Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Biogeosciences Discuss., 9, 6219–6259, 2012 www.biogeosciences-discuss.net/9/6219/2012/ Biogeosciences doi:10.5194/bgd-9-6219-2012 Discussions BGD © Author(s) 2012. CC Attribution 3.0 License. 9, 6219–6259, 2012 This discussion paper is/has been under review for the journal Biogeosciences (BG). Flagellate diversity Please refer to the corresponding final paper in BG if available. in the Arctic S. Balzano et al. Diversity of cultured photosynthetic flagellates in the North East Pacific and Title Page Arctic Oceans in summer Abstract Introduction Conclusions References S. Balzano1, P. Gourvil1, R. Siano2, M. Chanoine1, D. Marie1, S. Lessard3, D. Sarno4, and D. Vaulot1 Tables Figures 1UMR 7144, CNRS et Universite´ Pierre et Marie Curie (Paris-06), Station Biologique de J I Roscoff, 29680 Roscoff, France 2 IFREMER, centre de Brest, DYNECO, Pelagos, BP 70 29280, Plouzane,´ France J I 3Institut des Sciences de la Mer, Rimouski, Quebec,´ Canada 4Taxonomy and Identification of Marine Phytoplankton, Stazione Zoologica Anton Dohrn, Villa Back Close Comunale, 80121 Napoli, Italy Full Screen / Esc Received: 5 May 2012 – Accepted: 15 May 2012 – Published: 1 June 2012 Correspondence to: S. Balzano (balzano@sb-roscoff.fr) Printer-friendly Version Published by Copernicus Publications on behalf of the European Geosciences Union. Interactive Discussion 6219 Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Abstract BGD During the MALINA cruise (summer 2009) an extensive effort was undertaken to isolate phytoplankton strains from the North East (NE) Pacific Ocean, the Bering Strait, and 9, 6219–6259, 2012 the Beaufort Sea. Strains were isolated by flow cytometry sorting (FCS) and pipetting 5 before or after phytoplankton enrichment of seawater samples. Strains were isolated Flagellate diversity ◦ both onboard and back in the laboratory and cultured at 4 C under light/dark condi- in the Arctic tions. Overall, we isolated and characterised by light microscopy and 18S rRNA gene sequencing 104 strains of photosynthetic flagellates which grouped into 21 genotypes S. Balzano et al. (defined by 99.5 % 18S rRNA gene sequence similarity) mainly affiliated to Chloro- 10 phyta and Heterokontophyta. The taxon most frequently isolated was an Arctic eco- type of the green algal genus Micromonas (Arctic Micromonas) which was almost the Title Page only phytoplankter recovered within picoplankton (≤ 2 µm) size range. Strains of Arc- Abstract Introduction tic Micromonas as well as three unidentified strains related to the same genus were identified in further details by sequencing the Internal Transcribed Spacer (ITS) region Conclusions References 15 of the rRNA operon. The MALINA Micromonas strains share identical 18S rRNA and Tables Figures ITS sequences suggesting high genetic homogeneity within Arctic Micromonas. The unidentified strains form a genotype likely belonging to a new genus within the fam- ily Mamiellaceae to which Micromonas belongs. Other green algae genotypes from J I the genera Nephroselmis, Chlamydomonas, Pyramimonas were also isolated whereas J I 20 Heterokontophyta included Pelagophyceae, Dictyochophyceae and Chrysophyceae. Dictyochophyceae included Pedinellales which could not be identified to the genus Back Close level whereas Chrysophyceae comprised Dinobryon faculiferum. Moreover, we isolated Full Screen / Esc Rhodomonas sp. as well as a few Haptophyta and dinoflagellates. We identified the dinoflagellate Woloszynskia cincta by Scanning Electron Microscopy (SEM) and 28S Printer-friendly Version 25 rRNA gene sequencing. Our morphological analyses show that this species possess the diagnostic features of the genus Biecheleria, and the 28S rRNA gene topology Interactive Discussion corroborates this affiliation. We thus propose the transfer of W. cincta to the genus Biecheleria and its recombination as Biecheleria cincta. 6220 Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | 1 Introduction BGD Arctic phytoplankton undergoes a high seasonal variability with most of the biomass occurring during late summer (Sherr et al., 2003; Wang et al., 2005). During this pe- 9, 6219–6259, 2012 riod, freshwater inputs from rivers and ice melting in the Beaufort Sea lead to strong 5 stratification of the water column. Consequently phytoplankton depletes the surface Flagellate diversity layer in nutrients, especially inorganic nitrogen (Carmack and MacDonald, 2002). in the Arctic In the Canadian Arctic diatoms tend to dominate near the coast (Lovejoy et al., 2002; Sukhanova et al., 2009) and flagellates prevail in offshore waters, especially in mid and S. Balzano et al. late summer (Booth and Horner, 1997; Sherr et al., 2003). Arctic photosynthetic