Culturable diversity of Arctic phytoplankton during pack ice melting

Massive phytoplankton blooms develop at the Arctic ice edge, sometimes extending far under the pack ice. An extensive culturing effort was conducted before and during a phytoplankton bloom in Baffin Bay between April and July 2016. Different isolation strategies were applied, including flow cytometry cell sorting, manual single cell pipetting and serial dilution. Although all three techniques yielded the most common organisms, each technique retrieved specific taxa, highlighting the importance of using several methods to maximize the number and diversity of isolated strains. More than 1,000 cultures were obtained, characterized by 18S rRNA sequencing and optical microscopy and de-replicated to a subset of 276 strains presented in this work. Strains grouped into 57 genotypes defined by 100% 18S rRNA sequence similarity. These genotypes spread across five divisions: Heterokontophyta, Chlorophyta, Cryptophyta, Haptophyta and Dinophyta. Diatoms were the most abundant group (193 strains), mostly represented by the genera Chaetoceros and Attheya. The genera Rhodomonas and Pyramimonas were the most abundant non-diatom nanoplankton strains, while Micromonas polaris dominated the picoplankton. Diversity at the class level was higher during the peak of the bloom. Potentially new species were isolated, in particular within the genera Navicula, Nitzschia, Coscinodiscus, Thalassiosira, Pyramimonas, Mantoniella and Isochrysis. Submitted to: Elementa: Science of the Anthropocene Date: May 17, 2019


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The sequence of Cylindrotheca sp. RCC5216, also isolated from an IC ice 244 core sample (Supplementary Data S1), differed from that of Cylindrotheca sp.

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RCC5463 by two base pairs. Cells of RCC5216 are curved to sigmoid forming 246 coarse aggregates, with 17-20 µm apical and ∼ 4 µm transpical axes ( Figure 3B).  Figure 3I). Interestingly, none 260 of the Navicula sp. strains recovered in this study were related to previous po-261 lar strains or environmental sequences, despite this genus being diverse (Katsuki The Naviculales genotype represented by RCC5564 contains 12 strains from 270 all phases and sampling sites (Supplementary Data S1). Its sequence is 99.7% 271 similar to Naviculales strain CCMP2297 from northern Baffin Bay and to uncul-272 tured sequences from the Arctic (Figure 4). Cells have ∼ 3 µm apical and 5 µm 273 pervalvar axes. They are solitary or form short chains ( Figure 3AC).

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The Naviculales genotype represented by RCC5387 contains four strains from 275 IC water and ice samples (Supplementary Data S1). Its sequence has low simi-276 larity to sequences from GenBank or to the genotype represented by RCC5564,277 sharing only 96.9% similarity with strain CCMP2297 (Naviculales) (Figure 4).

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Cells are elongated, mainly solitary, with up to 6 µm apical and 3 µm pervalvar 279 axes ( Figure 3K).  Leu et al., 2015). Surprisingly, none of the Nitzschia sp. strains isolated in this 288 study had high 18S rRNA similarity to other known polar strains. They did, how-289 ever, have high similarity with Arctic environmental sequences ( Figure 4).

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Nitzschia sp. RCC5391 was isolated from an ice core sample during the pre-295 bloom period. Its sequence matches with only 97.8% similarity that of a strain 296 TA394 (Nitzschia paleaeformis) from the Yellow Sea ( Figure 4). Cells are solitary 297 lanceolate with bluntly rounded apices, measuring ∼ 10 µm and 2 µm for the 298 apical and transapical axes, respectively ( Figure 3N).

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Nitzschia sp. RCC5458 was also retrieved from an ice sample from the pre-300 bloom period and its sequence is 98.1% similar to Nitzschia sp. strain KSA2015-301 49 from the Red Sea ( Figure 4). Cells are linear to lanceolate and larger than 302 other Nitzschia strains retrieved in this study, with an apical axis up to 15 µm 303 ( Figure 3P).

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Nitzschia sp. RCC5510 was isolated from AM waters (Supplementary 305 Data S1). Its sequence is 98.6% similar to Nitzschia sp. strain KSA2015-38 from  The Pseudo-nitzschia arctica genotype represented by RCC5469 contains 311 nine strains of the recently described P. arctica (Percopo et al., 2016), all orig-312 inating from IC (Supplementary Data S1). Their sequence is 100% similar to P.  The Synedra sp. genotype represented by RCC5535 comprises ten strains of 342 which four were isolated from the Amundsen cruise and the other six from within 343 or under the IC ice ( Figure 4). Its sequence shares 100% identity with other Arctic 344 strains such as Fragilariales RCC2509. Cells vary considerably in shape, from 345 almost linear to lanceolate and sometimes asymmetrical in the valvar central area.

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The Actinocyclus sp. genotype represented by RCC5608 comprises two strains 349 isolated from AM waters during the bloom-peak (Supplementary Data S1). Its   The Shionodiscus bioculatus genotype represented by RCC5532 contains two 431 strains isolated from the Amundsen cruise (Supplementary Data S1). Its sequence ∼ 2 µm and 7 µm, respectively ( Figure 7J). Pelagophyceae may dominate surface 513 waters during the Arctic summer (Balzano et al., 2012) and yet undescribed strains 514 have been recovered previously from northern waters (Balzano et al., 2012).

