18S (SSU) rRNA gene; mixotrophy; North Sea; protists; seasonal succession; time-series; Oceanography; Global and Planetary Change; Aquatic Science; Water Science and Technology; Environmental Science (miscellaneous); Ocean Engineering
Abstract :
[en] Protists engaging in photo- and phago- mixotrophy (mixoplankton) are common members of the global plankton community. They are involved in primary production and contribute to the carbon and nutrient cycling. Two major mixoplankton functional types (MFTs) are considered based upon the origin of their photosynthetic abilities: innate for constitutive-mixoplankton (CM) and obtained from prey for non-constitutive mixoplankton (NCM). Regardless of their significance, little attention has been paid to their diversity and temporal succession. We performed a metabarcoding survey of the V4-18S rRNA gene in 92 surface water samples collected during 2018–2019 in five fixed stations of the Belgian Coastal Zone. Environmental data such as nutrients, sea surface temperature, salinity, Chl-a and light were collected to understand their influences over mixoplankton community changes. The temporal diversity of mixotrophs, autotrophs, and heterotrophs was analyzed and the distinct seasonal patterns were evidenced. Results showed that dinoflagellates and ciliates were the major mixoplankton contributors. There were no significant differences among protist communities between the stations sampled. The time-series showed high proportional abundances of CM, accounting in average for 24.4% of the reads, against the low contribution of NCM, 4.8%. CM dinoflagellates belonging to Heterocapsa, Alexandrium, Karlodinium, and Tripos genus were the most abundant, and co-occurred with strict autotrophic plankton. Strombidium genus ciliates were the most representative organisms for NCM. Mixoplankton showed lower diversity than autotrophs and heterotrophs throughout the time series, however, the environmental factors controlling the seasonal community shifts (β-diversity) were similar. Overall, the metabarcoding approach allowed to depict with high resolution the composition of mixoplankton and its diversity among auto- and heterotrophs in the Belgian Coastal Zone.
Disciplines :
Aquatic sciences & oceanology
Author, co-author :
Lapeyra Martin, Jon; Laboratoire d’Ecologie des Systèmes Aquatiques, Université Libre de Bruxelles, Brussels, Belgium
John, Uwe; Alfred Wegener Institute, Helmholtz Centre for Polar and Marine Research, Bremerhaven, Germany ; Helmholtz Institute for Functional Marine Biodiversity, Carlvon Ossietzky University of Oldenburg, Oldenburg, Germany
Royer, Colin ; Université de Liège - ULiège ; Laboratoire d’Ecologie des Systèmes Aquatiques, Université Libre de Bruxelles, Brussels, Belgium
Gypens, Nathalie; Laboratoire d’Ecologie des Systèmes Aquatiques, Université Libre de Bruxelles, Brussels, Belgium
Language :
English
Title :
Fantastic Beasts: Unfolding Mixoplankton Temporal Variability in the Belgian Coastal Zone Through DNA-Metabarcoding
This research was supported by the MixITiN (www.mixotroph. org) project, which received funding from the European Union?s Horizon 2020 Research and Innovation Programme under the Marie Sk?odowska-Curie Actions (MSCA) grant agreement no. 766327. JL was granted with MSCA funded ITN-ETN MixITiN Early Stage Researchers (ESRs) support. JL, NG, and CR received financial support from the Fonds David et Alice Van Buuren. CR was a Ph.D. grant from the FRIA (Fund for Research Training in Industry and Agriculture, FNRS). UJ was financially and logistically supported through the POF IV, topic 6 and subtopic 2 research program of the Alfred Wegener Institute, Helmholtz Centre for Polar and Marine Research.This research was supported by the MixITiN ( www.mixotroph. org ) project, which received funding from the European Union’s Horizon 2020 Research and Innovation Programme under the Marie Skłodowska-Curie Actions (MSCA) grant agreement no. 766327. JL was granted with MSCA funded ITN-ETN MixITiN Early Stage Researchers (ESRs) support. JL, NG, and CR received financial support from the Fonds David et Alice Van Buuren. CR was a Ph.D. grant from the FRIA (Fund for Research Training in Industry and Agriculture, FNRS). UJ was financially and logistically supported through the POF IV, topic 6 and subtopic 2 research program of the Alfred Wegener Institute, Helmholtz Centre for Polar and Marine Research.
