Abundance and diversity of pico- and nanophytoplankton in the South West Atlantic off Brazil

Abstract

Although they are important primary producers, the diversity of marine eukaryotic pico and nanophytoplankton still represents a gap in the global oceanographic data. Access these data through sequencing is complicated due to the massive presence of sequences related to heterotrophic organisms (MARIE et al., 2010). To overcome this methodological limitation, it was developed a protocol for sequencing autotrophic cells previously selected by their pigmentation (fluorescence) and size, with the aid of flow cytometry cell sorting (BALZANO et al, 2012;. MARIE et al, 2010;.SHI et al., 2009).This pioneering project will access the genetic diversity of pico and nanophytoeukaryotes using a novel approach that combine cell sorting by flow cytometry and next generation sequencing (NGS) of short regions of three selected genes: nuclear18S rRNA, plastid 16S rRNA and nifH, a gene involved in nitrogen fixation. The data obtained will be of high impact and interest. First, by the novelty of the approach applied in oceanographic studies, and second, due to the fact that the continental shelf off Brazil has unique features that will bring interesting insights about the pico/nanoplankton diversity and structure in the oceans. Our preliminaries results are from a small clone library recovered from sorted cells of fixed water samples collected in October of 2013. We were able to successfully amplify the nuclear 18S rRNA, plastidial 16S rRNA and the nifH gene. The sequences obtained were closely related to the Haptophyta group, specifically the recently discovered host of UCYN-A, a nitrogen fixing cyanobacteria.This research is fully integrated to PhD project "Structure of diazotrophic communities in the Brazilian coast and adjacent oceanic area" (Estrutura de comunidades diazotróficas na costa brasileira e zona oceânica adjacente, FAPESP 2012/04800-9), since it deals directly with nitrogen fixing organisms and the microbial community of the South West Atlantic off Brazil. Our objectives are: i) determine the diversity of nitrogen-fixing cyanobacteria in the samples; ii) estimate the diversity and composition of the picophytoplankton in the tropical waters of the SW Atlantic Ocean with metabarcoding technique (NGS), including the diversity of hosts (picophytoplankton) of N2-fixing cyanobacteria; iii) establish the relationship between hosts and symbionts.The samples were collected onboard the RV "Alpha Crucis", between 31/10/2013 and 23/11/2013. Simultaneously were obtained salinity, temperature and fluorescence data, with a CTD SeaBird®. The surveyed area lies between Lat 23°11'635"S - 30°52.735"S and Long 39°22.610"W - 49°09.62"W longitudes, up to the 3510 meters isobath. The samples were divided into cryotubes in accordance with their use: flow cytometer cell abundance, preserved with 0.1% glutaraldehyde, culturing and cell sorting, preserved with 5 and 10% of DMSO, respectively. Regarding the culturing efforts, so far we have obtained 4 strains of Synechococcus that will be subject to further characterization. The protocols for the culture media used (PCR11) can be found in http://roscoff-culture-collection.org.Due to the fact that the flow cytometry data does not provide information on the taxonomic composition of phytoplankton groups, we chose to select pico and nanoeukaryotes cells by cell sorting (as reported by Shi et al., 2009), in order to analyze them by metabarcoding of the V4 region of the 18S rRNA, plastid 16S rRNA and the nifH gene. The processing of the metabarcodes sequences will be held in the SBR, where there is a pipeline of necessary bioinformatic methods, including grouping (clustering by SWARM method, MAHÉ et al,.2014) and taxonomic annotation of sequences by BLAST against the database of 18S rRNA PR2 (GUILLOU et al. 2013). This semi-quantitative information on the distribution of different groups of microalgae will be interpreted in light of meta data acquired during the cruises. (AU)