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Polar Biol (2012) 35:519–533 DOI 10.1007/s00300-011-1097-8

ORIGINAL PAPER

Molecular diversity and temporal variation of picoeukaryotes in two Arctic fjords, Svalbard

N. Sørensen • N. Daugbjerg • T. M. Gabrielsen

Received: 24 March 2011 / Revised: 22 August 2011 / Accepted: 23 August 2011 / Published online: 21 September 2011 Ó Springer-Verlag 2011

Abstract Picoeukaryotes ( \3 lm) form an early onset of the growth season, whereas larger cells important component of Arctic marine , dominated the bloom itself. although knowledge of their diversity and functioning is limited. In this study, the molecular diversity Keywords Arctic Environmental cloning and autotrophic contribution of picoeukaryotes Picobiliphytes from January to June 2009 in two Arctic fjords at Svalbard were examined using 18S environmental cloning and size- fractioned a measurements. A total of 62 Introduction putative picoeukaryotic phylotypes were recovered from 337 positive clones. Putative picoeukaryotic In the Arctic, picoplankton can contribute significantly to were mostly limited to one species: pusilla, although larger dominate the while the putative heterotrophic picoeukaryote assemblage spring bloom (Degerlund and Eilertsen 2010). In a study of was more diverse and dominated by uncultured marine the Arctic from June to August, picoeukaryotes (MAST) and marine groups. One contributed 36% of autotrophic biomass (Booth and Horner MAST-1A phylotype was the only phylotype to be found in 1997), whereas in the Canadian Arctic from August to all clone libraries. The diversity of picoeukaryotes in September, 11–72% of total chlorophyll a could be general showed an inverse relationship with total auto- obtained from intact cells passing through a 1-lm filter trophic biomass, suggesting that the conditions dominating (Smith et al. 1985). However, temporal variation plays an during the peak of the spring bloom may have a negative important role as small cells in general appear to be most impact on picoeukaryote diversity. Picoplankton could important when total chlorophyll concentrations are low contribute more than half of total autotrophic biomass (Brewin et al. 2010), and in the Barent Sea, 46% of total before and after the spring bloom and benefited from an primary production was attributed to cells \10 lm during the entire bloom period, despite accounting for only 19% of the chlorophyll a during the peak of the bloom (Hodal and Kristiansen 2008). The near-absence of marine cya- Electronic supplementary material The online version of this nobacteria in the Arctic makes the dominant article (doi:10.1007/s00300-011-1097-8) contains supplementary component of autotrophic picoplankton, thus underlining material, which is available to authorized users. the importance of this relatively unknown ecosystem component (Gradinger and Lenz 1995; Booth and Horner N. Sørensen (&) N. Daugbjerg Marine Biological Section, Department of Biology, 1997;Li1998; Sherr et al. 2003). University of Copenhagen, Øster Farimagsgade 2D, An Arctic strain of Micromonas pusilla has been shown to 1353 Copenhagen K, Denmark be an abundant marine picoautotroph throughout the Arctic e-mail: [email protected] (Not et al. 2005; Lovejoy et al. 2007), although other N. Sørensen T. M. Gabrielsen Mamiellophyceae (previously known as , The University Centre in Svalbard, 9171 Longyearbyen, Norway Marin and Melkonian 2010), Haptophyta, Cryptophyceae 123 520 Polar Biol (2012) 35:519–533 and several Bacillariophyceae (diatoms) phylotypes of environmental 18S clone libraries from an open-ended Arctic picoeukaryotes have also been recovered (Lovejoy shallow fjord with partial ice cover throughout winter et al. 2006). Stramenopiles and Alveolata are common (Adventfjorden) and a deep two-silled fjord with annual phylotypes in Arctic picoeukaryotic clone libraries, and as fast ice (Billefjorden) in Isfjorden, western Spitsbergen. In seen elsewhere, the putative heterotrophic uncultured marine addition, size-fractioned chlorophyll a measurements were stramenopiles (MAST) and marine alveolate group (MAG) I used to assess the contribution of picoplankton to the total and II are numerous (Massana et al. 2004b; Lovejoy et al. autotrophic biomass. To the best of our knowledge, this is 2006; Guillou et al. 2008). the first molecular study of Arctic picoeukaryote diversity In spite of giving insight into the hidden world of Arctic including seasonal variability. picoeukaryotes, molecular studies have not yet addressed their seasonal variation. A recent study on the molecular diversity of freshwater protists using high-throughput Materials and methods sequencing showed that temporal variation can be of utmost importance as many phylotypes showed a Study sites and sampling high temporal turnover (Nolte et al. 2010). Using tradi- tional environmental cloning in a temporal study of pic- Seawater samples were collected from three sampling oeukaryote diversity in the English Channel, Romari and stations: Adventfjorden at 15 m depth (78°16 N 15°30 E) Vaulot (2004) also found high temporal variability, which and Adolfbukta in Billefjorden at 15 and 150 m depth was also the case for a molecular study of small (\5 lm) (78°38 N 16°32 E, Fig. 1). For each station, two clone photosynthetic eukaryotes in the Gulf of Naples (McDonald libraries were made from samples collected in winter-early et al. 2007). In the English Channel, the app- spring (defined as January to March, called ‘winter’ eared stable at class/division level except during the henceforth) and one or two clone libraries from samples summer bloom where picoeukaryote diversity decreased. collected in spring-early summer (April to June, called A study by Medlin et al. (2006) at Helgoland also found ‘spring’, Table 1). This seasonal division was used because high temporal variation at a monthly basis, but identified an autotrophic biomass first started increasing in April, i.e. annual pattern in the community composition, suggesting ‘spring’ (see Results). Sampling of chlorophyll a and ice seasonality in the picoeukaryotic community. As the Arctic thickness was more frequent. The fjords were sampled is subject to extreme seasonal variation, it is an ideal using a 5- or 10-L Niskin bottle (KC Denmark A/S, location to investigate the influence of seasonality on pic- Silkeborg, Denmark). The samples were kept cold and dark oeukaryote diversity. Additionally, as the Arctic is pre- until further processing, which was done within 36 h of dicted to show amplified responses to global warming sampling. Filtrations were done with a vacuum pump or a (IPCC 2007), obtaining baseline data on Arctic picoeuk- Pump drive PD 5001 with Pump head C4 (Heidolph, aryotes is of importance for future comparisons. Schwabach, Germany). In Billefjorden ice thickness, In this study, the molecular diversity and temporal freeboard and snow thickness were measured at three variation of eukaryotic picoplankton were examined using points in a triangular pattern, each point 10 m apart.