Adolf J. E. Stoecker D. K. Harding L. W. (2006). The balance of autotrophy and heterotrophy during mixotrophic growth of Karlodinium micrum (dinophyceae). J. Plankton Res. 28 737–751. 10.1093/plankt/fbl007
Aitchison J. (1986). The Statistical Analysis of Compositional Data. London: Chapman and Hall.
Anderson M. J. (2001). A new method for non-parametric multivariate analysis of variance. Austral. Ecol. 26 32–46.
Anderson R. Jürgens K. Hansen P. J. (2017). Mixotrophic phytoflagellate bacterivory field measurements strongly biased by standard approaches: a case study. Front. Microbiol. 8:1398. 10.3389/fmicb.2017.01398 28798734
Anschütz A. A. Flynn K. J. (2020). Niche separation between different functional types of mixoplankton: results from NPZ - style N - based model simulations. Mar. Biol. 167 1–21. 10.1007/s00227-019-3612-3
Armeli Minicante S. Piredda R. Quero G. M. Finotto S. Bernardi Aubry F. Bastianini M. et al. (2019). Habitat heterogeneity and connectivity: effects on the planktonic protist community structure at two adjacent coastal sites (the lagoon and the gulf of venice, northern adriatic sea, italy) revealed by metabarcoding. Front. Microbiol. 10:1–16. 10.3389/fmicb.2019.02736 32038505
Barton A. D. Pershing A. J. Litchman E. Record N. R. Edwards K. F. Finkel Z. V. et al. (2013). The biogeography of marine plankton traits. Ecol. Lett. 16 522–534. 10.1111/ele.12063 23360597
Beisner B. E. Grossart H. P. Gasol J. M. (2019). A guide to methods for estimating phago-mixotrophy in nanophytoplankton. J. Plankton Res. 41 77–89. 10.1093/plankt/fbz008
Berdjeb L. Parada A. Needham D. M. Fuhrman J. A. (2018). Short-term dynamics and interactions of marine protist communities during the spring-summer transition. ISME J. 12 1907–1917. 10.1038/s41396-018-0097-x 29599520
Bruhn C. S. Wohlrab S. Krock B. Lundholm N. John U. (2021). Seasonal plankton succession is in accordance with phycotoxin occurrence in disko bay, west greenland. Harm. Alg. 103:101978. 10.1016/j.hal.2021.101978 33980456
Callahan B. J. McMurdie P. J. Rosen M. J. Han A. W. Johnson A. J. A. Holmes S. P. (2016). DADA2: high-resolution sample inference from Illumina amplicon data. Nat. Methods 13 581–583. 10.1038/nmeth.3869 27214047
Chain F. J. J. Brown E. A. Macisaac H. J. Cristescu M. E. (2016). Metabarcoding reveals strong spatial structure and temporal turnover of zooplankton communities among marine and freshwater ports. Divers. Distrib. 22 493–504. 10.1111/ddi.12427
Connolly J. A. Oliver M. J. Beaulieu J. M. Knight C. A. Tomanek L. Moline M. A. (2008). Correlated evolution of genome size and cell volume in diatoms (bacillariophyceae). J. Phycol. 44 124–131. 10.1111/j.1529-8817.2007.00452.x 27041049
Da̧browska A. M. Wiktor J. M. Merchel M. Wiktor J. M. (2020). Planktonic protists of the eastern nordic seas and the fram strait: spatial changes related to hydrography during early summer. Front. Mar. Sci. 7:1–14. 10.3389/fmars.2020.00557
Das P. B. Gauns M. Naqvi S. W. A. (2019). Ecological diversity of planktonic protists in spatial regimes of the arabian sea revealed through next-generation sequencing. Reg. Stud. Mar. Sci. 25:100484. 10.1016/j.rsma.2018.100484
De Vargas C. Audic S. Henry N. Decelle J. Mahé F. Logares R. et al. (2015). Eukaryotic plankton diversity in the sunlit ocean. Science 348:6237. 10.1126/science.1261605 25999516
Desmit X. Ruddick K. Lacroix G. (2015). Salinity predicts the distribution of chlorophyll a spring peak in the southern north sea continental waters. J. Sea Res. 103 59–74. 10.1016/j.seares.2015.02.007
Dolan J. R. (1992). Mixotrophy in ciliates: a review of chlorella symbiosis and chloroplast retention. Mar. Microb. Food Webs 6 115–132.
