DataSheet1_Community dynamics and co-occurrence relationships of pelagic ciliates and their potential prey at a coastal and an offshore station in the ultra-oligotrophic Eastern Mediterranean Sea.docx

Ciliates have been recognized as one of the major components of the microbial food web, especially in ultra-oligotrophic waters, such as the Eastern Mediterranean Sea, where nutrients are scarce and the microbial community is dominated by pico- and nano-sized organisms. For this reason, ciliates play an important role in these ecosystems since they are the main planktonic grazers. Regardless the importance of these organisms, little is known about the community structure of heterotrophic and mixotrophic ciliates and how they are associated to their potential prey. In this study, we used 18S V4 rRNA gene metabarcoding to analyze ciliate community dynamics and how the relationship with potential prey changes according to different seasons and depths. Samples were collected seasonally at two stations of the Eastern Mediterranean Sea (HCB: coastal, M3A: offshore) from the surface and deep chlorophyll maximum (DCM) layers. The ciliate community structure varied across depths in HCB and across seasons in M3A, and the network analysis showed that in both stations, mixotrophic oligotrichs were positively associated with diatoms and showed few negative associations with ASVs annotated as marine Stramenopiles (MAST). On the other hand, heterotrophic tintinnids showed negative relationships in both HCB and M3A stations, mostly with Ochrophyta and Chlorophyta. These results showed, in first place that, although the two stations are close to each other, the ciliate dynamics differed between them. Moreover, mixotrophic and heterotrophic ciliates may have different ecological niches since mixotrophic ciliates may be more selective compared to heterotrophic species regarding their prey. These findings are the first glimpse into an understanding of the dynamics between heterotrophic and mixotrophic ciliates and their role in microbial assemblages and dynamics of ultra-oligotrophic environments.</p

Table1_Community dynamics and co-occurrence relationships of pelagic ciliates and their potential prey at a coastal and an offshore station in the ultra-oligotrophic Eastern Mediterranean Sea.xlsx

Ciliates have been recognized as one of the major components of the microbial food web, especially in ultra-oligotrophic waters, such as the Eastern Mediterranean Sea, where nutrients are scarce and the microbial community is dominated by pico- and nano-sized organisms. For this reason, ciliates play an important role in these ecosystems since they are the main planktonic grazers. Regardless the importance of these organisms, little is known about the community structure of heterotrophic and mixotrophic ciliates and how they are associated to their potential prey. In this study, we used 18S V4 rRNA gene metabarcoding to analyze ciliate community dynamics and how the relationship with potential prey changes according to different seasons and depths. Samples were collected seasonally at two stations of the Eastern Mediterranean Sea (HCB: coastal, M3A: offshore) from the surface and deep chlorophyll maximum (DCM) layers. The ciliate community structure varied across depths in HCB and across seasons in M3A, and the network analysis showed that in both stations, mixotrophic oligotrichs were positively associated with diatoms and showed few negative associations with ASVs annotated as marine Stramenopiles (MAST). On the other hand, heterotrophic tintinnids showed negative relationships in both HCB and M3A stations, mostly with Ochrophyta and Chlorophyta. These results showed, in first place that, although the two stations are close to each other, the ciliate dynamics differed between them. Moreover, mixotrophic and heterotrophic ciliates may have different ecological niches since mixotrophic ciliates may be more selective compared to heterotrophic species regarding their prey. These findings are the first glimpse into an understanding of the dynamics between heterotrophic and mixotrophic ciliates and their role in microbial assemblages and dynamics of ultra-oligotrophic environments.</p

Phylogena and multiple alignment of the truncated N-terminal region from dinoflagellate KS.

<p>A. Maximum likelihood phylogenetic analysis of truncated N-terminal regions with 100 Bootstrap repetions. B. Multiple alignment of a highly conserved motif within the N-terminal region and the sequence logo showing the degree of sequence conservation of the respective site.</p

Conserved active site motifs of the ketoacyl synthase domain from different evolutionary distinct supergroups.

<p>A special emphasis was thereby given to protistan Type I PKS (Chlorophyta, Haptophyta, Apicomplexa and Dinoflagellata). A sequence logo of the active site motifs is given beneath the alignment; the height of the letters thereby indicates the degree of conservation. The three <i>A. ostenfeldii</i> KS sequences (Aost ac0019, Aost ac0038, and Aost 10-x_J14) and the two <i>H. triquetra</i> KS sequences (Hcaps HTE 5908 and Hcaps HTE 6310) contain all four active site amino acids <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048624#pone.0048624-vonWettsteinKnowles1" target="_blank">[20]</a> as is indicated by an asterisk.</p

Phylogenetic tree of Type I and Type II KS domains from prokaryotic and eukaryotic PKS and FAS.

<p>Fifty-nine taxa representing Type I and Type II KS domains were analyzed by a maximum likelihood approach. The Type II KS and the acyl carrier protein synthases (ACPS) were used as outgroup. Approximate likelihood ratios ≥50% and bootstrap values ≥50% are marked on appropriate branches like (50/50). A. Overall tree topology is in accord with previous reports <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048624#pone.0048624-JenkeKodama1" target="_blank">[5]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048624#pone.0048624-John2" target="_blank">[10]</a>. Dinoflagellate KS is classified as a well supported group within the protistan Type I FAS/PKS clade. B. Within the dinoflagellate KS group, one clade containing sequences from different species and one only from <i>K. brevis</i> can be observed.</p

Sequence properties of the ketoacyl snthases transcripts identified in <i>A. ostenfeldii</i> and <i>H. triquetra.</i>

<p>Sequence properties of the ketoacyl snthases transcripts identified in <i>A. ostenfeldii</i> and <i>H. triquetra.</i></p

Results of the 2-factorial RM-ANOVA for the induction phase, comparing feeding rates affected by treatment (control vs. water-borne cues/direct grazing) and time.

<p>Consumption was assessed in two-choice feeding assays using either fresh or reconstituted <i>Fucus vesiculosus</i> (n = 10).</p><p>Results of the 2-factorial RM-ANOVA for the induction phase, comparing feeding rates affected by treatment (control vs. water-borne cues/direct grazing) and time.</p

Results of two-tailed paired <i>t</i>-tests comparing isopod consumption between different 3 day intervals of the induction phase (n = 10).

<p>Significant p-values, i.e. α≤0.05, in bold.</p><p>Results of two-tailed paired <i>t</i>-tests comparing isopod consumption between different 3 day intervals of the induction phase (n = 10).</p

Mean and 95% confidence intervals of <i>Fucus vesiculosus</i> consumption by <i>Idotea baltica</i> during 3 day intervals in the induction phase (n = 10).

<p>Stippled line marks level of no consumption. Confidence intervals overlapping with stippled line indicate intervals when consumption was statistically not significantly different from zero. Different letters indicate significant difference in consumption.</p

Sets and subsets of differential gene expression in <i>Emiliania huxleyi</i> in response to Ocean Acidification and high light intensity.

<p>Responses to Ocean Acidification (upper part) and high light intensity (lower part) are shown for the diploid (left part, shaded) and the haploid (right part) life-cycle stage. Numbers represent significantly regulated genes; arrows indicate up- or down-regulation (↑ or ↓).</p