Marine photosynthetic picoeukaryotes: community structure at different spatial scales

UNIVERSIDAD DE LAS PALMAS DE GRAN CANARIA Facultad de Ciencias del Mar ANEXO I Dª MARÍA ISABEL PADILLA LEÓN, SECRETARIA DE LA FACULTAD DE CIENCIAS DEL MAR, ÓRGANO RESPONSABLE DEL PROGRAMA DE DOCTORADO EN OCEANOGRAFÍA, DE LA UNIVERSIDAD DE LAS PALMAS DE GRAN CANARIA. CERTIFICA Que el Consejo de Doctores del Programa de Doctorado en Oceanografía, en su sesión de fecha 16 de noviembre de 2015, tomó el acuerdo de dar el consentimiento para su tramitación, a la tesis doctoral titulada: “Marine photosynthetic picoeukaryotes: Community structure at different spatial scales” presentada por la doctoranda: Dª Ana Mª Cabello Pérez dirigida por los Doctores D. Mikel Latasa Arcalís D. Ramón Massana Molera Y para que así conste, a efectos de lo previsto en el Artº 6 del Reglamento para la elaboración, tribunal defensa y evaluación de tesis doctorales de la Universidad de Las Palmas de Gran Canaria, firmo el presente en Las Palmas de Gran Canaria, a dieciséis de noviembre de dos mil quince. PÁGINA 1 / 1 ID. DOCUMENTO VkjLieJx6GmUKtkBiT.PgQ$$ FIRMADO POR FECHA FIRMA ID. FIRMA 43646105V ISABEL PADILLA LEÓN 13/11/2015 12:40:32 NTM4Njk= Documento firmado digitalmente. Para verificar la validez de la firma copie el ID del documento y acceda a / Digitally signed document. To verify the validity of the signature copy the document ID and access to https://sede.ulpgc.es:8443/VerificadorFirmas/ulpgc/VerificacionAction.action Marine photosynthetic picoeukaryotes: community structure at different spatial scales (Picoeucariotas fotosintéticos marinos: estructura de la comunidad a diferentes escalas espaciales) Ana María Cabello Pérez Tesis Doctoral presentada por Dª Ana María Cabello Pérez para obtener el grado de Doctor por la Universidad de las Palmas de Gran Canaria, Programa de Doctorado en Oceanografía Directores: Dr. Mikel Latasa Arcalís y Dr. Ramon Massana i Molera Universidad de las Palmas de Gran Canaria Institut de Cièncias del Mar (ICM-CSIC) Centro Oceanográfico de Xixón (IEO) En Barcelona, a de de 2015 La Doctoranda El Director El Codirector Ana María Cabello Pérez Mikel Latasa Arcalís Ramon Massana i Molera A mis padres, a mi hermano, a mi compañero de viaje, y a la familia y amigos que siempre me han apoyado. A un capitán de navío Sobre tu nave - un plinto verde de algas marinas, de moluscos, de conchas, de esmeralda estelar, capitán de los vientos y de las golondrinas, fuiste condecorado por un golpe de mar. Por ti los litorales de frentes serpentinas desenrollan, al paso de tu arado, un cantar: - Marinero, hombre libre, que las mares declinas, dinos los radiogramas de tu Estrella Polar. Buen marinero, hijo de los llantos del norte, limón del mediodía, bandera de la corte espumosa del agua, cazador de sirenas; todos los litorales amarrados del mundo, pedimos que nos lleves en el surco profundo de tu nave, a la mar, rotas nuestras cadenas. Rafael Alberti, Marinero en tierra (1924) Cover photograph: Irene Forn. Contents Summary 11 General Introduction 15 Aims and outline of the thesis 31 Chapter 1 Vertical distribution of major photosynthetic 37 picoeukaryotic groups in stratified marine waters Chapter 2 Distribution of phytoplankton groups within the deep 69 chlorophyll maximum Chapter 3 Global distribution and vertical patterns of a 101 prymnesiophyte-cyanobacteria obligate symbiosis Chapter 4 Global distribution and intraspecific variability 131 of marine pelagophytes Synthesis of results and general discussion 159 Conclusions 173 Spanish summary (Resumen de la tesis) 177 General references (Introduction, Discussion and Spanish summary) 227 Acknowledgements 239 16 General Introduction Planktonic microbes are operationally categorized into three logarithmic size classes: picoplankton (0.2 to 2 µm), nanoplankton (2 to 20 µm), and microplankton (20 to 200 µm) (Figure 2) (Sieburth et al:LWKDIHZH[FHSWLRQVVXFKDVWKH¿ODPHQWRXVTrichodesmium sp., marine cyanobacteria are smaller than 2 µm, so the prokaryotic phytoplankton belongs virtually only to the picoplankton. By contrast, eukaryotic phytoplankton presents single-celled organisms covering a wide range of cell sizes, from a few micrometers to hundreds, including also colonial forms, so it appears in the three size classes above mentioned (Figure 2). The actual cell size spectra of the phytoplankton, mostly due to the eukaryotic component, is related to the trophic status of the system and has direct impact on the fate of primary production. So, eutrophic systems with high nutrient loads DUHJHQHUDOO\GRPLQDWHGE\ODUJHSK\WRSODQNWHUVVXFKDVGLDWRPVZKLFKDUHHI¿FLHQWO\JUD]HG by copepods but also can fast sink down to the deep layers, whereas oligotrophic systems are dominated by small phytoplankters whose fate is to be mostly recycled in the microbial loop (Figure 1). The marine phytoplankton thus forms a functional group by itself, that of primary producers with a planktonic lifestyle. This wide group can in turn be subdivided in smaller functional types, which KDYHEHHQPRVWO\GH¿QHGLQEDVHRISDUWLFXODUELRJHRFKHPLFDOUROHVXVHIXOIRUPRGHOOLQJSXUSRVHV (Le Quéré et al7KHVHW\SHVFRPSULVHSK\WRSODQNWRQFDOFL¿HUVHJFRFFROLWKRSKRULGV DIIHFWLQJ RFHDQ DONDOLQLW\ DQG FDUERQDWH FKHPLVWU\  SK\WRSODQNWRQ VLOLFL¿HUV HJ GLDWRPV PDMRUFRQWULEXWRUVWRFDUERQH[SRUWGLPHWK\OVXO¿GHSURGXFHUVHJ Phaeocystis and small DXWRWURSKLFÀDJHOODWHVDIIHFWLQJWKHDWPRVSKHULFVXOIXUF\FOHSLFRDXWRWURSKVProchlorococcus, Synechococcus and picoeukaryotes), which form the component generally recycled; 5) N2¿[HUV (e.g. Trichodesmium and N2¿[LQJXQLFHOOXODUSURNDU\RWHVZKLFKFDQXVHGLQLWURJHQIURPWKH DWPRVSKHUHDQGPL[HGSK\WRSODQNWRQHJGLQRÀDJHOODWHVDQGRWKHUVPDOOÀDJHOODWHVZKLFK constitute the background phytoplankton biomass but do not have a direct biogeochemical role assigned. Each of these functional types presents particular ecophysiological requirements that Figure 2. A comparison of the size range (maximum linear dimension) of phytoplankton relative to macroscopic objects. From Finkel et al., 2010. General Introduction 17 control their biomass and activity and therefore deserve a careful ecological inspection. Two of these types that are particularly relevant in the context of this thesis are developed next. Picoautotrophic cells, also named picophytoplankton, are important contributors to total phytoplankton carbon biomass and primary production and constitute the ocean’s veil above which larger protists and metazoans might bloom (Smetacek, 2002). These small algae are found HYHU\ZKHUHDQGGRPLQDWHWKHELRPDVVDQGSURGXFWLRQLQROLJRWURSKLFFKORURSK\OOD>&KO a] <0.3 mg m-3), nutrient poor (NO3 + NO20DQGZDUP!Û&ZDWHUVZKHUHDVWKH\ represent <10% of autotrophic biomass and production in rich (Chl a >5 mg m-3DQGFROGÛ& waters (Agawin et al., 2000) (Figure 3a). As mentioned before, nutrient rich waters are generally dominated by larger phytoplankters from other functional groups such as coccolitophores, diatoms DQGGLQRÀDJHOODWHV7KXVLQDJOREDOODWLWXGLQDOUDQJHIURPWKHFROGHVWWRWKHZDUPHVWZDWHUV picophytoplankton biomass dominates in the central gyres of the oceans comprising tropical and subtropical waters, with minor contribution in temperate and cold latitudes where the nano and microphytoplankton are major contributors (Figure 3b). On a global view, picophytoplankton dominate in huge areas of the oceans. A % contribution of picoplankton Total phytoplankton primary production (mg C m-3 d-1) B Surface Chla (mg m-3) Latitude A % contribution of picoplankton Total phytoplankton primary production (mg C m-3 d-1) B Surface Chla (mg m-3) Latitude 1LWURJHQ¿[LQJF\DQREDFWHULDDOVRFDOOHGGLD]RWURSKVSOD\DFHQWUDOUROHLQRSHQRFHDQPLFURELDO FRPPXQLWLHVE\SURYLGLQJ¿[HGQLWURJHQWRWKHELRWDIURPDWPRVSKHULFGLQLWURJHQ12) gas. As a consequence, they control the total ocean inventory of reactive nitrogen (Thompson and Zehr, 2013). Marine planktonic diazotrophs are composed by a few major lineages within three major Figure 3. (A) The relationship between the percent contribution of picophytoplankton fraction to total phytoplankton production (mg C m-3 d-1). Solid symbols represent the mean percentage within increasing bins and WKHLUVWDQGDUGHUURUDQGWKHVROLGOLQHLQGLFDWHVWKHHQYHORSHGHWHUPLQHGE\¿WWLQJDFXUYHWRWKHPD[LPDRIWKHELQ intervals. 0RGL¿HGIURP$JDZLQet al., 2000. (B) Zonal average of the contribution of different phytoplankton functional types to the total chla (in mg Chl m-3) for the micro-, nano-, and pico-size classes estimated using the combination of the statistical analysis of an HPLC pigment database and monthly composite SeaWiFS scenes of the year 2000.0RGL¿HGIURP/H4XpUpet al., 2005. 18 General Introduction JURXSVD¿ODPHQWRXVDQGIUHHOLYLQJQRQKHWHURF\VWIRUPLQJF\DQREDFWHULDLHTrichodesmium sp.)E¿ODPHQWRXVKHWHURF\VWIRUPLQJF\DQREDFWHULDHJRichelia sp., Calotrix sp.) in symbiosis with diatoms, and (c) unicellular cyanobacterial forms (e.g. UCYN-A, UCYN-B). Recently, 8&<1$KDVEHHQGLVFRYHUHGLQV\PELRVLVZLWKDVPDOOPSU\PQHVLRSK\WHDVLQJOHFHOOHG eukaryotic algae (Thompson et al., 2012), while UCYN-B (i.e. Crocosphaera sp.), once thought to be free-living, seems also be living in association, in this case with diatoms. Thus, diazotrophic cyanobacteria showcase a continuum of interactions with other microbes that range from freeOLYLQJORRVHDWWDFKPHQWWRREOLJDWHV\PELRVLV)LJXUH12¿[DWLRQLVDKLJKHQHUJ\GHPDQGLQJ process and diazotrophs have advantages in well-lit, warm, and nutrient poor waters, but are outcompeted elsewhere. Although Trichodesmium has been extensively studied and is though to EHWKHPDMRUQLWURJHQ¿[HULQWURSLFDODQGVXEWURSLFDOZDWHUV&DSRQHet al., 2005), recent studies have demonstrated the ubiquity and abundance of UCYN-A in a greater latitudinal range than Trichodesmium (Moisander et al., 2010; Thompson et alDQGKLJKOLJKWHGWKHLPSRUWDQFH of unicellular diazotrophs in the global N2¿[DWLRQ Figure 4. The spectrum of cellular interactions engaged in by marine N2¿[LQJF\DQREDFWHULD Dashed lines show features that are uncertain, such us the location of symbionts relative to host inner or outer membranes and the presence of calcareous plates on the Candidatus Atelocyanobacterium thalassa (UCYN-A) host. From left to right, other features include: free-living Crocosphaera cells (double cells are dividing); Trichodesmium with associated microbiota (gray); Calothrix with terminal heterocyst (brown) and vegetative cells (green); UCYN-A in relation to its host membranes; colonial aggregates of Crocosphaera-like cells in association with a diatom; and Richelia with terminal heterocyst (brown) and vegetative cells (green). From Thompson and Zehr, 2013. General Introduction 19 THE PLACE OF THE EUKARYOTIC PICOPHYTOPLANKTON 7KHSLFRSODQNWRQZDVWHFKQLFDOO\GH¿QHGDVFHOOVWKDWSDVVHGWKURXJKD¿OWHURIµm in pore size and initially this size class was thought to contain only prokaryotes. Nevertheless, very small eukaryotic algae were soon observed in natural