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The Pelagophyceae genotype represented by RCC5251 contains three strains 516 from the peak of the bloom (Supplementary Data S1) and its representative se-517 quence shares 100% similarity with that of the undescribed Arctic Pelagophyceae 518 RCC2040 ( Figure 6B). Cells are elongated with ∼ 7 µm in side view ( Figure 7B).  The Pyramimonas sp. genotype represented by RCC5252 is formed by two 580 IC strains from samples taken at 20 m depth at the peak of the bloom on differ-581 ent sampling days (Supplemntary Data S1). The representative sequence is 100% 582 similar to that of the Arctic strain Pyramimonas sp. RCC1987. These strains were 583 lost and no images are available.

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Pyramimonas australis RCC5269 strain from IC water has a sequence match-585 ing with 100% similarity that of P. australis (GenBank AJ404886) from the 586 subgenus Trichocystis, an Antarctic species described based on light/electron 587 microscopy, nuclear-encoded small-subunit ribosomal DNA and chloroplast-588 encoded rbcL gene sequences, but with no representative sequence from cultures 589 until now (Moro et al., 2002). Cells are pear-like to almost oval, ∼ 10 µm long 590 and 6 µm wide with four flagella ( Figure 7E).

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Pyramimonas sp. RCC5483 strain was recovered from IC surface waters dur-592 ing the pre-bloom phase and its sequence shares 100% similarity with that of the 593 Arctic strain RCC669 (Figure 8). This strain was lost and no images are available.

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Pyramimonas sp. RCC5453 was isolated from an IC ice core sample during 595 the pre-bloom phase and its sequence matches with 99.7% similarity that of the 596 Arctic strain Pyramimonas sp. RCC1987. Cells are pear-like to round, from 4 to 597 7 µm long and with four flagella ( Figure 7K).  (Comeau et al., 2013;Majaneva et al., 2017). The 821 same is true for the stages of ice formation (Kauko et al., 2018;Olsen et al., 2017).

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In the present work, ice core samples yielded most of the novel taxa, for all groups 823 from diatoms to green algae. It is important that culturing efforts continue in the 824 Arctic, as ongoing and predicted loss in ice coverage and thickness (Perovich 825 and Richter-Menge, 2009) will certainly impact plankton diversity, dynamics and 826 community structure (Blais et al., 2017;Comeau et al., 2011;Horvat et al., 2017).

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As the diversity within culture collections improves to reflect the complexity of 828 the environment, the increased amount of validated reference sequences will help 829 scientists to better access eukaryotic plankton distribution patterns across the Arc-830 tic. In addition, the availability of polar strains will enable experimental studies 831 to observe physiological and metabolic impacts of current changes such as global 832 warming on polar phytoplankton communities. 1 "cultured" corresponds to genotypes which 18S rRNA sequence is 100% similar to that of a culture that has been isolated previously, "detected -uncultured" correspond to genotypes which 18S rRNA sequence is 100% similar to that of a sequence detected in the environment but for which no culture existed prior to this work and "undetected" corresponds to genotypes for which no 100 % similar 18S rRNA sequence had been detected previously in the environment.   Overall diversity of the strains retrieved from ice and water samples assigned at the class level. Diatoms genera and most abundant strains are marked with as asterisk.  18S rRNA phylogenetic tree inferred by maximum likelihood (ML) analysis for pennate diatom strains obtained during the Green Edge campaign (in bold), using an alignment of 59 sequences with 406 positions. Circles mark strains retrieved from the Ice Camp ice (open) and water samples (solid); triangles (solid) mark Amundsen cruise water samples. The origin, sampling substrate and phase of the bloom from which they were recovered are provided along with their names and RCC code in Supplementary Data S1. When one genotype is represented by more than one strain, the number of strains is indicated between parenthesis. For the reference sequences, the strain (whenever available) and the Genbank ID number are displayed. Environmental sequences are marked in blue. 18S rRNA phylogenetic tree inferred by maximum likelihood (ML) analysis for centric diatoms strains. Legend is the same as in Figure 4, using an alignment of 55 sequences with 593 positions.  18S rRNA phylogenetic tree inferred by maximum likelihood (ML) analysis for the Chlorophyta strains. Legend is the same as in Figure 4, using an alignment of 70 sequences with 360 positions.  Figure S1. Novelty of genotypes.
Percentage of similarity of genotype representative 18S rRNA sequence to best BLAST hit from GenBank (see Supplementary Data S2). Figure S2. Strains from Amundsen cruise as a function of isolation method and depth.
Strain class distribution for the Amundsen cruise separated according to the method of isolation (cell sorting, serial dilution and single cell isolation) and sampling depth range. Figure S3. Genotype as a function of isolation method.
Treemap of the number of strains isolated as function of the isolation method.