1. Ramon Massana Institut de Ciències del Mar (CSIC), Barcelona MEDOCEAN meeting, 28 November 2013 Looking for the dominant marine bacterivores

2. Smetacek 2002. Nature

3. Unicellular eukaryotes, colorless, and with flagella Ubiquitous in the plankton at abundances around 1000 cells ml -1 Most are 1-3 µm in size Exhibit phylogenetic and functional diversity Heterotrophic flagellates - Main bacterial grazers Azam et al. 1983. Mar. Ecol. Progr. Ser Beyond the simplified microbial loop … Heterotrophic flagellates as algivorous and osmotrophs Mixotrophic flagellates as bacterivorous Viruses as a factor for bacterial mortalityå

4. 5 µm DAPI-stained epifluorescence microscopy The only quantitative approach Very poor identification power Heterotrophic flagellates - Microscopy

5. The most common in pelagic marine waters (by culturing, enrichments and direct observations) Stramenopiles Bicosoecida Caecitellus parvulus Cafeteria roenbergensis Boroka karpovii Chrysopytes Paraphysomonas imperforata Pedinellids Pteridomonas danica Kinetoplastids Neobodo designis Rynchomonas nasuta Inserta sedis Ancyromonas sigmoides Amastigomonas debruynei Paraphysomonas imperforata Dominates (up to 98% of cells) in flagellate enrichments in the US East Coast Represents <1% in natural samples Lim et al. 1999. Limnol. Oceanogr. Heterotrophic flagellates – Culturing bias

6. Massana 2011. Annu Rev. Microbiol. Analysis of 18S rDNA sequences retrieved from the environment 40 studies on marine microeukaryotes since 2001 8719 environmental sequences Analysis of 18S rDNA sequences retrieved from the environment 40 studies on marine microeukaryotes since 2001 8719 environmental sequences Heterotrophic flagellates – Environmental molecular surveys

7. Massana 2011. Annu Rev. Microbiol. Groups with cultured heterotrophic flagellates

8. Massana et al. 2002. Appl. Environ. Microbiol. Massana et al. 2004. Appl. Environ. Microbiol. Marine Stramenopiles - MAST (novel, uncultured groups) Marine Stramenopiles - MAST (novel, uncultured groups)

9. MAST-4 as a model heterotrophic flagellate FISH DAPI 10 µm Massana et al. 2006. Environ. Microbiol. A bacterivorous heterotrophic picoeukaryote DAPIFISHIngested FLB Grazing rate: 1–3 bacteria h -1 Massana et al. 2009. ISME J. Bacterial mortality: 1 to 10% of production Bacterial mortality: 1 to 10% of production

10. MAST-4 as a model heterotrophic flagellate Widely distributed and abundant Rodríguez-Martínez et al. 2009. Environ. Microbiol. q-PCR FISH

11. MAST-4 as a model heterotrophic flagellate Limited genetic diversity GenBank sequences

12. MAST-4 as a model heterotrophic flagellate Global distribution and environmental selection ~550 bp – 123 ARISA fingerprints Rodríguez-Martínez et al. submitted

13. MAST-4 as a model heterotrophic flagellate Rodríguez-Martínez et al. submitted Global distribution and environmental selection

14. Summary The diversity of heterotrophic flagellates is poorly investigated The culturing bias a main drawnback in the ecology of heterotrophic flagellates The novel MAST lineages are important marine heterotrophic flagellates Single Amplified Genomics provide new opportunities for ecological and evolutionary studies MAST-4 is a nice model of heterotrophic flagellates Widespread in euphotic waters Accounts for ~10% of cells Active bacterial grazer Limited diversity (only 5 clades) The diversity of heterotrophic flagellates is poorly investigated The culturing bias a main drawnback in the ecology of heterotrophic flagellates The novel MAST lineages are important marine heterotrophic flagellates Single Amplified Genomics provide new opportunities for ecological and evolutionary studies MAST-4 is a nice model of heterotrophic flagellates Widespread in euphotic waters Accounts for ~10% of cells Active bacterial grazer Limited diversity (only 5 clades)

15. Acknowledgments Institut de Ciències del Mar Raquel Rodríguez-Martínez Ramiro Logares Javier del Campo Massimo Pernice Caterina Rodríguez-Giner Irene Forn Vanessa Balagué Station Biologique de Roscoff, France Fabrice Not Colomban de Vargas Instituto Tecnológico de Chascomús, Argentina Fernando Unrein University of Washington, USA Gabrielle Rocap Bigelow Laboratory for Ocean Sciences, USA Mike Sieracki Ramunas Stepanauskas Institut de Ciències del Mar Raquel Rodríguez-Martínez Ramiro Logares Javier del Campo Massimo Pernice Caterina Rodríguez-Giner Irene Forn Vanessa Balagué Station Biologique de Roscoff, France Fabrice Not Colomban de Vargas Instituto Tecnológico de Chascomús, Argentina Fernando Unrein University of Washington, USA Gabrielle Rocap Bigelow Laboratory for Ocean Sciences, USA Mike Sieracki Ramunas Stepanauskas you for your attention !