Relative abundance of NGS V4 Leptocylindraceae sequences at the six stations.

<p>Data for plankton samples were inferred from the surface cDNA results normalised over the total number of sequences obtained for the sample and the average of the three size fractions (mean relative frequency). Data for sediment samples were inferred from cDNA template based sequences (relative frequency).</p

Image_1_Nitrogen and phosphorus significantly alter growth, nitrogen fixation, anatoxin-a content, and the transcriptome of the bloom-forming cyanobacterium, Dolichospermum.JPEG

While freshwater cyanobacteria are traditionally thought to be limited by the availability of phosphorus (P), fixed nitrogen (N) supply can promote the growth and/or toxin production of some genera. This study characterizes how growth on N2 (control), nitrate (NO3–), ammonium (NH4+), and urea as well as P limitation altered the growth, toxin production, N2 fixation, and gene expression of an anatoxin-a (ATX-A) – producing strain of Dolichospermum sp. 54. The transcriptomes of fixed N and P-limited cultures differed significantly from those of fixed N-deplete, P-replete (control) cultures, while the transcriptomes of P-replete cultures amended with either NH4+ or NO3– were not significantly different relative to those of the control. Growth rates of Dolichospermum (sp. 54) were significantly higher when grown on fixed N relative to without fixed N; growth on NH4+ was also significantly greater than growth on NO3–. NH4+ and urea significantly lowered N2 fixation and nifD gene transcript abundance relative to the control while cultures amended with NO3– exhibited N2 fixation and nifD gene transcript abundance that was not different from the control. Cultures grown on NH4+ exhibited the lowest ATX-A content per cell and lower transcript abundance of genes associated ATX-A synthesis (ana), while the abundance of transcripts of several ana genes were highest under fixed N and P - limited conditions. The significant negative correlation between growth rate and cellular anatoxin quota as well as the significantly higher number of transcripts of ana genes in cultures deprived of fixed N and P relative to P-replete cultures amended with NH4+ suggests ATX-A was being actively synthesized under P limitation. Collectively, these findings indicate that management strategies that do not regulate fixed N loading will leave eutrophic water bodies vulnerable to more intense and toxic (due to increased biomass) blooms of Dolichospermum.</p

Table_1_Nitrogen and phosphorus significantly alter growth, nitrogen fixation, anatoxin-a content, and the transcriptome of the bloom-forming cyanobacterium, Dolichospermum.docx

While freshwater cyanobacteria are traditionally thought to be limited by the availability of phosphorus (P), fixed nitrogen (N) supply can promote the growth and/or toxin production of some genera. This study characterizes how growth on N2 (control), nitrate (NO3–), ammonium (NH4+), and urea as well as P limitation altered the growth, toxin production, N2 fixation, and gene expression of an anatoxin-a (ATX-A) – producing strain of Dolichospermum sp. 54. The transcriptomes of fixed N and P-limited cultures differed significantly from those of fixed N-deplete, P-replete (control) cultures, while the transcriptomes of P-replete cultures amended with either NH4+ or NO3– were not significantly different relative to those of the control. Growth rates of Dolichospermum (sp. 54) were significantly higher when grown on fixed N relative to without fixed N; growth on NH4+ was also significantly greater than growth on NO3–. NH4+ and urea significantly lowered N2 fixation and nifD gene transcript abundance relative to the control while cultures amended with NO3– exhibited N2 fixation and nifD gene transcript abundance that was not different from the control. Cultures grown on NH4+ exhibited the lowest ATX-A content per cell and lower transcript abundance of genes associated ATX-A synthesis (ana), while the abundance of transcripts of several ana genes were highest under fixed N and P - limited conditions. The significant negative correlation between growth rate and cellular anatoxin quota as well as the significantly higher number of transcripts of ana genes in cultures deprived of fixed N and P relative to P-replete cultures amended with NH4+ suggests ATX-A was being actively synthesized under P limitation. Collectively, these findings indicate that management strategies that do not regulate fixed N loading will leave eutrophic water bodies vulnerable to more intense and toxic (due to increased biomass) blooms of Dolichospermum.</p

Number of reads and OTUs obtained by clustering the BioMarKs sequences at 97% similarity cut-off.

a<p>percentage over all BioMarKs sequences.</p>b<p>percentage over all BioMarKs diatom sequences.</p>c<p>ambiguously distinguishable in V4 sequences.</p>d<p>identified only among V4 sequences.</p

Image_2_Nitrogen and phosphorus significantly alter growth, nitrogen fixation, anatoxin-a content, and the transcriptome of the bloom-forming cyanobacterium, Dolichospermum.JPEG