Fig. 1 The study took place on Svalbard. Samples were collected from Adventfjorden (78°16 N 15°30) and n e d Billefjorden (78°38 N 16°32 E), r o j f which are part of the Isfjorden e l l system. The sampling locations 78°30 N i are marked with stars B

n de or fj Is 78°15 N Adventfjorden

14°00 E 16°00 E

123 Polar Biol (2012) 35:519–533 521

Table 1 Observed and estimated OTU number and number of clones 60 rpm. The 0.22-lm filters were cut in half and stored at sampled for each clone library -80°C until DNA extraction. Divided 0.22-lm filters were Station (m) Date OTUs Clones incubated in 594 lL CTAB and 6 lL b-mercaptoethanol found sampled for 45 min at 65°C, being vortexed every 15 min. The samples were frozen for C30 min at -80°C, heated at Adventfjorden 15 20–02–2009 10 26 65°C for 45 min and vortexed every 15 min. After adding Adventfjorden 15 25–03–2009 12 42 500 lL of chloroform mix (24:1 chloroform to isoamyl- Adventfjorden 15 19–05–2009 5 24 alcohol), the samples were vortexed twice, shaken contin- Adventfjorden 15 12–06–2009 14 32 uously for 10 min and then centrifuged at 12,000 rpm for Billefjorden 15 14–01–2009 17 24 5 min on an Eppendorf Centrifuge 5415D (Eppendorf AG, Billefjorden 15 30–03–2009 14 35 Hamburg, Germany). The water phases were transferred to Billefjorden 15 14–05–2009 9 22 new Eppendorf tubes, 500 lL chloroform mix was added Billefjorden 15 10–06–2009 9 26 again and the procedure repeated. A volume of 4 lLof Billefjorden 150 14–01–2009 20 39 RNAse was added to the transferred water phase and the Billefjorden 150 14–05–2009 20 31 samples were incubated for 30 min at 37°C. The samples Billefjorden 150 10–06–2009 15 36 were centrifuged briefly, and two-thirds of the sample volume of ice cold isopropanol was added and the samples were kept at -20°C for C30 min. The samples were cen- trifuged at 12,000 rpm for 10 min and washed twice in Chlorophyll a 600 lL 70% ethanol. Residual ethanol was evaporated by putting opened sample tubes on a heating block at 65°C for Seawater samples for chlorophyll a measurements were a few minutes. A volume of 30 lL Milli-Q water was collected in 10-L plastic bottles, which were rinsed with added to the sample and it was kept at room temperature distilled water between sampling. A volume of 0.60–1.70 L for one hour before being stored at ?5°C overnight. of water was filtered on 3-lm isopore membrane polycar- Extracted DNA was then stored at -20 or -80°C until bonate filters (Millipore, Billerica, USA) for non-pico- further processing. planktonic autotrophic biomass and a similar volume on 0.7-lm GF/F glass microfiber filters (Whatman, Maidstone, Polymerase chain reaction England) for total autotrophic biomass using a vacuum pump. The 0.7-lm filtrate was refiltered through 0.22-lm Polymerase chain reactions (PCR) were carried out on Durapore membrane hydrophilic PVDF filters (Millipore, an Eppendorf Mastercycler Ep Gradient S PCR cycler Billerica, USA) to test whether filtration through 0.7 lm (Eppendorf AG, Hamburg, Germany) in volumes of 25 lL was adequate for total chlorophyll a measurements. All containing 19 buffer with 2.5 mM MgCl (5 PRIME, filtrations were replicated three times, and filters were 2 Hamburg, Germany), 0.2 mM dNTP, 0.1 lg BSA, 0.2 lM stored in aluminium foil at -80°C. The equipment used for of each primer and 1 U HotMaster Taq DNA polymerase (5 filtration was rinsed with distilled water between uses. PRIME, Hamburg, Germany). For colony PCR, only half Chlorophyll a was extracted from the filters in 100 mL of the volumes were used per reaction, polymerase was methanol for 20–24 h at ?5°C (Holm-Hansen and Riemann reduced to 0.7 U and replaced with DreamTaq DNA 1978) and the concentration measured on a 10-AU-005-CE polymerase (Fermentas, Burlington, Canada) and BSA was Fluorometer (Turner Designs, Sunnyvale, USA). excluded. The following thermal cycling programme was used: 94°C for 3 min, 30 or 40 cycles of (94°C for 45 s, DNA extraction 57°C for 60 s, 72°C for 120 s) and 72°C for 10 min. An aliquot of 5 lL extracted DNA was amplified by an initial Seawater samples for DNA extraction were collected in PCR (EukA and EukB primers, 40 cycles). When neces- 2-L plastic bottles, which were washed with sample water sary, the correct band was isolated by gel extraction before collection. Containers and filtration units were (21.8 lL PCR product, 0.7% TAE gel, 90 V, 45 min) using washed with 1% chlorine for 12–24 h and repeatedly with Agarose GelExtract Mini Kit (5 PRIME, Hamburg, distilled water between uses. A volume of 2 L of sample Germany). A template of 2 lL 1,000-fold dilution of the water was prefiltered with a 3-lm isopore membrane PCR product or gel extract was used for a nested PCR polycarbonate filter and cells were collected on a 0.22-lm (30 cycles, Euk528f and EukB primers). DNA was purified Durapore membrane hydrophilic PVDF filter (both from using the E.Z.N.A. Cycle Pure Kit (Omega Bio-Tek, Millipore, Billerica, USA) on a Pump drive PD 5001 with Norcross, USA). An Eppendorf Centrifuge 5424 (Eppen- Pump head C4 (Heidolph, Schwabach, Germany) at dorf AG, Hamburg, Germany) was used for all post-PCR 123 522 Polar Biol (2012) 35:519–533 centrifugation. The initial PCR was insufficient for were manually adjusted. Alignments are available upon amplifying DNA from winter samples, explaining the need request to NS. for a total of 70 PCR cycles. The nested PCR was also used Bayesian analyses were done with MrBayes 3.1.2 on spring samples to impose the same PCR bias in all clone (Huelsenbeck et al. 2001; Ronquist and Huelsenbeck 2003). libraries. Bayesian posterior probabilities were calculated with 2,000,000 generations (5,000,000 for and Cloning stramenopiles), sampling trees every 50 generations. Tree convergence was checked with AWTY (Wilgenbusch et al. Samples were cloned with CloneJET PCR cloning kit 2004) and burn-in was found manually: the first 101 (Fermentas, Burlington, Canada) using TOP10 cells (Qia- (Choanoflagellida) or 401 (all other groups) trees were gen, Hilden, Germany) or 10G cells (Lucigen, Middleton, discarded. Posterior probabilities were calculated using a England) and ampicillin as selective media. Inserts were post-burn-in 50% majority rule consensus tree. ModelTest amplified by PCR (Euk528f and EukB, 30 cycles) and 3.7 (Posada and Crandall 1998) was used to find the best-fit digested with HaeIII (50-GG;CC-30, Fermentas, Burling- model (TrN ? I ? G for all alignments, Tamura and Nei ton, Canada) following the manufacturer’s protocol. Clones 1993) and neighbour-joining bootstrap values (1,000 with unique restriction fragment length polymorphism replicates), using maximum likelihood to estimate all (RFLP) patterns were grown overnight at 37°Cin*1mL pairwise comparisons, were calculated with PAUP* 4.b10 liquid LB–ampicillin medium, and the plasmids were iso- (Swofford 2003). Rarefaction curves were created using lated using the E.Z.N.A. Plasmid Miniprep Kit I (Omega Rarefaction Calculator (http://www.2.biology.ualberta.ca/ Bio-Tek, Norcross, USA) using 500 lL DNA wash buffer jbrzusto/rarefact.php). Sequences reported in this study for the second optional wash. The isolated plasmids were have been deposited in GenBank under accession numbers sequenced at the CEES DNA lab, Centre for Ecological HQ156808-HQ156856 and HQ156858-HQ156897 and the and Evolutionary Synthesis, University of Oslo, on a 3730 frequency of each of these in the different clone libraries DNA Analyzer (Applied Biosystems, Foster City, USA) can be found in the supplementary table supplied online. using various sequencing primers. Primers used for PCR and sequencing reactions are listed in Table 2 (Elwood et al. 1985; Medlin et al. 1988; Ekelund et al. 2004). Results

