CATHERINE GÉRIKAS RIBEIRO Pico- and nanoplankton abundance and biomass in the Southwest Atlantic Ocean off Brazil Tese apresentada ao Instituto Oceanográfico da Universidade de São Paulo, como parte dos requisitos para obtenção do título de Doutora em Ciências, área de Oceanografia Biológica Orientador: Prof. Dr. Frederico Pereira Brandini São Paulo 2016 UNIVERSIDADE DE SÃO PAULO INSTITUTO OCEANOGRÁFICO Pico- and nanoplankton abundance and biomass in the Southwest Atlantic Ocean off Brazil Catherine Gérikas Ribeiro Tese apresentada ao Instituto Oceanográfico da Universidade de São Paulo, como parte dos requisitos para obtenção do título de Doutora em Ciências, área de Oceanografia Biológica Julgada em ____/____/____ _____________________________________ _______________ Prof(a). Dr(a). Conceito _____________________________________ _______________ Prof(a). Dr(a). Conceito _____________________________________ _______________ Prof(a). Dr(a). Conceito _____________________________________ _______________ Prof(a). Dr(a). Conceito _____________________________________ _______________ Prof(a). Dr(a). Conceito i SUMMARY LIST OF FIGURES ............................................................................................. iii LIST OF TABLES ............................................................................................... ix ACKNOWLEDGMENTS / AGRADECIMENTOS ................................................ x RESUMO............................................................................................................ 1 ABSTRACT ........................................................................................................ 2 GENERAL INTRODUCTION .............................................................................. 3 GENERAL OBJECTIVES ................................................................................... 9 SPECIFIC OBJECTIVES.................................................................................. 10 REFERENCES ................................................................................................. 11 CHAPTER 1 ..................................................................................................... 14 Abbreviations .................................................................................................... 15 RESUMO.......................................................................................................... 16 ABSTRACT ...................................................................................................... 17 1.1. INTRODUCTION ....................................................................................... 18 1.2. OBJECTIVES ............................................................................................ 20 1.3. MATERIALS AND METHODS ................................................................... 21 1.3.1. Sampling ....................................................................................................... 21 1.3.2. Flow Cytometry Analysis ............................................................................... 23 1.3.3. Clone libraries ............................................................................................... 24 1.4. RESULTS .................................................................................................. 26 1.4.1. Environmental conditions............................................................................... 26 1.4.2. Abundance of microbe populations ................................................................ 32 1.4.3. Carbon biomass of microbe populations ........................................................ 40 1.4.4. Pico and nano-phytoplankton phylogenetic composition ................................ 46 1.5. DISCUSSION ............................................................................................ 49 1.6. CONCLUSIONS ........................................................................................ 55 1.7. REFERENCES .......................................................................................... 56 1.8. SUPPLEMENTARY MATERIAL ................................................................ 61 ii CHAPTER 2 ..................................................................................................... 69 RESUMO.......................................................................................................... 70 ABSTRACT ...................................................................................................... 71 2.1. INTRODUCTION ....................................................................................... 72 2.2. OBJECTIVES ............................................................................................ 76 2.3. MATERIAL AND METHODS ..................................................................... 77 2.3.1. Trichodesmium spp. bloom ............................................................................ 77 2.3.2. Mesodinium rubrum bloom ............................................................................ 78 2.3.3. Epifluorescence microscopy .......................................................................... 82 2.4. RESULTS .................................................................................................. 83 2.4.1. Trichodesmium spp. bloom ............................................................................ 83 2.4.2. Mesodinium rubrum bloom ............................................................................ 85 2.5. DISCUSSION ............................................................................................ 95 2.6. CONCLUSIONS ...................................................................................... 100 2.7. REFERENCES ........................................................................................ 