Validating Metabarcoding As a Tool for Eukaryotic Plankton Monitoring
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Validating Metabarcoding as a Tool for Eukaryotic Plankton Monitoring in Estuaries David Abad1, Mikel Aguirre1, Aitor Albaina1, Aitor Laza2, Ibon Uriarte3, Andone Estonba1 1Department of Genetics, Physical Anthropology & Animal Physiology. Faculty of Science and Technology. University of the Basque Country, UPV/EHU. Leioa, Bizkaia, Spain. 2Phytoplankton group,Department of Plant Biology and Ecology. Faculty of Science and Technology. University of the Basque Country, UPV/EHU. Leioa, Bizkaia, Spain. 3Zooplankton group, Department of Plant Biology and Ecology. Faculty of Science and Technology. University of the Basque Country, UPV/EHU. Leioa, Bizkaia, Spain. Introduction Results Nowadays metabarcoding arises as a good The percentage of not assigned reads was lower at the higher size-fractions. While maxillopoda alternative for biodiversity assessment because predominated at those size fractions, a more diverse assemblage characterized the 0.22-20 µm one. it combines extreme sensibility with, potentially, Copepods represented 48.6, 36 and 2.3 % while phytoplankton groups <0.1, 1.6 and 32.7 % for each the highest taxonomic resolution in a both cost- size-fraction respectively. and time-effective methodology. To evaluate its The CCA explained 57.7 % of variance. Main environmental factors were salinity and date. While a capacity for estuarine plankton monitoring we reduced number of brackish water species, such as the copepods A. tonsa and Calanipeda aquaedulcis performed a comparison between the 18S V9 characterized the 30 ‰ community, a higher number of OTUs encompassing mostly neritic taxa rRNA metabarcoding approach and microscopy. conformed the 35 ‰ water mass. This region was selected because of its broad Correlations were significant in most of the cases and with no noticeable effect when comparing amplification range among eukaryotes and against microscopy-based counts or biomass. previous success in marine plankton global Regarding A. tonsa, although similar relative abundances were found in the 30 ‰ water mass by both studies (Tara Oceans and Biomarks initiatives) approaches, with one and three stations having favourable detection for the metabarcoding and [1, 2]. The estuary of Bilbao was one of the most microscopy approaches respectively (Fig 4a), the species was only detected by metabarcoding in the contaminated in Europe until the late 80s and 35 ‰ one (Fig 4b). With respect to P. marinus, in six out of the eight stations, detection was favourable since then has undergone a water recovery for metabarcoding while it was reduced to two for microscopy (Fig 4c, d). program; this transition has allowed the Figure 1 Figure 2 recolonization by a mixture of neritic and estuarine species. Among them, there are NIS such as Acartia tonsa, that was first 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 first time in the Bilbao estuary [5] and whose effect on the community cannot yet be predicted. Material and methods Sampling was carried out during summer and autumn in the 30‰ and 35‰ salinities of the estuary of Bilbao. Water was filtered through 200, 20 and 0.22 μm meshes and a ~150bp Figure 4 fragment of the 18S rDNA gene amplified and sequenced according to the Earth Microbiome Blue arrows and green ellipses indicate temporal and spatial cycles respectively. Project protocols. Qiime v1.9 was used to assign Figure 3 reads to Operational Taxonomic Units (OTUs); 780 were identified with a 99% similarity threshold. 1) OTUs were classified into 33 categories, including one for not assigned reads (Fig 1). 2) Forty-one OTUs were included in the multivariate analysis condensing the three size- factions together (Fig 2). 3) Correlations between metabarcoding and microscopy when comparing relative abundances of every taxon within a particular sample (Fig 3). 4) We compared the performance of metabarcoding and microscopy to detect two NIS species: Acartia tonsa and Pseudodiaptomus Conclusions marinus (Fig 4). The somewhat reduced performance of this approach for the lowest size fractions is to be mainly related with 18s V9 database incompleteness for these organisms. This highlights that DNA-barcoding is necessary and complementary to metabarcoding [6]. Metabarcoding was able to replicate the Bilbao estuary plankton community temporal and spatial patterns. The lack of correlation between relative abundances could be explained by technical biases introduced Environmental Sample DNA PCR during the DNA extraction [7] or PCR amplification step [8], the Copy Number Variation (CNV) (e.g. Filtered Seawater) extraction Amplification 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 multi- copy genes will remain as a semi-quantitative approach [10]. The present study showed that metabarcoding has more sensitivity than microscopy (Fig 4), confirming previous studies [11, 12]. The reasons behind this are a) the ability to analyze higher sample volumes (microscopy-based methods would take a lot longer to screen all the sample) and b) the capacity to take into account individuals at any life stage, such as eggs or nauplius larvae (identification of which Taxonomic Compositon Bioinformatics Sequencing is complicated). Bibliography 1. Massana R, Gober A, Audic S, Bass D, Bittner L, Boutte C, et al. Marine protist diversity in European coastal waters and sediments as revealed by high-throughput sequencing. Environmental Microbiology. 2015. DOI: 10.1111/1462-2920.12955 2. de Vargas C, Audic S, Henry N, Decelle J, Mahé F, Logares R, et al. Eukaryotic plankton diversity in the sunlit ocean. Science. 2015. DOI: 10.1126/science.1261605 3. Albaina A, Villate F, Uriarte I. 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