pi- 10 coplankton is dominated by the green algal class Mamiellophyceae (Not et al., 2005; Lovejoy et al., 2007), specifically by a Micromonas ecotype (Arctic Micromonas) genet- Title Page ically and physiologically distinct from Micromonas genotypes typically found in warmer Abstract Introduction oceans (Slapeta et al., 2006; Lovejoy et al., 2007). This ecotype occurs in the Arctic throughout the year (Sherr et al., 2003) replacing the cyanobacteria as the baseline Conclusions References 15 community (Li, 1998). In contrast, larger (≥ 2 µm) photosynthetic flagellates fluctuate Tables Figures during the year and are more diverse (Booth et al., 1982; Booth and Horner, 1997; Lovejoy et al., 2002). The summer composition of photosynthetic pico and nanoplankton has been investi- J I gated in greater details from the North East (NE) Pacific to the Beaufort Sea during the J I 20 MALINA cruise in summer 2009 (Balzano et al., 2012). Terminal restriction fragment length polymorphism (T-RFLP) and cloning/sequencing approaches have confirmed Back Close the ubiquity of Arctic Micromonas which occurred in the NE Pacific, dominated the Full Screen / Esc Bering Strait and was almost the unique photosynthetic picoplankter found throughout the Beaufort Sea in both nitrogen-depleted surface waters and nitrogen-replete deep Printer-friendly Version 25 chlorophyll maximum (DCM) waters. It is not known whether such ubiquity and ex- clusivity covers intraspecific differences between populations occurring under different Interactive Discussion seawater conditions or whether populations are rather homogeneous and all adapted to variable conditions. In contrast, nanoplankton was more diverse and dominated by 6221 Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | cultured microorganisms mainly belonging to diatoms, Chrysophyceae, and Pelago- phyceae. BGD Despite obvious biases, culturing approaches permit a better characterisation of the 9, 6219–6259, 2012 strains isolated by the combination of microscopy and molecular methods (Le Gall 5 et al., 2008). To date existing datasets on Arctic phytoplankton are based either on light microscopy (Okolodkov and Dodge, 1996; Booth and Horner, 1997; Lovejoy et al., Flagellate diversity 2002; Sukhanova et al., 2009) or cloning/sequencing (Lovejoy et al., 2006; Luo et al., in the Arctic 2009; Lovejoy and Potvin, 2011) but few studies have performed large scale isolation efforts in the Arctic. S. Balzano et al. 10 Our study aims to isolate and culture Arctic phytoplankton for a genetic characterisa- tion of the main species present and to assess if the genotypes present in the Beaufort Title Page Sea are endemic or occur in other oceans. From the MALINA 2009 cruise we isolated about 200 strains from the NE Pacific, the Bearing Strait, the Arctic Ocean and the Abstract Introduction Beaufort Sea using different approaches (flow cytometry sorting, single cell pipetting). Conclusions References 15 About half of the strains belong to diatoms and will be investigated in a parallel study, here we characterise 104 strains of photosynthetic flagellates by 18S rRNA gene se- Tables Figures quencing. We also sequenced the internal transcribed spacer (ITS) region of the rRNA operon from our strains of Mamiellophyceae to understand whether Arctic Micromonas J I is genetically homogeneous or consists of several distinct genotypes, and if the other 20 Mamiellophyceae strains isolated here correspond to a new genus. Finally we charac- J I terised in further details by scanning electron microscopy (SEM) and 28S rRNA gene Back Close sequencing, two dinoflagellate strains belonging to Woloszynskia cincta, a recently de- scribed species (Siano et al., 2009) and propose the transfer of W. cincta to the genus Full Screen / Esc Biecheleria as B. cincta. Printer-friendly Version Interactive Discussion 6222 Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | 2 Materials and methods BGD 2.1 Sampling 9, 6219–6259, 2012 The MALINA cruise took place on board the Canadian research vessel CCGS Amund- sen during summer 2009 from Victoria (British Columbia, Canada) to Beaufort Sea Flagellate diversity 5 (Table 1, Leg 1b) and then throughout Beaufort Sea (Leg 2b). Seawater samples were in the Arctic collected with a bucket from surface during Leg 1b and at different depths with Niskin bottles mounted on a CTD frame during Leg 2b (Fig. 1). Water temperature, salin- S. Balzano et al. ity, nutrient concentrations, and the phytoplankton composition were obtained from the MALINA database (http://www.obs-vlfr.fr/Malina/data.html). Title Page 10 2.2 Strain isolation Abstract Introduction Phytoplankton strains were isolated both onboard and back in the laboratory. Onboard, Conclusions References strains