assemblages (Jonhnson and Sieburth, 1982) and are well represented in cultured species (Vaulot et al., 2008), so it became clear that the picoplankton also contained eukaryotic cells. Furthermore, inspections of natural assemblages revealed that for these cells the upper threshold of 3 µm (instead of 2 µm) was a more natural boundary (Massana et al., 2011). Photosynthetic picoeukaryotes (PPEs), considered as cells from 0.8 to 3 µm in size, show a clear seasonality and generally explain the majority of eukaryotic phytoplankton counts (>80%) in marine systems (Figure 5) (Not et al., 2008; Massana et al., 2011; Piwosz et al., 2015). This 3 µm threshold KDVEHHQZLGHO\DSSOLHGLQ¿HOGVWXGLHVWKDWWDUJHWWKHGLYHUVLW\RI33(WR separate this community from larger cells. Nevertheless, some studies have considered cells up to 5 microns (the so-called ultraphytoplankton; Murphy and Haugen, 1985) to evaluate the ecological relevance of the tiniest algae (Li et al0F'RQDOGet al., 2007). In fact, some abundant and widespread algal lineages, such as the prymnesiophytes, display a natural size continuity towards this 5 µm-boundary (Cuvelier et al., 2010). Figure 5. 3HUFHQWDJHRISLJPHQWHGHXNDU\RWHVH[SODLQHGE\33(DQGPFHOOVDWWKH%ODQHV%D\ 0LFURELDO2EVHUYDWRU\GXULQJQLQH\HDUVRIPRQWKO\VDPSOLQJ0RGL¿HGIURP0DVVDQDet al., 2011. Julian day Pigmented eukaryotes (%) 20 General Introduction 33(VDEXQGDQFHVWKDQNVWRWKHUHGDXWRÀXRUHVFHQFHRIWKHFKORURSK\OODDUHXVXDOO\HVWLPDWHG E\GLUHFWFRXQWVWKURXJKHSLÀXRUHVFHQFHPLFURVFRS\+REELHet al., 1977; Murphy and Haugen, RULQDIDVWHUDXWRPDWHGZD\WKURXJKÀRZF\WRPHWU\2OVRQ)LJXUH7\SLFDO abundances of PPEs in the global ocean are between 103 to 10 cells ml-1, being more abundant than heterotrophic eukaryotes of the same size fraction (Jürgens and Massana 2008; Not et al., 2008; Massana et al., 2011), and usually less abundant than prokaryotic picophytoplankton. A general trend was observed of PPEs plus Synechococcus dominating in high-nutrient situations (coastal systems) and Prochlorococcus being more abundant in low-nutrient oligotrophic environments (open sea). On a global latitudinal scale, cyanobacteria tend to disappear at higher latitudes, so PPEs remain as the unique picophytoplankters. Red fluorescence (FL3) 90º light scatter (SSC) A B Figure 6. (A)(SLÀXRUHVFHQFHPLFURVFRS\LPDJHRIDSU\PQHVLRSK\WHDOJDHWDNHQXQGHUEOXH OLJKWH[FLWDWLRQIRUFKORURSK\OODUHGDXWRÀXRUHVFHQFH.0RGL¿HGIURP0DVVDQDet al., 2011. (B) Example of a picophytoplankton sample observed with blue light excitation. The sample corresponds to the Bay of Xixón (Southern Bay of Biscay, Cantabrian Sea). The beads (B) used are invitrogen 1 µm beads. The populations that can be distinguished are as follows: P, Prochlorococcus; S, Synechococcus3NSLFRHXNDU\RWHV0RGL¿HGIURP*DVRODQG0RUiQ Numerous picophytoplankton studies have shown the importance of the picoeukaryotic component in terms of biomass and primary production. Data from the landmark study of Li and colleagues in LQWKH1RUWK$WODQWLFVKRZHGWKDW33(VDOWKRXJKOHVVDEXQGDQWWKDQWKHF\DQREDFWHULDa of total picophytoplankton counts), accounted for more than three-quarters of carbon biomass and more than half of the primary production (Li et al.,/L7KLVGRPLQDQFHZDVDOVR observed in coastal sites (Worden et al.,&DOYR'LD]et al., 2008) (Figure 7). Thus, despite a much lower abundance and due to their larger size, PPE can indeed rival marine cyanobacteria in terms of biomass and activity. General Introduction 21 )RUGHFDGHV33(VKDYHEHHQWUHDWHGDVDEODFNER[RIGLI¿FXOWDFFHVVEHFDXVHXQGHUHSLÀXRUHVFHQFH PLFURVFRSLFREVHUYDWLRQVRUÀRZF\WRJUDPVWKH\IRUPVHHPLQJO\KRPRJHQHRXVSRSXODWLRQVRI cells. New insights into this