101 CHAPTER 3 ................................................................................................... 106 RESUMO........................................................................................................ 107 ABSTRACT .................................................................................................... 108 3.1. INTRODUCTION ..................................................................................... 109 3.2. OBJECTIVES .......................................................................................... 114 3.3. MATERIAL AND METHODS ................................................................... 115 3.3.1. Sampling ..................................................................................................... 115 3.3.2. Flow Cytometry Analysis ............................................................................. 116 3.4. RESULTS ................................................................................................ 119 3.4.1. HNA and LNA bacteria ................................................................................ 119 3.4.2. Prochlorococcus and Synechococcus ......................................................... 119 3.4.3. Pico- and nanoeukaryotes ........................................................................... 126 3.5. DISCUSSION .......................................................................................... 129 3.6. CONCLUSIONS ...................................................................................... 133 3.7. REFERENCES ........................................................................................ 134 iii LIST OF FIGURES Figure 1. General schematic figure showing the main systems of a flow cytometer, including fluidics, optics, electronics and sorting systems . Reprinted from PICOT et al., (2012). .................................................................................. 5 Figure 2. Oceanic food web showing the role of picoplankton on the paths of organic carbon flux. Reprinted from BARBER (2007). ....................................... 6 Figure 3. Planktonic groups analyzed in the present work (purple) and their subdivisions. Subgroups are marked in orange (heterotrophic), and green (autotrophic). ...................................................................................................... 8 Figure 1.1. Location of sampling stations in the SAO off Brazil (11-18 November 2013). Profiles: transect 1 (TR1, St.81 to 92) and transect 2 (TR2, St.96 to 114), represented by black dots; Surface sampling: transect 3 (TR3, St.133 to 147), represented by black triangles. Sampling stations where 16S rRNA partial sequences were retrieved are marked with a circle. The asterisk represent the TRICHO station, inside a Trichodesmium spp. bloom. ............... 22 Figure 1.2. Vertical distribution of temperature (T°C) and salinity, for transect 1 (A, B) and transect 2 (C, D); numbers indicate sampling stations; black dots indicate sampling depths; water masses are delimitated in white: Tropical Water (TW); South Atlantic Central Water (SACW) and Coastal Water (CW). ........... 28 Figure 1.3. Vertical distribution of fluorescence (RFU) and nitrate (µM.L-1), for transect 1 (A, B) and transect 2 (C, D); numbers indicate sampling stations; black dots indicate sampling depths; water masses are delimitated in white: Tropical Water (TW); South Atlantic Central Water (SACW) and Coastal Water (CW). ................................................................................................................ 29 Figure 1.4. Vertical distributions of phosphates (µM.L-1), for transect 1 (A) and transect 2 (B); numbers indicate sampling stations; black dots indicate sampling depths; water masses are delimitated in white: Tropical Water (TW); South Atlantic Central Water (SACW) and Coastal Water (CW). ............................... 30 Figure 1.5. Surface distribution of temperature (T°C) (A), salinity (B), fluorescence (RFU) (C), nitrates (µM.L-1) (D) and phosphates (µM.L-1) (E) for transect 1, 2 and 3; numbers indicate the beginning and the end of each transect; black dots indicate each station. ........................................................ 31 iv Figure 1.6. Vertical distribution of total heterotrophic bacteria and Prochlorococcus (in cells.mL-1), for transect 1 (A, B) and transect 2 (C, D); numbers indicate sampling stations; black dots indicate sampling depths; water masses are delimitated in white: Tropical Water (TW); South Atlantic Central Water (SACW) and Coastal Water (CW). ......................................................... 35 Figure 1.7. Vertical distribution of Synechococcus and picoeukaryotes (in cells.mL-1), for transect 1 (A, B) and transect 2 (C, D); numbers indicate sampling stations; black dots indicate sampling depths; water masses are delimitated in white: Tropical Water (TW); South Atlantic Central Water (SACW) and Coastal Water (CW). ................................................................................. 36 Figure 1.8. Vertical distribution of: nanoeukaryotes (in cells.mL-1), for transect 1 (A) and transect 2 (B); numbers indicate sampling stations; black dots indicate sampling depths; water masses are delimitated in white: Tropical Water (TW); South Atlantic Central Water (SACW) and Coastal Water (CW). ..................... 37 Figure 1.9. Surface distribution of total heterotrophic bacteria (A), Prochlorococcus (B), Synechococcus (C), picoeukaryotes (D) and nanoeukaryotes (E) (in cells.mL-1) for transect 1, 2 and 3; numbers indicate the beginning and the end of each transect; black dots indicate each station. ....... 