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Validating Metabarcoding as a Tool for Eukaryotic

Plankton Monitoring in Estuaries

David Abad1, Mikel Aguirre1, Aitor Albaina1, Aitor Laza-Martnez2, Ibon Uriarte3, Andone Estonba1
Department of Genetics, Physical Anthropology & Animal Physiology. Faculty of Science and Technology. University of the Basque Country, UPV/EHU. Leioa, Spain.
Phytoplankton group,Department of Plant Biology and Ecology. Faculty of Science and Technology. University of the Basque Country, UPV/EHU. Leioa, Spain.
Zooplankton group, Department of Plant Biology and Ecology. Faculty of Science and Technology. University of the Basque Country, UPV/EHU. Gasteiz, Spain.



Nowadays metabarcoding arises as a valuable

alternative for biodiversity assessment because
it combines extreme sensitivity with, potentially,
the highest taxonomic resolution in a both costand time-eective methodology. In order to
evaluate its capacity for estuarine plankton
monitoring we performed a comparison between
this approach and microscopy; the V9 region of
the 18S rDNA gene was selected because of its
broad amplication range among eukaryotes
and previous success in marine plankton global
studies, such as Tara Oceans and Biomarks
initiatives [1, 2]. The estuary of Bilbao was one
of the most contaminated in Europe but since
1979 it has undergone a water recovery
program; this transition has allowed the
recolonization by a mixture of neritic and
estuarine species. Among them, there are NonIndigenous Species (NIS) such as Acartia tonsa,
that was rst described in the this estuary in
2001 and became dominant the following year
displacing other congeneric species [3, 4], and
Pseudodiaptomus marinus, which was recently
cited for the rst time in this estuary [5] and
whose eect cannot be predicted yet.

The percentage of not assigned reads was lower at the higher size-fractions. While maxillopoda
predominated at those size fractions, a more diverse assemblage characterized the 0.22-20 m one.
Copepods represented 48.6, 36 and 2.3% while phytoplankton groups <0.1, 1.6 and 32.7% for each
size-fraction respectively.
The CCA explained 57.7% of variance. Main environmental factors were salinity and date. While a
reduced number of brackish water species, such as the copepods A. tonsa and Calanipeda aquaedulcis
characterized the 30 community, a higher number of OTUs encompassing mostly neritic taxa
conformed the 35 water mass.
Correlations were signicant in most of the cases and with no noticeable eect when comparing
against microscopy-based counts or biomass.
Whilst similar relative abundances were found for A. tonsa in the 30 water mass by both approaches
(Fig 4a), it was only detected by metabarcoding in the 35 salinity (Fig 4b). Regarding P. marinus,
detection was favorable to metabarcoding in six out of eight cases meanwhile only in two its presence
was detected by microscopy (Fig 4c, d).
Figure 1. Proportion of taxonomic groups in each sample based on the metabarcoding approach.

Figure 2. CCA of the most abundant OTUs.

Material and methods

Sampling was carried out during summer and
autumn in the 30 and 35 salinities of the estuary
of Bilbao. Water was ltered through 200, 20
and 0.22 m meshes and the 18S V9 (~150 bp)
amplied and sequenced according to the Earth
Microbiome Project protocols. Qiime v1.9 was
used to assign reads to Operational Taxonomic
Units (OTUs); 831 were identied with a 99%
similarity threshold.
1) OTUs were classied into 33 categories,
including one for not assigned reads (Fig 1).
2) Forty-one OTUs were included in the
multivariate analysis condensing the three sizefactions together (Fig 2).
3) Correlations between metabarcoding and
abundances of every taxon within a particular
sample (Fig 3).
microscopy when assessing abundances for two
NIS: Acartia tonsa and Pseudodiaptomus
marinus (Fig 4).

Environmental Sample
(e.g. Filtered Seawater)


Taxonomic Compositon




Figure 4. Comparison of metabarcoding and microscopy when assessing abundances for NIS.

Blue arrows and green ellipses indicate temporal and spatial cycles respectively.

Figure 3. Among taxa comparisons.

The somewhat reduced performance of this approach for the lowest size fractions is mainly related to
18s V9 database incompleteness for these organisms. This highlights that DNA-barcoding is necessary
and complementary to metabarcoding [6].
Metabarcoding replicated the Bilbao estuary plankton community temporal and spatial patterns.
The lack of correlation between relative abundances could be explained by technical biases introduced
during the DNA extraction [7] or PCR amplication step [8]. The Copy Number Variation (CNV)
associated to multi-copy genes, such as rRNA ones, has been suggested as one of the main factors
limiting the quantitative value of metabarcoding [9]. In the meantime, metabarcoding targeting multicopy genes will remain as a semi-quantitative approach [10].
The present study demonstrated the suitability of metabarcoding for early detection of NIS at
extremely low abundances (Fig 4), conrming previous studies [11, 12]. The reasons behind this are a)
the ability to analyze higher sample volumes and b) the capacity to take into account individuals at
any life stage, such as eggs or nauplius larvae.
All of this suggests that metabarcoding could be a powerful tool, if implemented in plankton
monitoring, for early detection of NIS or plankton biodiversity shifts.

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