Molecular analyses Study sites

The sequences were divided into major taxonomical groups The fast ice thickness in Billefjorden varied from 72 cm in based on an initial NCBI BLAST search (Altschul et al. late February to 100 cm in mid-May and broke up in mid- 1990). Suspected chimeras were checked using Chimera June just after the final sampling. The snow thickness Detection (http://www.35.8.164.52/cgis/chimera.cgi?su= varied between 12 and 28 cm throughout the season. SSU) and KeyDNATools (http://www.keydnatools.com/) Adventfjorden was partially ice covered until May, with and by performing BLASTn searches using different ends more complete ice cover in late March and late April. See of the 18S rDNA sequence. Non-eukaryotic, fungal and supplementary material for more detailed information on \500-bp sequences were discarded. Clones with distinct ice thickness, CTD profiles and nutrient concentrations. RFLP patterns and \99% sequence similarity were treated as separate operational taxonomical units (OTUs). Chlorophyll a Taxonomical subdivisions were aligned against selected 18S sequences from NCBI GenBank in CLC Main In Adventfjoden at 15 m, the phototrophic biomass was low Workbench 5 (CLC bio, A˚ rhus, Denmark) and alignments during winter and early spring (0.02–0.03 lg Chl a L-1),

Table 2 Primers used for PCR Primer Sequence References and sequencing EukA 50-AACCTGGTTGATCCTGCCAGT-30 Medlin et al. (1988) EukB 50-TGATCCTTCTGCAGGTTCACCTAC-30 Medlin et al. (1988) Euk528f 50-GCGGTAATTCCAGCTCCAA-30 Elwood et al. (1985) ND5F 50-GGTGGTGCATGGCCGTTC-30 Ekelund et al. (2004) pJETf and pJETr primers ND7R 50-GAACGGCCATGCACCACC-30 Ekelund et al. (2004) (specific for the pJET cloning ND8R 50-TCTGAGAATTTCACCTCT-30 Ekelund et al. (2004) vector) were also used 123 Polar Biol (2012) 35:519–533 523 increased from April and reached a maximum of 5.7 lg metazoan, fungal, chimeric, non-eukaryotic and sequences Chl a L-1 in May (Fig. 2). Picoplankton made up over half of poor quality (\500 bp). Of the phylotypes, [75% of the total autotrophic biomass in April, but this decreased belonged to stramenopiles and Syndiniales (MAG-I and to 10–16% in May when total chlorophyll a was highest. In MAG-II). Only two phylotypes of Mamiellophyceae were June, total chlorophyll a dropped to 0.3 lgL-1, and found. Four sequences clustered within the novel lineage picoplankton again made up a substantial amount (62%) of picobiliphytes (Not et al. 2007b). Two sequences belonging the autotrophic biomass. In Billefjorden at 15 m, the to Haptophyta and three to Choanoflagellida were also autotrophic biomass also increased from April, but the found, as well as two unidentified Alveolata (sister to initial concentrations were lower (for both total and pico- Dinokaryota) and a single Cryptophyceae. Additionally, 72 planktonic chlorophyll a) and values [1 lgL-1 were not clones representing 18 phylotypes of Ciliophora, Dino- reached until June (Fig. 2). In April and May, picoplankton karyota and Rhizaria were found as well as 9 phylotypes of made up 24–51% of total autotrophic biomass, but this Metazoa (see supplementary material). decreased to 12% in June when total chlorophyll a was at Rarefaction curves showed Billefjorden at 150 m to its highest. Chlorophyll a concentration of the refiltrate generally have high molecular diversity (Fig. 3). For both (0.22–0.7 lm) was on average 1.0% of the total ([0.7 lm) fjords at 15 m, the lowest picoeukaryote diversity was with a maximum of 2.8%. found at the peak of the bloom, although the diversity was also relatively low in Billefjorden 15 m just Molecular diversity before the bloom. Following the decline in chlorophyll a in Adventfjorden, molecular diversity rose again. Most The 11 clone libraries yielded 337 clones belonging to 62 phylotypes showed C95% similarity to sequences already different putative picoeukaryotic phylotypes (Tables 1, 3) deposited in GenBank; some stramenopiles and picobili- when excluding , dinoflagellate, rhizarian (all three phytes had lower similarities (Fig. 4). There was no rela- groups unlikely to be picoplanktonic, see Discussion), tionship between the seasonal distribution of a phylotype (whether it was found in winter, spring or both seasons) a and its similarity to deposited sequences (ANOVA, F = 0.43, P = 0.65).

Mamiellophyceae

Both prasinophyte phylotypes clustered within the Mami- ellales (Fig. 5). The most widespread one (100609_23) was identified as M. pusilla and clustered within the Arctic subclade as defined by Lovejoy et al. (2007). It was found in all clone libraries except from Billefjorden 150 m in January and May. The other prasinophyte phylotype, 010809_04, formed a sister group to the genus b

µ Table 3 Number of operational taxonomical units (OTU) found for different taxonomical groups based on partial 18S sequencing Taxonomic group Number of OTUs Proportion (%)

Syndiniales 27 44 Stramenopiles 21 34 Picobiliphytes 4 6 Choanoflagellida 3 5 Mamiellophyceae 2 3 Haptophyta 2 3 Unidentified Alveolata 2 3 Cryptophyceae 1 2 Fig. 2 a Chlorophyll a concentrations in Adventfjorden 2009 at 15 m and b in Billefjorden 2009 15 m for phytoplankton [3 lm(black Sequences were defined as different OTUs if they had different RFLP bars) and picoplankton (white bars). In Adventfjorden, the bloom patterns and \99% sequence similarity. Ciliate, dinoflagellate, rhiz- occurred in mid-May and chlorophyll a decreased drastically arian, fungal, metazoan and non-eukaryotic sequences are not afterwards, and in Billefjorden, the bloom occurred in June included 123 524 Polar Biol (2012) 35:519–533