38 Figure 1.10. Principal Component Analysis (PCA) showing (A) total heterotrophic bacteria, Prochlorococcus, Synechococcus, picoeukaryotes, nanoeukaryotes and chlorophyll fluorescence distributions (supplementary variables) in relation with temperature, salinity, nitrates and phosphates; (B) distribution of the stations. Dashed ellipse indicates samples from Coastal Water (CW), dashed square indicates the zoom window; #96 refers to samples from St. 96. ....................................................................................................... 39 Figure 1.11. Biomass (µgC.mL-1) estimated for total heterotrophic bacteria (BACT), Prochlorococcus (PRO), Synechococcus (SYN) and picoeukaryotes (PEUK) for TR1(A), TR2 (B) and TR3 (C). Note that the scale for TR1 is different from TR2 and TR3. ............................................................................. 41 Figure 1.12. Vertical distribution of total autotrophic biomass (μgC.L-1) and relative contribution to total biomass (%) of total heterotrophic bacteria for transect 1 (A, B) and transect 2 (C, D); numbers indicate sampling stations; black dots indicate sampling depths; water masses are delimitated in white: Tropical Water (TW); South Atlantic Central Water (SACW) and Coastal Water (CW). ................................................................................................................ 42 v Figure 1.13. Vertical distribution of relative contribution to total biomass (%) of Prochlorococcus and Synechococcus for transect 1 (A, B) and transect 2 (C, D); numbers indicate sampling stations; black dots indicate sampling depths; water masses are delimitated in white: Tropical Water (TW); South Atlantic Central Water (SACW) and Coastal Water (CW). ......................................................... 43 Figure 1.14. Vertical distribution of relative contribution to total biomass (%) of picoeukaryotes for transect 1 (A) and transect 2 (B); numbers indicate sampling stations; black dots indicate sampling depths; water masses are delimitated in white: Tropical Water (TW); South Atlantic Central Water (SACW) and Coastal Water (CW). ..................................................................................................... 44 Figure 1.15. Surface distribution of total autotrophic biomass (μgC.L-1) (A), relative contribution to total biomass (%) of total heterotrophic bacteria (B), Prochlorococcus (C), Synechococcus (D) and picoeukaryotes (E) for transect 1, 2 and 3; numbers indicate the beginning and the end of each transect; black dots indicate each station. ................................................................................ 45 Figure 1.16. Phylogenetic tree of plastid 16S rRNA gene sequences. Sequences were retrieved from photosynthetic pico- and nanoeukaryotes sorted by flow cytometry in the Southwest Atlantic Ocean.The tree is inferred from 537 positions of an alignment of 25 partial sequences. The phylogenetic tree was constructed by the maximum likelihood (ML) method based on a TN93 (Tamura-Nei) model of nucleotides substitution. Bootstrap values over 50% are indicated on the internal branches. Sequences in bold are representative of OTUs obtained in the present study. Red and blue labeling refers to sequences retrieved from the pico- and nanoplankton, respectively. Numbers with symbols correspond to the number of clones retrieved for each OTU. ........................... 47 Figure 1.17. Composition of 16S rRNA sequences (total of 30 sequences) from pico- and nanoplanktonic sorted cells at three stations. ................................... 48 Figure S1. Temperature-Salinity (T-S) diagram generated with CTD dataset showing the water masses sampled in this study (open black boxes), with the depth on which they were found (Z axis, colorbar): Tropical Water (TW); South Atlantic Central Water (SACW) and Coastal Water (CW). ............................... 61 Figure S2. Representation of the seasonal intrusion of South Atlantic Central Water (SACW) over the bottom of the continental shelf, which is enhanced during summer, due to offshore Ekman transport of surface waters. CW: Coastal Water; TW: Tropical Water. Reprinted from (CAMPOS; VELHOTE; SILVEIRA, 2000). ............................................................................................. 62 vi Figure 2.1. Schematic model of the impact of a Trichodesmium spp. bloom in the water column community (reproduced from Hynes et al., 2009). ............... 73 Figure 2.2. Location of sampling stations in the SAO off Brazil, corresponding to two cruises: Trichodesmium spp. bloom (St. T100 and St. T101) in November/2013 (shaded dots) and Mesodinium rubrum bloom (St. M1, St. M3, St. M5, St. M7 and St. M9) in July/2014 (shaded squares). Sampling stations right above the blooms are marked with a star (TRICHO and MR1/MR3). ....... 80 Figure 2.3. A) Satellite imagery (MODIS - Moderate Resolution Imaging Spectroradiometer) showing mean surface chlorophyll concentration (mg.m3) in the Trichodesmium spp. bloom area, during its occurrence (3-days compilation). B) Satellite imagery (MODIS) showing the extension of the M. rubrum in the Brazilian coast. ................................................................................................. 81 Figure 2.4. A) Abundance (in cells.mL-1) and B) relative contribution (in percentage) of HNA (dark purple) and LNA (light purple), for both M. rubrum (MR1 to MR15) and Trichodesmium spp. bloom (TRICHO and adjacent sampling stations, T100 and T101). Shaded stars represent samples immediately above the blooms. ........................................................................ 88 Figure 2.5. A) Abundance (in cells.mL-1) and B) relative contribution (in percentage) of Prochlorococcus (dark green) and Synechococcus (lime green), for both M. rubrum (MR1 to MR15) and Trichodesmium spp. bloom (TRICHO and adjacent sampling stations, T100 and T101). Shaded stars represent samples immediately above the blooms. .......................................................... 