Dolan J. R. Pérez M. T. (2000). Costs, benefits and characteristics of mixotrophy in marine oligotrichs. Freshw. Biol. 45 227–238. 10.1046/j.1365-2427.2000.00659.x
Duffy J. E. Godwin C. M. Cardinale B. J. (2017). Biodiversity effects in the wild are common and as strong as key drivers of productivity. Nat. Publ. Gr. 549 261–264. 10.1038/nature23886 28869964
Ebenezer V. Medlin L. K. Ki J. (2011). Molecular detection quantification and diversity evaluation of microalgae molecular. Mar. Biotechnol. 14 129–142. 10.1007/s10126-011-9427-y 22200918
Edwards K. F. (2019). Mixotrophy in nanoflagellates across environmental gradients in the ocean. Proc. Natl. Acad. Sci. U.S.A. 116 6211–6220. 10.1073/pnas.1814860116 30760589
Elbrecht V. Leese F. (2015). Can DNA-based ecosystem assessments quantify species abundance? Testing primer bias and biomass-sequence relationships with an innovative metabarcoding protocol. PLoS One 7:e0130324. 10.1371/journal.pone.0130324 26154168
Faure E. Not F. Benoiston A. S. Labadie K. Bittner L. Ayata S. D. (2019). Mixotrophic protists display contrasted biogeographies in the global ocean. ISME J. 13 1072–1083. 10.1038/s41396-018-0340-5 30643201
Fischer R. Giebel H. Hillebrand H. Ptacnik R. (2017). Importance of mixotrophic bacterivory can be predicted by light and loss rates. Oikos 126 713–722.
Flynn K. J. Hansen P. J. (2013). Cutting the canopy to defeat the ‘selfish gene’; conflicting selection pressures for the integration of phototrophy in mixotrophic protists. Protist 164 1–13. 10.1016/j.protis.2013.09.002 24189043
Flynn K. J. Mitra A. (2009). Building the ‘perfect beast’: modelling mixotrophic plankton. J. Plankton Res. 31 965–992. 10.1093/plankt/fbp044
Flynn K. J. Mitra A. Anestis K. Anschütz A. A. Calbet A. Ferreira G. D. et al. (2019). Mixotrophic protists and a new paradigm for marine ecology: where does plankton research go now? J. Plankton Res. 41 375–391. 10.1093/plankt/fbz026
Galluzzi L. Penna A. Bertozzini E. Vila M. Garce E. Magnani M. (2004). Development of a real-time PCR assay for rapid detection and quantification of Alexandrium minutum (a dinoflagellate). Appl. Environ. Microbiol. 70 1199–1206. 10.1128/AEM.70.2.1199
Genitsaris S. Monchy S. Viscogliosi E. Sime-Ngando T. Ferreira S. Christaki U. (2015). Seasonal variations of marine protist community structure based on taxon-specific traits using the eastern english channel as a model coastal system. FEMS Microbiol. Ecol. 91 1–15. 10.1093/femsec/fiv034 25873460
Ghyoot C. Lancelot C. Flynn K. J. Mitra A. Gypens N. (2017). Introducing mixotrophy into a biogeochemical model describing an eutrophied coastal ecosystem: The Southern North Sea. Prog. Oceanogr. 157 1–11. 10.1016/j.pocean.2017.08.002
Glibert P. M. Berdalet E. Burford M. A. Pitcher G. C. Zhou M. (eds) (2018). Global Ecology and Oceanography of Harmful Algal Blooms, Vol. 232. Cham: Springer.