Chrysophyceae and one could not be assigned to any specific clade (Fig. 6). Those within MAST-1A, MAST- 1B, MAST-1C, MAST-3 and MAST-4 clades matched the probes designed by Massana et al. (2002, 2006b), except for 010609_04 (MAST-1A, 1 mismatch). MAST-1A, B and C clades are in accordance with Massana et al. (2006a), while the remaining MAST clades are as defined by Massana et al. (2004b) and Kolodziej and Stoeck (2007). Of the 21 phylotypes, 16 were absent from Adventfjorden (including the entire MAST-1C and MAST-4 clade). The phylotypes were evenly distributed between those found in winter, spring or both of these seasons (8, 7 and 7 phylotypes, respectively). The vast majority of closely related environmental sequences from other studies were from picoplanktonic size fraction and they were geographically diverse. The phylotype 100609_22 (MAST-1A) was found in all clone libraries and two other phylotypes, 130609_13 (MAST-1B) and 100609_12 (Paraphysomonas), were found in both winter and spring at all stations, although they were not present in Fig. 3 Rarefaction curves of putative picoeukaryotic OTUs for the all clone libraries. eleven clone libraries. The number of OTUs was found by the restriction enzyme HaeIII, and treating sequences with [99% similarity as belonging to the same phylotype. Ciliophora, Dino- Syndiniales karyota and Rhizaria are not included Ten phylotypes clustered within MAG-I and 17 within MAG-II (Fig. 7), both clades as defined by Skovgaard et al. (2005). Two phylotypes (190609_12 and 300709_06) could not be assigned to any specific clade, but showed weak support as a sister group to Dinokaryota together with their respective sister taxa (clade A). Both of these phyl- otypes were only found in one clone library each. MAG-I phylotypes were more often found in winter (90% of phylotypes) than in spring (30%). MAG-II did not exhibit such a temporal pattern (70 and 65%, respectively). The majority of the closest related environmental sequences stemmed from picoplanktonic size fraction (\3 lm), and they were collected from geographically diverse regions. Two of the related environmental sequences were from ship ballast water (BW-dinoclone1 and BW-dinoclone22). Phylotypes 210609_12 and 100609_24, belonging to MAG-I and MAG-II respectively, were found in both winter and spring at all stations, although not in all clone Fig. 4 Novelty histogram of phylotypes from this study by taxo- libraries. nomic affiliation. Phylotypes are binned by 0.5% identity to sequences in GenBank Picobiliphytes

Crustomastix with DSGM-81, although with limited sup- In Fig. 8, four phylotypes clustered within the clade port. It was only observed in spring in Adventfjorden. defined as picobiliphytes by Not et al. (2007b). One of the phylotypes was found in both winter and spring and at all Stramenopiles three sampling sites, while the others had a more limited distribution. Most of the closest related environmental Of the 21 stramenopile phylotypes obtained, 18 clustered sequences from other studies are from picoplanktonic size within a MAST clade, two clustered within the fractions (\3 lm) and were collected from the North 123 Polar Biol (2012) 35:519–533 525

0.52 Cyanophora paradoxa UTEX 555 (AY823716) Glaucophyceae (outgroup) Prasinococcus capsulatus CCMP1193 (AF203399) 0.99/91 Coccoid prasinophyte CCMP1413 (AF203402) 0.77/- Pseudoscourfieldia marina (AF122888) Pseudoscourfieldiales Pycnococcus provasolii CCMP1198 (AJ010406) Dolichomastix tenuilepis (AF509625) 0.57/- DSGM-81 (AB275081) North Pacific Ocean, sediment 0.62/- 010809_04 (HQ156812) S00

0.60/- Crustomastix sp. MBIC10709 (AB183628) Crustomastix 1.00/82 Crustomastix stigmatica (AJ629844)

1.00/93 Mamiella sp. (AB017129) Mantoniella squamata (X73999) Mantoniella antarctica (AB017128) 1.00/75 Micromonas pusilla CCMP490 (AY955003) D Micromonas pusilla CCMP1764 (AY954998) B Micromonas pusilla CCMP489 (AJ010408) A 0.67/- Mamiellales Micromonas pusilla CCMP1195 (AY954993) C 0.86/59 Micromonas pusilla CCMP1646 (AY954995) 100609_23 (HQ156835) BBS

0.99/87 Micromonas pusilla CCMP2099 (DQ025753) NW414.34 (DQ055151) Arctic Ocean, 0.22-3 µm E Ea 0.90/87 NOR46.10 (DQ055150) Arctic Ocean, 0.22-3 µm NW614.40 (DQ055158) Arctic Ocean, 0.22-3 µm MD65.21 (DQ055155) Arctic Ocean, 0.22-3 µm Bathycoccus prasinos (AY425315)

0.89/90 sp. MBIC10636 (AB058376) Ostreococcus tauri (Y15814) Cymbomonas tetramitiformis (AB017126)

Pterosperma cristatum NIES221 (AJ010407) Pyramimonadales 0.59/52 Pyramimonas olivacea (AB017122)

0.1

Fig. 5 Phylogenetic tree of Mamiellophyceae based on Bayesian stations: Adventfjorden 15 m (1st letter), Billefjorden 15 m (2nd inference. The clades A–E are the different clades of M. pusilla as letter) and Billefjorden 150 m (3rd letter). For each sampling station, defined by Slapeta et al. (2006), while Ea is the Arctic subclade phylotypes were categorized as found in winter (W), spring (S), both investigated by Lovejoy et al. (2007). Phylogenetic trees based on seasons (B), or absent (0); for example, W0B means a phylotype was Bayesian inference with posterior probability and bootstrap support found in winter in Adventfjorden at 15 m, absent from Billefjorden at values in per cent at relevant nodes. Filled circle indicates 1.00/100% 15 m and found in spring and winter in Billefjorden at 150 m. Winter and open circle indicate C0.95/C95%. Hyphen indicates posterior was defined as January to March and spring was defined as May and probability \0.50 or bootstrap \50%. Sequences from this study are June. For environmental sequences from other studies, location of in bold. The three-character code after the accession number tells the sampling and size fraction of clone library is given if applicable seasonal distribution of phylotypes at each of the three sampling

Atlantic or Arctic regions. Two phylotypes (100609_15 probe PICOBI02 also by Not et al. (2007b) had 6 mis- and 210609_18) represented a distinct subclade within one matches with each of the 4 phylotypes. of the clades (subclade A) and contained 4 identical mis- matches to the 18-bp picobiliphyte probe PICOBI01 Choanoflagellida designed by Not et al. (2007b). Phylotype 130609_16 had no mismatches while 100609_40_pJETf_b had 1 mismatch Three phylotypes clustered within the Choanoflagellida to the same probe. None of the mismatches were similar to (Fig. 9). The support for phylotype 100609_37 as a member mismatches reported by Not et al. (2007b) for sequences of Choanoflagellida was limited, especially when consider- within the picobiliphyte clade. The 18-bp picobiliphyte ing that Metazoa clustered within the Choanoflagellida.

