleon wrasse (Cheilinus
undulatus , Endangered, IUCN redlist,www.iucnredlist.org), the
Scalloped hammerhead shark (Sphyrna lewini , Endangered) and
several shark species being classified as Near Threatened (NT) (C.
brevipinna , C. Leucas , C. sorrah , C. melanopterus ,T. obesus ).
Even if not assigned at species-level, OTUs can be defined as distinct
entities for which their distribution and temporal variability can be
assessed and monitored ((41) Cordier et al 2017). Moreover, the OTUs and
their associated sequences can remain in public repositories until they
are assigned to a species, subspecies or complex as databases improve
((42) Wangensteen et al 2018). However, the major caveat of using OTUs
for diversity inventories is that they cannot be directly considered as
species with complete certainty. Species with intra-specific genetic
variability can produce two separate OTUs, overestimating species
diversity. Conversely, two species phylogenetically close to each other
with low genetic variability can be grouped into a single OTU, thus
underestimating species diversity. The accuracy of diversity inventories
using eDNA metabarcoding is thus directly based on the taxonomic
resolution of the barcode used and genetic variability among families
but also the number of samples.
Here we also reveal the gap of biodiversity that remains to be detected
using OTU accumulation curves. The effort can be massive for some
families (Fig. 6) and more ambitious eDNA sampling campaigns should be
on the agenda in species-rich regions like the Coral Triangle. OTU
accumulation curves can also serve to evaluate the efficiency of a
sampling method (e.g. punctual filtration, transect filtration), the
sampled area or the diversity of habitats that are required (e.g. depth,
complexity, distance from the seafloor) and their location (e.g.
proximity of reefs, hotspots) especially when targeting rare, elusive,
highly mobile or cryptobenthic families of fish.
The contrasts between assigned taxa diversity, OTU diversity and OTU
asymptote diversity show that the detectability varies strongly among
fish families. These contrasts can be related to the ecology of the
species but also to the state of the retrieved DNA fragments (intra or
extracellular), their sources (e.g. gametes, larvae, feces), their
release rate (different and their diffusion in the water column (limited
or wide). For instance, a benthic fish species such as gobies with a
small movement range would release DNA fragments through skin and feces
on a small area. However, such species could release a massive number of
gametes carried through the water column ((13) Brandl et al. 2019) so
may appear highly detectable during breeding season. Further comparative
works are urgently needed between visual, camera and eDNA metabarcoding
surveys to better estimate the level of detectability of each species or
family in order to provide reliable biodiversity assessments. For
instance, coupling eDNA metabarcoding and video surveillance allows the
detection of eighty-two fish genera from 13 orders on reefs and seagrass
with only 24 genera in common ((43) Stat et al 2019). Investigating
biodiversity should also consider its multiple components including
functional and phylogenetic diversity that are key for reef ecosystem
functioning ((44) Duffy et al 2016). Associating OTUs to species might
allow to fill this gap but it will require massive sampling and
sequencing efforts.