Gloor G. B. Macklaim J. M. Pawlowsky-Glahn V. Egozcue J. J. (2017). Microbiome datasets are compositional: and this is not optional. Front. Micr. 8:2224. 10.3389/fmicb.2017.02224 29187837
Godhe A. Asplund M. E. Härnström K. Saravanan V. Tyagi A. Karunasagar I. (2008). Quantification of diatom and dinoflagellate biomasses in coastal marine seawater samples by real-time PCR. Appl. Environ. Microbiol. 74 7174–7182. 10.1128/AEM.01298-08 18849462
Gong J. Dong J. Liu X. Massana R. (2013). Extremely high copy numbers and polymorphisms of the rDNA operon estimated from single cell analysis of oligotrich and peritrich ciliates. Protist 164 369–379. 10.1016/j.protis.2012.11.006 23352655
Gran-Stadniczeñko S. Egge E. Hostyeva V. Logares R. Eikrem W. Edvardsen B. (2019). Protist diversity and seasonal dynamics in skagerrak plankton communities as revealed by metabarcoding and microscopy. J. Eukaryot. Microbiol. 66 494–513. 10.1111/jeu.12700 30414334
Gu Z. Eils R. Schlesner M. (2016). Complex heatmaps reveal patterns and correlations in multidimensional genomic data. Bioinformatics 32 2847–2849. 10.1093/bioinformatics/btw313 27207943
Guillou L. Bachar D. Audic S. Bass D. Berney C. Bittner L. et al. (2012). The protist ribosomal reference database (PR2): a catalog of unicellular eukaryote small sub-unit rRNA sequences with curated taxonomy. Nucl. Acad. Res. 41 D597–D604. 10.1093/nar/gks1160 23193267
Gypens N. Lacroix G. Lancelot C. (2007). Causes of variability in diatom and phaeocystis blooms in belgian coastal waters between 1989 and 2003: a model study. J. Sea Res. 57 19–35. 10.1016/j.seares.2006.07.004
Hörstmann C. Raes E. J. Buttigieg P. L. Lo Monaco C. John U. Waite A. M. (2021). Hydrographic fronts shape productivity, nitrogen fixation, and microbial community composition in the southern indian ocean and the southern ocean (2021). Biogeoscience 18 3733–3749. 10.5194/bg-18-3733-2021
Illumina (2013). 16S Metagenomic Sequencing Library. 1–28. Available online at: http://support.illumina.com/content/dam/illuminasupport/documents/documentation/chemistry_documentation/16s/16s-metagenomic-library-prep-guide-15044223-b.pdf. (accessed March 15, 2020)
Jeong H. J. du Yoo Y. Kim J. S. Seong K. A. Kang N. S. Kim T. H. (2010). Growth, feeding and ecological roles of the mixotrophic and heterotrophic dinoflagellates in marine planktonic food webs. Ocean Sci. J. 45 65–91. 10.1007/s12601-010-0007-2
Jones H. L. J. (1997). A classification of mixotrophic protists based on their behaviour. Freshw. Biol. 37 35–43. 10.1046/j.1365-2427.1997.00138.x
Kase L. Kraberg A. C. Metfies K. Neuhaus S. Sprong P. A. A. Fuchs B. M. et al. (2020). Rapid succession drives spring community dynamics of small protists at helgoland roads, north sea. J. Plankton Res. 42 305–319. 10.1093/plankt/fbaa017 32494090
Lathi L. Shetty S. (2017). Tools for Microbiome Analysis in R. Available online at: https://microbiome.github.io/tutorials/ (accessed November 20, 2022).
Lancelot C. Spitz Y. Gypens N. Ruddick K. Becquevort S. Rousseau V. et al. (2005). Modelling diatom and phaeocystis blooms and nutrient cycles in the southern bight of the north sea: the MIRO model. Mar. Ecol. Prog. Ser. 289 63–78. 10.3354/meps289063
Leles S. Bruggeman J. Polimene L. Blackford J. Flynn K. J. Mitra A. (2020). Differences in physiology explain succession of mixoplankton functional types and affect carbon fluxes in temperate seas. Prog. Oceanogr. 190:102481. 10.1016/j.pocean.2020.102481
Leles S. G. Mitra A. Flynn K. J. Stoecker D. K. Hansen P. J. Calbet A. et al. (2017). Oceanic protists with different forms of acquired phototrophy display contrasting biogeographies and abundance. Proc. R. Soc. B Biol. Sci. 284:20170664. 10.1098/rspb.2017.0664 28768886
Leles S. G. Mitra A. Flynn K. J. Tillmann U. Stoecker D. Jin H. et al. (2019). Sampling bias misrepresents the biogeographical significance of constitutive mixotrophs across global oceans. Glob. Eco. Biogeo. 28 418–428. 10.1111/geb.12853
Leles S. G. Polimene L. Bruggeman J. Blackford J. Ciavatta S. Mitra A. et al. (2018). Modelling mixotrophic functional diversity and implications for ecosystem function. J. Plankton Res. 40 627–642. 10.1093/plankt/fby044
Lim A. S. Jeong H. J. Ok J. H. (2019). Five alexandrium species lacking mixotrophic ability. Algens 34 289–301.