123 526 Polar Biol (2012) 35:519–533

Gyrodinium helveticum (AB120004) turbo (DQ146405) (outgroup) 0.91/- 130609_07 (HQ156850) 00S 0.69/- ME1-21 (AF363190)* Mediterranean Sea, 0.2-5 µm OLI11026 (AJ402339)* Equatorial Pacific Ocean, 0.7-3 µm 0.87/- DH144-EKD10 (AF290063)* Southern Ocean, 0.2-5 µm 010609_04 (HQ156808) 0WW MAST-1A 1.00/- 020609_06 (HQ156815) 00W 1.00/- 100609_22 (HQ156834) BBB 130609_20 (HQ156858) S0S 0.51/- 130609_17 (HQ156856) 00S Hyphochytrium catenoides (AF163294) Rhizidiomyces apophysatus (AF163295) 010609_06 (HQ156809) 00W 0.91/90 300709_16 (HQ156897) 0SS RA000412.91 (AY295431)* North Atlantic Ocean, 0.22-3 µm MAST-1B NW414.07 (DQ062485)* Arctic Ocean, 0.22-3 µm 130609_13 (HQ156854) BBB 0.95/- 0.96/78 0.70/- 100609_12 (HQ156829) BBB Paraphysomonas (AF174376) Paraphysomonas 0.64/- Paraphysomonas imperforata (AF109324) 0.99/75 Ochromonas sp. CCMP1278 (OMU42382) Chrysophyceae 1.00/- Synura uvella (SUU73222) 300709_10 (HQ156894) 0WS Aureococcus anophagefferens CCMP1184 (AF118443) Pelagomonas calceolata (EF455763) 0.58/- 0.63/- Pelagococcus subviridis (PSU14386) 0.61/- Pinguiococcus pyrenoidosus (AF438324) 0.72/- Glossomastix chrysoplasta (AF438325) Dictyocha speculum (DSU14385) 0.99/89 Tribonema ulotrichoides (AM490817) 0.97/- 0.73/- 1.00/- Sargassum thunbergii (EF634360) Chattonella subsalsa (CSU41649) salina (FJ896223) Bolidomonas pacifica (AF123595) 0.94/- Bolidomonas mediterranea (AF123596) Minutocellus polymorphus CCMP497 (AY485478) 1.00/92 Minidiscus trioculatus 0.92/- CCMP495 (DQ093363) Bacillariophyceae (diatoms) 0.73/- Skeletonema japonicum CCMP784 (DQ396518) CCMP1007 (DQ093367) Pirsonia verrucosa (AJ561113) Pirsonia punctigera (AJ561115) 0.78/- 130609_01 (HQ156847) 0WB 130609_10 (HQ156852) 00S 1.00/74 130609_02 (HQ156848) 0WS ANT12-11 (AF363197)* Southern Ocean, 0.2-1.6 µm MAST-1C 1.00/51 NOR26.19 (DQ029013)* Arctic Ocean, 0.22-3 µm ME1-22 (AF363191)* Mediterranean Sea, 0.2-5 µm BAQA232 (AF372760)* North Pacific Ocean, sediment DH148-5-EKD53 (AF290083)* ME1-17 (AF363186)* MAST-2 080609_17 (HQ156822) 00S ANT37-16 (AF363206) Southern Ocean, 0.2-1.6 µm ANT12-8 (AF363208)* Southern Ocean, 0.2-1.6 µm MAST-8 0.93/85 ANT12-26 (AY116220)* Southern Ocean, 0.2-1.6 µm 1.00/83 210609_16 (HQ156879) 0S0 0.93/- ANT12-10 (AF363196) Southern Ocean, 0.2-1.6 µm BL010320.6 (AY381207)* Mediterranean Sea, 0.2-3 µm MAST-7 0.60/- BL000921.11 (AY381191)* Mediterranean Sea, 0.2-3 µm 0.96/- OLI11150 (AJ402355)* Equatorial Pacific Ocean, 0.7-3 µm 1.00/75 BL010625.32 (AY381217) 0.93/- BL000921.26 (AY381196) 1.00/83 100609_34 (HQ156840) 00W 1.00/85 170609_20 (HQ156871) 00S 1.00/52 ME1-20 (AF363189)* Mediterranean Sea, 0.2-5 µm ME1-19 (AF363188)* Mediterranean Sea, 0.2-5 µm MAST-4 1.00/91 OLI11066 (AJ402356)* Equatorial Pacific Ocean, 0.7-3 µm 0.90/- 150609_03 (HQ156860) 0W0 0.72/56 100609_36 (HQ156841) 00W 1.00/92 ME1-28 (AY116221)* Mediterranean Sea, 0.2-5 µm MAST-3 0.98/70 BL000921.38 (AY381200)* Mediterranean Sea, 0.2-3 µm 0.91/- M1_18B12 (DQ103774)* 0.94/- M2_18B03 (DQ103795)* MAST-13 OLI51105 (AF167414)* BAQD21 (AF372753)* MAST-12 0.72/- BAQD38 (AF372759)* Phytophthora undulata (AJ238654) Achlya apiculata (AJ238656) ME1-24 (AF363207)*MAST-6 300709_11 (HQ156895) 0WB Aplanochytrium kerguelense (AB022103) Labyrinthuloides minuta (LADDLRRNA) 0.84 DH147-EKD10 (AF290070)* MAST-5 Cepedea virguloidea (AF141969) Opalina ranarum (AF141970) 0.1

Fig. 6 Phylogenetic tree of stramenopiles based on Bayesian inference. Sequences marked with an asterisk (*) are used by Massana et al. (2004b), or Massana et al. (2006a), Kolodziej and Stoeck (2007) to define the respective clades. The explanations are given in Fig. 5

Two of the phylotypes were only represented by one clone Ciliophora, Dinokaryota, and Rhizaria each, while the last phylotype (230609_07) was present in 6 of 11 clone libraries. Among the Ciliophora, six phylotypes clustered within Spirotrichea, 170609_14 within , and 2306 Haptophyta and Cryptophyceae 09_05 together with RD010517.29 and Urotricha sp. as sister to and (Fig. A5, The two haptophycean phylotypes both clustered within the supplementary material). Ciliophora were found at all genus Chrysochromulina (Fig. 10). Both phylotypes were sampling stations but only in spring. All the related envi- only represented by one clone each in spring in Billefjorden ronmental sequences had been found in a \5-lm fraction. at 150 m. The single cryptophyceaen phylotype obtained in Six phylotypes clustered within Dinokaryota (Fig A6, this study showed 99.38% similarity to Geminigera cryo- supplementary material). Dinokaryota and its sister group phila (accession number AB058368). It was only repre- Clade A were more often found in winter (87.5% of sented by a single clone in June in Billefjorden at 15 m. phylotypes) than in spring (37.5%).

123 Polar Biol (2012) 35:519–533 527

Tetrapyle octacantha (AB246680) Spongodiscus resurgens (AB246696) Rhizaria (outgroup)

0.65/0.58 mikimotoi CCMP429 (FJ587220) Prorocentrum minimum CCMP696 (FJ587221) 0.80/- Cochlodinium polykrikoides (EU418965) Gyrodinium helveticum (AB120004) Dinokaryota Azadinium spinosum (FJ217814) catenatum CCMP414 (DQ779990) Heterocapsa rotundata CCCM680 (AF274267) 1.00/100 190609_12 (HQ156874) 0W0 SS1_E_02_04 (EU050981) Kongsfjorden, sediment Clade A 0.60/- 300709_06 (HQ156892) 0W0 0.73/85 BW-dinoclone1 (FJ832099) ballast water from Singapore

1.00/74 080609_01 (HQ156818) W0S 100609_05 (HQ156825) SWB SSRPC38 (EF172859) North Atlantic Ocean, 2-5 µm