While freshwater cyanobacteria are traditionally thought to be limited by the availability of phosphorus (P), fixed nitrogen (N) supply can promote the growth and/or toxin production of some genera. This study characterizes how growth on N2 (control), nitrate (NO3–), ammonium (NH4+), and urea as well as P limitation altered the growth, toxin production, N2 fixation, and gene expression of an anatoxin-a (ATX-A) – producing strain of Dolichospermum sp. 54. The transcriptomes of fixed N and P-limited cultures differed significantly from those of fixed N-deplete, P-replete (control) cultures, while the transcriptomes of P-replete cultures amended with either NH4+ or NO3– were not significantly different relative to those of the control. Growth rates of Dolichospermum (sp. 54) were significantly higher when grown on fixed N relative to without fixed N; growth on NH4+ was also significantly greater than growth on NO3–. NH4+ and urea significantly lowered N2 fixation and nifD gene transcript abundance relative to the control while cultures amended with NO3– exhibited N2 fixation and nifD gene transcript abundance that was not different from the control. Cultures grown on NH4+ exhibited the lowest ATX-A content per cell and lower transcript abundance of genes associated ATX-A synthesis (ana), while the abundance of transcripts of several ana genes were highest under fixed N and P - limited conditions. The significant negative correlation between growth rate and cellular anatoxin quota as well as the significantly higher number of transcripts of ana genes in cultures deprived of fixed N and P relative to P-replete cultures amended with NH4+ suggests ATX-A was being actively synthesized under P limitation. Collectively, these findings indicate that management strategies that do not regulate fixed N loading will leave eutrophic water bodies vulnerable to more intense and toxic (due to increased biomass) blooms of Dolichospermum.</p

RAxML tree inferred from the alignment of 165 representative V9 sequences of leptocylindracean OTUs from the BioMarKs data, six leptocylindracean sequences from GenBank, and 96 reference sequences of bolidomonads, leptocylindraceans and other diatoms, utilizing the GTRGAMMA base substitution model and Hill Climbing algorithm.

<p><i>Bolidomonas pacifica</i> and <i>B. mediterranea</i> were designated as outgroups. All non-leptocylindracean sequences were pruned from the tree following tree construction (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0103810#pone.0103810.s002" target="_blank">Figure S2</a> for tree with outgroups included). Bootstrap values were inferred from 100 distinct alternative runs and values <50 are deleted. OTU labels follow same principle as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0103810#pone-0103810-g001" target="_blank">Figure 1</a>.</p

Venn diagrams showing the number of site-specific and shared OTUs among the six sampling stations.

<p>(A) V4 at Naples, Oslo Fjord, Gijon and Blanes (B) V9 at Varna, Oslo Fjord, Naples and Roscoff (C) V9 at Oslo Fjord, Gijon, Blanes and Naples. Venn diagrams for V9 have been split into two figures to compare OTU distribution among the sequence-abundant stations Naples and Oslo and the other four stations.</p

Image_3_Nitrogen and phosphorus significantly alter growth, nitrogen fixation, anatoxin-a content, and the transcriptome of the bloom-forming cyanobacterium, Dolichospermum.JPEG

While freshwater cyanobacteria are traditionally thought to be limited by the availability of phosphorus (P), fixed nitrogen (N) supply can promote the growth and/or toxin production of some genera. This study characterizes how growth on N2 (control), nitrate (NO3–), ammonium (NH4+), and urea as well as P limitation altered the growth, toxin production, N2 fixation, and gene expression of an anatoxin-a (ATX-A) – producing strain of Dolichospermum sp. 54. The transcriptomes of fixed N and P-limited cultures differed significantly from those of fixed N-deplete, P-replete (control) cultures, while the transcriptomes of P-replete cultures amended with either NH4+ or NO3– were not significantly different relative to those of the control. Growth rates of Dolichospermum (sp. 54) were significantly higher when grown on fixed N relative to without fixed N; growth on NH4+ was also significantly greater than growth on NO3–. NH4+ and urea significantly lowered N2 fixation and nifD gene transcript abundance relative to the control while cultures amended with NO3– exhibited N2 fixation and nifD gene transcript abundance that was not different from the control. Cultures grown on NH4+ exhibited the lowest ATX-A content per cell and lower transcript abundance of genes associated ATX-A synthesis (ana), while the abundance of transcripts of several ana genes were highest under fixed N and P - limited conditions. The significant negative correlation between growth rate and cellular anatoxin quota as well as the significantly higher number of transcripts of ana genes in cultures deprived of fixed N and P relative to P-replete cultures amended with NH4+ suggests ATX-A was being actively synthesized under P limitation. Collectively, these findings indicate that management strategies that do not regulate fixed N loading will leave eutrophic water bodies vulnerable to more intense and toxic (due to increased biomass) blooms of Dolichospermum.</p

RAxML tree inferred from the alignment of 12 representative V4 sequences of leptocylindracean OTUs from the BioMarKs data, 46 leptocylindracean sequences from GenBank, and 134 reference sequences of bolidomonads, Leptocylindraceae and other diatoms, utilizing the GTRGAMMA base substitution model and Hill Climbing algorithm.

<p><i>Bolidomonas pacifica</i> and <i>B. mediterranea</i> were designated as outgroups. All non-leptocylindracean reference sequences were pruned from the tree following tree construction (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0103810#pone.0103810.s001" target="_blank">Figure S1</a> for tree with outgroups included). Bootstrap values were inferred from 100 distinct alternative runs and values <50 are deleted. Each OTU is labelled as follows: the first letter denotes the first letter of the genus, the second letter, the first one of the species; the number denotes the cluster number (numbering starts from zero); the number after the underscore denotes the abundance of the OTU.</p

Distribution maps of Leptocylindraceae species inferred from NGS V4 and V9 sequences in the BioMarKs and GenBank datasets (blue dots), plus reliable microscopy images (red dots).

<p>Absence of finding in the BioMarKs dataset is represented by grey dots. Records for the microscopic observation reports of species presence are provided in supplementary material.</p