Lin Y. Gifford S. Ducklow H. Schofield O. Cassara N. (2018). Towards quantitative microbiome community profiling using internal standards. Appl. Environ. Microbiol. 85 1–14. 10.1128/AEM.02634-18 30552195
Mäki A. Salmi P. Mikkonen A. Kremp A. Tiirola M. (2017). Sample preservation, DNA or RNA extraction and data analysis for high-throughput phytoplankton community sequencing. Front. Microbiol. 8:1–13. 10.3389/fmicb.2017.01848 29018424
Martínez L. A. Mortelmans J. Dillen N. Debusschere E. Deneudt K. (2020). LifeWatch observatory data: phytoplankton observations in the belgian part of the north sea. Biodivers. Data J. 8 1–18. 10.3897/BDJ.8.E57236 33376438
Masquelier S. Foulon E. Jouenne F. Ferréol M. Brussaard C. P. D. Vaulot D. (2011). Distribution of eukaryotic plankton in the english channel and the north sea in summer. J. Sea Res. 66 111–122. 10.1016/j.seares.2011.05.004
Massana R. Gobet A. Audic S. Bass D. Bittner L. Boutte C. et al. (2015). Marine protist diversity in european coastal waters and sediments as revealed by high-throughput sequencing. Environ. Microbiol. 17 4035–4049. 10.1111/1462-2920.12955 26119494
Medlin L. K. Kooistra W. H. (2010). Methods to estimate the diversity in the marine photosynthetic protist community with illustrations from case studies: a review. Diversity 2 973–1014. 10.3390/d2070973
Millette N. C. Pierson J. J. Aceves A. Stoecker D. K. (2017). Mixotrophy in Heterocapsa rotundata: a mechanism for dominating the winter phytoplankton. Limnol. Oceanogr. 62 836–845. 10.1002/lno.10470
Millette N. C. Stoecker D. K. Pierson J. J. (2015). Top-down control by micro- and mesozooplankton on winter dinoflagellate blooms of Heterocapsa rotundata. Aquat. Microb. Ecol. 76 15–25. 10.3354/ame01763
Mitra A. Flynn K. J. (2010). Modelling mixotrophy in harmful algal blooms: more or less the sum of the parts? J. Mar. Syst. 83 158–169. 10.1016/j.jmarsys.2010.04.006
Mitra A. Flynn K. J. Burkholder J. M. Berge T. Calbet A. Raven J. A. et al. (2014). The role of mixotrophic protists in the biological carbon pump. Biogeosciences 11 995–1005. 10.5194/bg-11-995-2014
Mitra A. Flynn K. J. Tillmann U. Raven J. A. Caron D. Stoecker D. K. et al. (2016). Defining planktonic protist functional groups on mechanisms for energy and nutrient acquisition: incorporation of diverse mixotrophic strategies. Protist 167 106–120. 10.1016/j.protis.2016.01.003 26927496
Mortelmans J. Goossens J. Amadei Martínez L. Deneudt K. Cattrijsse A. Hernandez F. (2019a). LifeWatch observatory data: zooplankton observations in the Belgian part of the North Sea. Geosci. Data J. 6, 76–84. 10.1002/gdj3.68
Mortelmans J. Deneudt K. Cattrijsse A. Beauchard O. Daveloose I. Vyverman W. et al. (2019b). Nutrient, pigment, suspended matter and turbidity measurements in the belgian part of the north sea. Sci. Data 6 1–9. 10.1038/s41597-019-0032-7 30967554
Muylaert K. Gonzales R. Franck M. Lionard M. (2006). Spatial variation in phytoplankton dynamics in the belgian coastal zone of the north sea studied by microscopy, HPLC-CHEMTAX and underway fluorescence recordings. J. Sea Res. 55 253–265. 10.1016/j.seares.2005.12.002
Nohe A. Gof A. Tyberghein L. Lagring R. De Cauwer K. Vyverman W. et al. (2020). Marked changes in diatom and dinoflagellate biomass, composition and seasonality in the belgian part of the north sea between the 1970s and 2000s. Sci. Tot. Env. 716:136316. 10.1016/j.scitotenv.2019.136316 32036126
Nohe A. Knockaert C. Goffin A. Dewitte E. De Cauwer K. Desmit X. et al. (2018). Marine phytoplankton community composition data from the belgian part of the north sea, 1968–2010. Sci. Date. 5 1–9.