0.79/- 080609_18 (HQ156823) 00S 100609_38 (HQ156843) 00W PROSOPE.ED-50m.30 (EU793718) Mediterranean Sea, 1-3 µm 0.8/- 300509_08 (HQ156888) W00 DH147-EKD3 (AF290069)* Southern Ocean, 0.2-5 µm 170609_12 (HQ156866) 00S 0.51/- SCM37C36 (AY664989) North Atlantic 100609_14 (HQ156831) S0B 0.93/- He001005_12 (AJ965153) North Sea, 0.2-5 µm 210609_05 (HQ156876) BWS 0.98/- 130609_12 (HQ156853) 00S SSRPD58 (EF172942) North Atlantic, 0.45-2 µm 0.88/- 130609_08 (HQ156851) 00S Marine Alveolate Group II Amoebophrya sp. (AF069516)* 0.51/- 170609_10 (HQ156865) S0S BW-dinoclone22 (FJ832120) ballast water from Singapore Amoebophrya sp. ex Prorocentrum minimum (AY208894)* 300709_01 (HQ156891) 0W0 PROSOPE.EM-50m.44 (EU793863) Mediterranean Sea, 1-3 µm DH148-EKD14 (AF290079)* Southern Ocean, 0.2-5 µm 0.98/67 100609_24 (HQ156836) BBB 300509_11 (HQ156889) W00 0.52/- ZZ0053321 (EU817941) North Atlantic Ocean, 0.2-2 µm 210609_04 (HQ156875) SWS 1.00/- 0.98/74 100609_07 (HQ156826) WWW 0.93/64 300709_12 (HQ156896) 0W0 RA071004T.058 (FJ431861) North Atlantic Syndinium turbo (DQ146405)* 1.00/77 sp. MF-2000 (AF286023)* 020609_07 (HQ156816) W00 Uncultured marine alveolate isolate 68 (DQ916405) Sognefjorden (Norway) 040609_01 (HQ156817) 0W0 0.92/86 100609_48 (HQ156846) 00W 1.00/60 170609_09 (HQ156864) 00S 0.99/- 0.98/87 DH148-EKD22 (AF290078)* Southern Ocean, 0.2-5 µm OLI11033 (AJ402353)* Equatorial Pacific Ocean, 0.7-3 µm OLI11038 (AJ402328)* Equatorial Pacific Ocean, 0.7-3 µm 080609_06 (HQ156820) W00 1.00/95 100609_13 (HQ156830) 0W0 Marine Alveolate Group I DH144-EKD3 (AF290064)* Southern Ocean, 0.2-5 µm 130609_03 (HQ156849) SB0 0.40 150609_01 (HQ156859) 0W0 210609_12 (HQ156878) BBB 1.00/- p15B2 (AY882471) Caribbean Sea, 0.65 µm PROSOPE.ED-50m.202 (EU793695) Mediterranean Sea, 1-3 µm 300509_13 (HQ156890) W00 0.90/- infectans (AF133909) mediterraneus (AY517647) (X75762) 0.94/77 colobi (AF111186) (TOXRRE) 0.1

Fig. 7 Phylogenetic tree of Syndiniales. Sequences marked with an in this study. See supplementary material for a phylogeny including asterix (*) are used by Skovgaard et al. (2005) to define the respective Dinokaryota sequences obtained in this study. The explanations are clades; these clades have been expanded to include other phylotypes given in Fig. 5

Of the four Rhizaria phylotypes found, one sequence, found, comparable to other picoplankton communities both 170609_19, clustered within the Nassellaria, while the within and outside of Arctic waters (Romari and Vaulot remaining three sequences did not cluster with morpholog- 2004; Massana et al. 2004a; Lovejoy et al. 2006; Medlin ically well-characterized Rhizaria (Fig. A7, supplementary et al. 2006; Not et al. 2007a, 2009). The MAST clades material). Instead, they clustered with sequences of unknown described so far (MAST-1A, MAST-1B, MAST-1C, cellular identity. However, group A had a bootstrap support MAST-2, MAST-3, MAST-4 and MAST-12) appear to be of 79% as a monophyletic clade, although being unresolved strictly heterotrophic based on incubations, grazing in the Bayesian analysis. All phylotypes were found in winter experiments and taxonomical affiliations (Massana et al. and two were also found in spring. The related environmental 2002, 2004b, 2006a, b; Kolodziej and Stoeck 2007; sequences were geographically diverse and half of them Massana et al. 2009). MAG-I and II are part of Syndiniales, were obtained from\3-lm fractions. which are obligate marine parasitoids (i.e. parasites that kill or castrate their host) able to produce picoplanktonic spores (Guillou et al. 2008). Of the five phylotypes found at both Discussion seasons and all stations, four belonged to MAST (100609_22 and 130609_13, Fig. 6) and MAG (100609_24 dominated picoeukaryote diversity and 210609_12, Fig. 7). The last phylotype (100609_12, throughout the Arctic spring Fig. 6) most likely belong to the strictly heterotrophic genus Paraphysomonas (Caron et al. 1999). So not only do Stramenopiles (including MAST) and Syndiniales (i.e. heterotrophs dominate picoeukaryote diversity, they also MAG-I and II) accounted for [75% of the phylotypes contain the phylotypes with the widest spatial and temporal

123 528 Polar Biol (2012) 35:519–533

Cyanophora paradoxa UTEX 555 (AY823716) Glaucophyceae (outgroup) Glaucocystis nostochinearum (X70803) Cryptomonas pyrenoidifera CCAP979/61 (AJ421147) Chilomonas (L28811) Cryptophyceae Geminigera cryophila MBIC10575 (AB058368) 1.00/- 0.60/86 Hanusia phi (U53126) Leucocryptos marina (AB194980) Katablepharidophyta Katablepharis japonica (AB231617) OR000415.159 (DQ222875) North Atlantic Ocean, <3 µm RA010613.144 (DQ222880) North Atlantic Ocean, <3 µm NOR46.24 (DQ060526) Arctic Ocean, 0.22-3 µm 1.00/84 HE001005.148 (DQ222874) North Sea, <3 µm 100609_15 (HQ156832) W0W 1.00/84 Subclade A 210609_18 (HQ156881) W00 RA000907.33 (DQ222876) North Atlantic Ocean, <3 µm HE000803.72 (DQ222873) North Sea, <3 µm RA001219.38 (DQ222878) North Atlantic Ocean, <3 µm 0.92/- RA080215T.063 (FJ431633) North Atlantic Ocean RA000907.54 (DQ222877) North Atlantic Ocean, <3 µm Picobiliphytes RA080215T.037 (FJ431615) North Atlantic Ocean RA080215T.066 (FJ431635) North Atlantic Ocean NW414.27 (DQ060524) Arctic Ocean, 0.22-3 µm HE000427.214 (DQ222872) North Sea, <3 µm NW617.02 (DQ060525) Arctic Ocean, 0.22-3 µm RA000907.18 (DQ222879) North Atlantic Ocean, <3 µm 130609_16 (HQ156855) 0SS 0.81/86 NOR46.29 (DQ060523) Arctic Ocean, 0.22-3 µm 05M100r.12 (EU682624) Arctic Ocean, 0.22-3 µm 100609_40 (HQ156845) WBW 05M100n.04 (EU682625) Arctic Ocean, 0.22-3 µm NOR50.52 (DQ060527) Arctic Ocean, 0.22-3 µm NOR50.25 (DQ060528) Arctic Ocean, 0.22-3 µm 0.86/- NOR50.14 (DQ060529) Arctic Ocean, 0.22-3 µm Chrysochromulina polylepis B11 (AJ004866) 1.00/84 Phaeocystis globosa (X77476) Haptophyceae Pavlova virescens HAP16 (AJ515248) Pavlova salina (L34669) 1.00/82 Oogamochlamys gigantea (AJ410465) Ulva rigida (AJ005414) Trebouxia asymmetrica (Z21553) Viridiplantae 0.89/- Mesostigma viride (AJ250109) Porphyra suborbiculata (AB013180) 1.00/82 Bangia atropurpurea (L36066) Gracilaria lemaneiformis (M54986) Rhodophyta Chondrus crispus (Z14140)