89 Figure 2.6. A) Abundance (in cells.mL-1) and B) relative contribution (in percentage) of picoeukaryotes (dark blue), nanoeukaryotes (light blue) and phycoerythrin-containing eukaryotes (red), for both M. rubrum (MR1 to MR15) and Trichodesmium spp. bloom (TRICHO and adjacent sampling stations, T100 and T101). Shaded stars represent samples immediately above the blooms. . 90 Figure 2.7. Cytograms of A) Phycoerythrin versus Chlorophyll and B) SSC (side scatter) versus Chlorophyll data of sample MR3, showing the following gating patterns: Prochlorococcus (pink), Synechococcus (green), picoeukaryotes (blue), nanoeukaryotes (yellow) and phycoerythrin-containing eukaryotes (presumably Cryptophyta) (orange). Calibrations beads are marked in black. . 91 Figure 2.8. A) Biomass (in µgC.L-1) and B) its relative contribution (in percentage) of picoeukaryotes (light green), Synechococcus (red), Prochlorococcus (yellow), HNA (purple) and LNA (blue) bacteria, for both M. rubrum (MR1 to MR15) and Trichodesmium spp. bloom (TRICHO and adjacent sampling stations, T100 and T101). Shaded stars represent samples immediately above the blooms. ........................................................................ 92 vii Figure 2.9. Epifluorescence microscopy images from the Trichodesmium spp. bloom, showing the following features: A) different pigmentation; B) tuft morphology of the colonies; C) different terminations; D) diazocytes; E, F) accumulation of genetic material and G, H) epibiont community. ..................... 93 Figure 2.10. Possible kleptoplasty occurrence between Mesodinium rubrum and cryptophyte captured on light microscopy image from the M. rubrum cruise. Credits: Catherine G. Ribeiro. Samples were courtesy of Salvador Airton Gaeta and Mayza Pompeu (LaPP/IOUSP). ................................................................ 94 Figure 2.11. Phycoerythrin fluorescence from Mesodinium rubrum captured on epifluorescence microscopy image from the M. rubrum cruise. Credits: Catherine G. Ribeiro. Samples were courtesy of Salvador Airton Gaeta and Mayza Pompeu (LaPP/IOUSP). ....................................................................... 94 Figure 3.1. Cytograms of Phycoerythrin versus Chlorophyll of sample 137 (St. 100, 110 meters depth) from A) BD FACSCanto™ and B) BD Accuri™ C6 readings, showing the following gating patterns: Prochlorococcus (pink), Synechococcus (green), picoeukaryotes (blue) and nanoeukaryotes (yellow). Calibrations beads are marked in black. ........................................................ 111 Figure 3.2. Location of sampling stations in the SAO off Brazil. Profiles: transect 1 (TR1, shaded circles) ; transect 2 (TR2, shaded triangles) and surface sampling, transect 3 (TR3, shaded squares). The color bar in the left indicates bottom depths. ................................................................................ 118 Figure 3.3. Examples of red fluorescence distribution (number of events versus fluorescence intensity) from different depths of A) Prochlorococcus and B) Synechococcus recorded with BD FACSCanto™. Samples and their respective depths are described in the right. ................................................................... 121 Figure 3.4. Examples of red fluorescence distribution (number of events versus fluorescence intensity) from different depths of A) Prochlorococcus and B) Synechococcus recorded with BD Accuri™ C6. Samples and their respective depths are described in the right. ................................................................... 122 Figure 3.5. Relationship between abundance measurements performed with BD Accuri™ C6 e BD FACSCanto™ (in cells.mL-1): (A) HNA bacteria; (B) LNA bacteria; (C) Prochlorococcus; (D) Synechococcus; (E) picoeukaryotes and (F) nanoeukaryotes. Regarding correction in red fluorescence distribution, points are marked as: 'no correction': black shaded circles, 'correction': grey shaded squares; outliers: open triangles. The coefficient of determination and the equation are indicated on each graphic. The regression line is marked in grey for points with no correction and in black for 'correction' samples, although being totally superimposed in Prochlorococcus regression (C). ..................... 123 viii Figure 3.6. Examples of discriminated depth profiles (St. 100 and St. 114) of normalized red fluorescence distribution (chlorophyll versus relative cell number) and respective cell abundance of Prochlorococcus (A:L) and Synechococcus (M:X) on BD FACSCanto™ and BD Accuri™ C6. In the depth profiles (F, L, R, X), black shaded represents 'no correction' samples; grey shaded squares indicates 'correction' samples; samples within the noise were suppressed. .................................................................................................... 124 Figure 3.7. Comparison between measurements with BD FACSCanto™ (A, B) and BD Accuri™ C6 (C, D) regarding the vertical abundance distribution (in cells.mL-1) of Prochlorococcus; numbers in the top indicate sampling stations; sampling points are marked as: 'no correction': black shaded circles, 'correction': grey shaded squares; outliers: open triangles, 'cells in noise': open circles. ............................................................................................................ 127 Figure 3.8. Comparison between measurements with BD FACSCanto™ (A, B) and BD Accuri™ C6 (C, D) regarding the vertical abundance distribution (in cells.mL-1) of Synechococcus; numbers in the top indicate sampling stations; sampling points are marked as: 'no correction': black shaded circles, 'correction': grey shaded squares; outliers: open triangles, 'cells in noise': open circles. ............................................................................................................ 128