Oksanen J. Blanchet F. G. Kindt R. Legendre P. Minchin P. R. (2019). Package ‘vegan’. Commun. Ecol. Package 2 1–295.
Passy P. Gypens N. Billen G. Garnier J. Thieu V. Rousseau V. et al. (2013). A model reconstruction of riverine nutrient fluxes and eutrophication in the belgian coastal zone since 1984. J. Mar. Syst. 128 106–122. 10.1016/j.jmarsys.2013.05.005
Piwosz K. Shabarova T. Pernthaler J. Posch T. Šimek K. Porcal P. et al. (2020). Bacterial and eukaryotic small-subunit amplicon data do not provide a quantitative picture of microbial communities, but they are reliable in the context of ecological interpretations. mSphere. 10.1128/msphere.00052-20 32132159
Porter K. G. Feig Y. S. (1980). The use of DAPI for identifying and counting aquatic microflora1. Limnol. Oceanogr. 25 943–948. 10.4319/lo.1980.25.5.0943
R Core Team (2018). R: A Language and Environment for Statistical Computing. Vienna: R Foundation for Statistical Computing.
Romano F. Symiakaki K. Pitta P. (2021). Temporal variability of planktonic ciliates in a coastal oligotrophic environment: mixotrophy, size classes and vertical distribution. Front. Mar. Sci. 8:641589. 10.3389/fmars.2021.641589
Rousseau V. Becquevort S. Parent J. Y. Gasparini S. Daro M. H. Tackx M. et al. (2000). Trophic efficiency of the planktonic food web in a coastal ecosystem dominated by Phaeocystis colonies. J. Sea Res. 43 357–372. 10.1016/S1385-1101(00)00018-6
Rousseau V. Leynaert A. Daoud N. Lancelot C. (2002). Diatom succession, silicification and silicic acid availability in belgian coastal waters (southern north sea). Mar. Ecol. Prog. Ser. 236 61–73. 10.3354/meps236061
Rousseau V. Park Y. Ruddick K. Vyverman W. Parent J.-Y. Lancelot C. (2006). “Phytoplankton blooms in response to nutrient enrichment in curr,” in Status Eutrophication Belgian Coast. Zo, eds Rousseau V. Lancelot C. Cox D. 45–59.
Santi I. Kasapidis P. Karakassis I. Pitta P. (2021). A comparison of DNA metabarcoding and microscopy methodologies for the study of aquatic microbial eukaryotes. Diversity 13:180. 10.3390/d13050180
Santoferrara L. F. (2019). Current practice in plankton metabarcoding: optimization and error management. J. Plankton Res. 41 571–582. 10.1093/plankt/fbz041
Schneider L. K. Flynn K. J. Herman P. M. J. Troost T. A. Stolte W. (2020b). Exploring the trophic spectrum: placing mixoplankton into marine protist communities of the southern north sea. Front. Mar. Sci. 7:586915. 10.3389/fmars.2020.586915
Schneider L. K. Anestis K. Mansour J. Anschütz A. A. Gypens N. Hansen P. J. et al. (2020a). A dataset on trophic modes of aquatic protists. Biodivers Data J. 8:e56648. 10.3897/BDJ.8.e56648 33177947
Selosse M. A. Charpin M. Not F. (2017). Mixotrophy everywhere on land and in water: the grand écart hypothesis. Ecol. Lett. 20 246–263. 10.1111/ele.12714 28032461
Seong K. A. Jeong J. H. Kim S. Hoon Kim G. Hoon Kang J. (2006). Bacterivory by co-occurring red-tide algae, heterotrophic nanoflagellates, and ciliates. Mar. Ecol. Prog. Ser. 322 85–97. 10.3354/meps322085
Simon M. Azam F. (1989). Protein content and protein synthesis rates of planktonic marine bacteria. Mar. Ecol. Prog. Ser. 51 201–213. 10.3354/meps051201
Stern R. Kraberg A. Bresnan E. Kooistra W. H. C. F. Lovejoy C. Montresor M. et al. (2018). Molecular analyses of protists in long-term observation programmes - current status and future perspectives. J. Plankton Res. 40 519–536. 10.1093/plankt/fby035
Stoeck T. Bass D. Nebel M. Christen R. Meredith D. (2010). Multiple marker parallel tag environmental DNA sequencing reveals a highly complex eukaryotic community in marine anoxic water. Mol. Ecol. 19 21–31. 10.1111/j.1365-294X.2009.04480.x 20331767
Stoecker D. K. (1998). Conceptual models of mixotrophy in planktonic protists and some ecological and evolutionary implications. Eur. J. Protistol. 34 281–290. 10.1016/S0932-4739(98)80055-2
Stoecker D. K. Hansen P. J. Caron D. A. Mitra A. (2017). Mixotrophy in the marine plankton. Ann. Rev. Mar. Sci. 9 311–335. 10.1146/annurev-marine-010816-060617 27483121
Stoecker D. K. Johnson M. D. De Vargas C. Not F. (2009). Acquired phototrophy in aquatic protists. Aquat. Microb. Ecol. 57 279–310. 10.3354/ame01340
Strickland J. D. H. Parsons T. R. (1972). A Practical Handbook of Seawater Analysis. Ottawa: Fisheries Research Board of Canada. 293.