0.1

Fig. 8 Phylogenetic tree of picobiliphytes based on Bayesian inference. Sequences marked with asterix (*) are used by Not et al. (2007b)to define the respective clades; these clades have been expanded to include other phylotypes in this study. The explanations are given in Fig. 5 distributions. Thus, the diversity of the Arctic picoeuk- et al. 2006b). In the fjords of this study, MAST-2 was not aryotic community is dominated by organisms with a het- found, but MAST-4 was. The latter clade was found during erotrophic or parasitic lifestyle throughout the investigated winter in Billefjorden, where temperatures were around period. -1.5°C, indicating a distribution not as limited by cold The MAST clades 1A, 1B, 1C, 2, 3 and 7 have previ- temperatures as previously suspected. MAST-3 was not ously been found in Arctic open-ocean environments in abundant, only being found once in winter in Billefjorden late August and early October, MAST-3 being especially at 150 m depth. Furthermore, although the MAST-3 abundant (Lovejoy et al. 2006), while MAST clades 1A, 3 phylotype from this study did match the probe by Massana and 7 have been found in the North Water in August et al. (2006b), it only clustered within the MAST-3 clade (Hamilton et al. 2008). MAST-4 has been suggested to be with weak support (Fig. 6). mostly absent from polar waters below 5°C (Rodriguez- In line with other studies (Lovejoy et al. 2006; Guillou Martinez et al. 2009), but have previously been found in et al. 2008; Hamilton et al. 2008), MAG-I and II were the Arctic, at temperatures [4°C, in late August (Massana ubiquitous in the clone libraries, with MAG-II being more

123 Polar Biol (2012) 35:519–533 529

Rhizophlyctis rosea (AY635829) Fungi (outgroup) Saccharomyces cerevisia (EU011664)

Homo sapiens (HSU13369) Metazoa 0.54/- Calanus finmarchicus (AF367719)

Salpingoeca infusionum (AF100941) 0.82/- Proterospongia sp. (EU011924) Monosiga brevicollis (AF174375)

0.95/83 Choanoeca perplexa (AF084232)

Codonosiga gracilis (AY149897)

Monosiga ovata (AF271999) 1.00/73 0.98/58 Salpingoeca amphoridium (DQ059032)

Salpingoeca napiformi (EU011929) 0.58/- Salpingoeca pyxidium (EU011930)

Diaphanoeca grandis (DQ059033) 0.81/58 Stephanoeca diplocostata (EU011926)

0.59/- Stephanoeca diplocostata (EU011927)

010809_03 (HQ156811) 0S0

0.93/- DB25_BASS (EU154974) Drake Passage, 0.2-5 µm Choanoflagellida 0.58/- 230609_07 (HQ156886) SBS 0.93/79 0.72/- CS050S19 (FJ169746) Yellow Sea 0.73/- 0.81/- 1.00/91 MO010_1.00381 (GQ382459) North Pacific Ocean Choanoflagellida sp. (EF432538) 0.61/- Volkanus costatus (EU011923) 99/58 Choanoflagellida sp. (EF432540)

Helgoeca nana (EF523335)

1.00/56 Savillea micropora (EU011928) 0.91/- Acanthoeca spectabilis (EU011922)

Salpingoeca urceolata (EU011931) 100609_37 (HQ156842) 00W

NIF_3G4 (EF526821) North Sea, >0.65 µm

Lagenoeca sp. Antarctica (DQ995807) 0.1

Fig. 9 Phylogenetic tree of Choanoflagellida based on Bayesian inference. The explanations are given in Fig. 5 diverse and widely distributed. Syndiniales can play a role (Lovejoy et al. 2007). In fact, of the putative autotrophic stronger than herbivory in regulating the abundance of their phylotypes found, M. pusilla was the only one to have a host species (Chambouvet et al. 2008), but the ecological widespread distribution supportive of significant primary impact of each phylotype is bound to be strongly linked to production. its choice of host species. However, inferring host species Picobiliphytes were originally thought to be photosyn- based on phylogenetic data can be difficult for Syndiniales, thetic (Not et al. 2007b), although a newer study using as they in general do not appear to co-evolve with their whole-genome shotgun sequencing has challenged this host, and some may be opportunistic in host choice notion (Yoon et al. 2011). In the investigated fjords, their (Guillou et al. 2008). The association of MAG-I phylotypes distribution does not support extensive phototrophy, as with winter libraries could indicate that their hosts are most they were all absent from Adventfjorden in spring and 2 of abundant in winter. the 4 phylotypes were also absent from Billefjorden in spring. Indeed, Hamilton et al. (2008) found picobiliphytes Autotrophs and other protists in the North Water to be associated with deep Arctic waters with low chlorophyll concentration, making them unlikely The widespread abundance (especially in spring) of M. primary producers of importance in the Arctic. pusilla in our clone libraries indicates that it is likely to be No picoplanktonic Bacillariophyceae were found, an important picoplanktonic primary producer in the although they have previously been recovered from the investigated fjords, as found elsewhere in the Arctic Arctic (Lovejoy et al. 2006) and several picoplanktonic

123 530 Polar Biol (2012) 35:519–533

Diacronema vlkianum (AJ515246) Pavlovales (outgroup) Pavlova gyrans (PGU40922)

080609_15 (HQ156821) 00S 0.75/65 170609_17 (HQ156869) 00S

0.76/79 Chrysochromulina simplex (AM491021) 0.82/94 0.76/70 Chrysochromulina sp. (DQ980478)

Chrysochromulina cymbium (AM491018)

0.90/80 Chrysochromulina acantha (AJ246278) 0.99/94 Chrysochromulina trondsenii (AJ246279) Chrysochromulina 0.72/76 Chrysochromulina polylepis (AJ004866)

Chrysochromulina cf. polylepis (AM491009)

Prymnesium parvum (AJ246269)

Prymnesium nemamethecum (AM491004) 0.18 0.92/- 0.54/- Prymnesium calathiferum (AM491008) 0.99/77 Platychrysis simplex (AM491028)

Imantonia rotunda (AM491014)

Chrysochromulina hirta (AJ246272)

Phaeocystis pouchetii (X77475)

Phaeocystis antarctica (X77481)

0.1

Fig. 10 Phylogenetic tree of Haptophyta based on Bayesian inference. The explanations are given in Fig. 5 bacillariophyceaen species have been described, many et al. (2010) have suggested that salinity, influenced by belonging to the genus Minidiscus (Vaulot et al. 2008; glacial melt water, may play an important role in shaping Kaczmarska et al. 2009). the microbial eukaryotic community of Arctic fjords, but clogging of the 3-lm prefiltration filter due to a high Temporal patterns of picoeukaryote diversity concentration of phytoplankton could potentially withhold some picoplanktonic species, thus decreasing the pico- The inverse relationship between picoeukaryote diversity planktonic diversity observed during the bloom. and autotrophic biomass indicates that the conditions found Although the biomass of picoautotrophs also increased during the spring bloom have a negative impact on pic- during the bloom, it is unclear to what degree this pic- oeukaryote diversity; likewise, a negative correlation oeukaryote bloom influenced the heterotrophic picoeuk- between phytoplankton biomass and diversity has also been aryotes. For instance, phylotypes of the MAST-1C and found for larger protists (Moustaka-Gouni 1993). Although MAST-4 clades, which have been observed to ingest M. single picoeukaryote species may flourish, the negative pusilla in experimental set-ups (Massana et al. 2009), were impact from the Arctic spring bloom on the picoeukaryote not found in Adventfjorden in spring. diversity suggests an important connection between the Despite the proliferation of marine picoeukaryote diatom bloom and the picoeukaryote community, which molecular diversity studies over the past years (e.g. Diez requires further investigation. A similar result was found in et al. 2001; Moon-van der Staay et al. 2001; Romari and a study from the English Channel where the lowest pic- Vaulot 2004; Massana et al. 2004a; Lovejoy et al. 2006; oeukaryote diversity was found at the peak of the diatom- Medlin et al. 2006; Worden et al. 2006; Not et al. 2007a, dominated spring bloom (Romari and Vaulot 2004). Piquet 2009; Piquet et al. (2010)), many novel sequences were

123 Polar Biol (2012) 35:519–533 531 found in this study: of 62 sequences, 26 (42%) had \93% The absence of Choanoflagellida, Cryptophyceae and similarity with sequences available in GenBank. Fjords Haptophyta from most clone libraries could suggest a may contain more novel sequences than the open ocean due limited ecological impact. However, both Haptophyta and to the focus on open in earlier molecular studies, Cryptophyceae have been observed to contribute signifi- and Arctic regions have not yet been well studied. More cantly to phytoplankton in Adventfjorden (Dobrzyn et al. novel sequences were not found as a consequence of 2009) and elsewhere in the Arctic (Booth and Smith 1997). sampling in winter, as there was no relationship between While Choanoflagellida, G. cryophila and Chrysochromu- seasonal distribution of a phylotype and its similarity to lina spp. can all have dimensions that may facilitate pas- previously deposited sequences. Thus, it is the location sage through a 3-lm filter (Taylor and Lee 1971; Rhodes rather than the addition of winter sampling that best and Burke 1996; Nitsche et al. 2007), the prefiltration may accounts for the novelty found in this study. That this little have reduced their frequency in the clone libraries if the sampled environment provided many novel sequences organisms in the fjords are not strictly picoplankton. These supports the idea of endemic Arctic picoeukaryotes as organisms may play an important ecological role in the discussed by Lovejoy and Potvin (2011). investigated ecosystems, but are unlikely to be important It proved difficult to obtain amplified 18S rDNA PCR members of the picoplankton size fraction. Finally, the products from the winter samples, as a total of 70 PCR relative low occurrence of Haptophyta may be explained cycles were required. Potentially, this could have caused a by poor amplification during PCR, as they are in general severe PCR bias. However, the diversity dominated by underrepresented in environmental clone libraries (Liu stramenopiles and Syndiniales in this study is in concor- et al. 2009; Marie et al. 2010). dance with other molecular studies, which suggests limited GF/F filters proved sufficient to estimate total chloro- additional PCR bias. In addition, Not et al. (2009), using a phyll a as refiltration with 0.22-lm filters only yielded 1% PCR-free approach for investigating the molecular diver- additional chlorophyll a on average, but picoplanktonic sity of picoeukaryotes, found that PCR steps do not impose autotrophic biomass may still have been underestimated as major biases. the fraction of phytoplankton [3 lm could have been overestimated as a result of the 3-lm filter clogging during Size fractions filtration, thus retaining smaller particles.

Mamiellophyceae (Slapeta et al. 2006), stramenopiles Open versus ice-covered ecosystems (Kaczmarska et al. 2009), Syndiniales (Guillou et al. 2008) and picobiliphytes (Not et al. 2007b) all have picoplank- The less extensive ice cover likely caused the bloom to tonic species, while Choanoflagellida (Nitsche et al. 2007), start earlier and higher pre-bloom chlorophyll a concentra- Haptophyta (Rhodes and Burke 1996) and Cryptophyceae tions in the open Adventfjorden compared to the ice-cov- (Taylor and Lee 1971), as well as several MAST cells ered Billefjorden. As the ratio of picoplanktonic to total (Massana et al. 2002, 2006b; Kolodziej and Stoeck 2007; chlorophyll a was greatest before the bloom, as seen in Massana et al. 2009), can have dimensions that may other studies (Hodal and Kristiansen 2008; Brewin et al. facilitate occasional passage through a 3-lm filter. This is 2010), it seems that picophytoplankton benefit from con- not the case for Rhizaria, Ciliophora, or Dinokaryota as we ditions that support early growth. Indeed, Li et al. (2009) currently understand the range of cell sizes in each of these found that warming of the surface water in the Canada groups. Basin caused an increase in picophytoplankton. The A recent molecular study found that, although present in predicted warming of the Arctic may therefore result the\0.8-lm fraction, Ciliophora and Rhizaria were absent in pelagic picophytoplankton forming an increasingly from the 0.8- to 3-lm fraction and the occurrence of important component of Arctic marine ecosystems. This Dinokaryota greatly reduced (Not et al. 2009). This indicates could have serious cascading effects on higher trophic that extracellular DNA is the source of these sequences. levels. Despite being represented by a total of 18 phylotypes and 72 clones, sequences belonging to Ciliophora, Dinokaryota Acknowledgments This work was supported by Svalbard Science and Rhizaria may not represent picoplankton in this study, Forum grant 3371 and a UNIS grant to TMG. The authors would like although as yet undescribed picoplanktonic members of to thank Bioportal at the University of Oslo for providing CPU these groups may exist. Sampling of ‘too large’ organisms resources, Lilith Kuckero and Emma Johansson-Karlsson for chlo- rophyll a measurements and Svein Kristiansen for measuring nutri- is a well-known phenomenon when working with size- ents. The authors would also like to thank Eike Mu¨ller for useful fractioned clone libraries (Lovejoy et al. 2011); this was discussion when planning the molecular work and Sofia Isabel dos also evident from the fact that 9 metazoan phylotypes were Santos Ribeiro, Daniel Vaulot and three anonymous reviewers for recovered from the fjords. helpful criticism on earlier versions of this manuscript. 123 532 Polar Biol (2012) 35:519–533

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