Suzuki M. T. (1999). Effect of protistan bacterivory on coastal bacterioplankton diversity. Aquat. Microb. Ecol. 20 261–272. 10.3354/ame020261
Taylor J. D. Cunliffe M. (2014). High-throughput sequencing reveals neustonic and planktonic microbial eukaryote diversity in coastal waters. J. Phycol. 50 960–965. 10.1111/jpy.12228 26988649
Unrein F. Gasol J. M. Not F. Forn I. Massana R. (2014). Mixotrophic haptophytes are key bacterial grazers in oligotrophic coastal waters. ISME J. 8:132. 10.1038/ismej.2013.132 23924785
van der Loos L. M. Nijland R. (2021). Biases in bulk: DNA metabarcoding of marine communities and the methodology involved. Mol. Eco. 30 3270–3288. 10.1111/mec.15592 32779312
Vasselon V. Bouchez A. Rimet F. Jacquet S. Trobajo R. Corniquel M. et al. (2017). Avoiding quantification bias in metabarcoding: application of a cell biovolume correction factor in diatom molecular biomonitoring. Methods Ecol. Evol. 9 1060–1069. 10.1111/2041-210X.12960
Wang J. Shen J. Wu Y. Tu C. Soininen J. Stegen J. C. et al. (2013). Phylogenetic beta diversity in bacterial assemblages across ecosystems: deterministic versus stochastic processes. ISME J. 7 1310–1321. 10.1038/ismej.2013.30 23446837
Weiss S. Xu Z. Z. Peddada S. Amir A. Bittinger K. Gonzalez A. et al. (2017). Normalization and microbial differential abundance strategies depend upon data characteristics. Microbiology 5 1–18. 10.1186/s40168-017-0237-y 28253908
Weisse T. Anderson R. Arndt H. Calbet A. Hansen P. J. Montagnes D. J. S. (2016). Functional ecology of aquatic phagotrophic protists – concepts, limitations, and perspectives. Eur. J. Protistol. 55 50–74. 10.1016/j.ejop.2016.03.003 27094869
Wilken S. Huisman J. Naus-Wiezer S. Van Donk E. (2013). Mixotrophic organisms become more heterotrophic with rising temperature. Ecol. Lett. 16 225–233. 10.1111/ele.12033 23173644
Xue Y. Chen H. Yang J. R. Liu M. Huang B. Yang J. (2018). Distinct patterns and processes of abundant and rare eukaryotic plankton communities following a reservoir cyanobacterial bloom. ISME J. 2 2263–2277. 10.1038/s41396-018-0159-0 29899512
Zhu F. Massana R. Not F. Marie D. Vaulot D. (2005). Mapping of picoeukaryotes in marine ecosystems with quantitative PCR of the 18S rRNA gene. EMS Microb. Eco. 52 79–92. 10.1016/j.femsec.2004.10.006 16329895
Zubkov M. V. Tarran G. A. (2008). High bacterivory by the smallest phytoplankton in the north atlantic ocean. Nature 455 224–226. 10.1038/nature07236 18690208