assemblage came with direct molecular studies of natural samples that revealed PPEs as taxonomically very diverse, with members from all known algal groups that are placed in most major lineages of the eukaryotic tree of life (Figure 8). This wide diversity was unveiled at the beginning of the XXI century by sequencing the 18S rDNA gene from environmental samples from the picoplankton size fraction (Díez et al.,/ySH]*DUFtDet al., 2001; Moonvan der Staay et al., 2001). These seminal studies revealed the presence in this small size fraction of a wide array of algal classes such as Bacillariophyceae, Bolidophyceae, Chrysophyceae, &U\SWRSK\FHDH 'LFW\RFKRSK\FHDH 'LQRSK\FHDH (XVWLJPDWRSK\FHDH *ODXFRF\VWRSK\FHDH 3HODJRSK\FHDH3UDVLQRSK\FHDHDQG3U\PQHVLRSK\FHDH5RPDULDQG9DXORW3DUWRIWKLV diversity has been brought into culture (Vaulot et al., 2008) and some strains are preserved in reference collections for picoplankton cultures (Andersen et al.,/H*DOOet al., 2008). About hundred species of photosynthetic picoeukaryotes have been formally described (Vaulot et al., 2008), which represent a minor fraction compared to those species or entire new lineages found by molecular culture-independent tools. Relative abundance (%)Relative carbon biomass (%) Figure 7. (A) The relative contribution to picophytoplankton abundance (%) and (B) the relative contribution to picophytoplankton standing stock carbon ELRPDVVIRUHDFKJURXSIURP$XJXVWWKURXJK-DQXDU\LQD3DFL¿F coastal site. From Worden et al 22 General Introduction The use of ‘universal’ 18S rDNA gene primers targeting both phototrophs and heterotrophs has been extremely useful, but may also prevent to characterize adequately the diversity of the phototrophic component since for this purpose an extensive screening of clone libraries is required. Subsequent VWXGLHVWDUJHWLQJWKHFKORURSODVW6U'1$JHQHVXSSRVHGDVLJQL¿FDQWSURJUHVVLQWKHDVVHVVPHQW of PPE diversity (Fuller et al., 2006a; McDonald et al.,7KHVHVWXGLHV\LHOGHGDVLJQL¿FDQW dataset of 16S chloroplast sequences from the environment and from cultures, and allowed the design of marine algal-plastid PCR primers that excluded picocyanobacteria. With the same intention of avoiding underrepresentation of phototrophs, the diversity of the PPE community has EHHQDQDO\VHGE\WKHLU6DQG6U'1$JHQHVLQSRSXODWLRQVVRUWHGE\ÀRZF\WRPHWU\6KLet al., 2009, 2011). Other plastid encoded genes have also been used in a minor extent, including the psbA (encoding the D1 protein of photosystem II) (Zeidner et al., 2003; Man-Aharonovich et al., 2010) or the rbcL (encoding the large subunit of the CO2¿[LQJHQ]\PH58%,6&23DXO et al., 2000; Bhadury and Ward, 2009). As the result of all these combined molecular studies, three algal lineages come up as major representatives of PPE assemblages: chlorophytes (mainly mamiellophytes and prasinophytes), stramenopiles (mainly pelagophytes and chrysophytes) and haptophytes or prymnesiophytes (Figure 9). )LJXUH6FKHPDWLFSK\ORJHQHWLFWUHHUHSUHVHQWLQJWKHGLVWULEXWLRQRISK\WRSODQNWRQLFWD[DDFURVVHXNDU\RWH lineages (in color). Illustrations of (a) Chlorophyceae, (b) 3VHXGRVFRXU¿HOGLD sp., (c) Porphyridium cruentum, (d) Gymnochlora dimorphaH'LQRÀDJHOODWHVI Odontella sp., (g) %ROLGRPRQDVSDFL¿FD, (h) Dictyocha sp., (i) Aureococcus anophagefferens, (j) Heterosigma akashima, (k) Pinguiochrysis pyriformis, (l) Ochromonas sp., (m) Nannochloropsis salina, (n) Calcidiscus sp., (o) Cryptomonas sp., (p) Euglenids. From Not et al., 2012. General Introduction 23 TOOLS FOR EVALUATING COMMUNITY STRUCTURE It is important to characterize PPEs community structure over temporal and spatial scales and to LGHQWLI\WKHHQYLURQPHQWDOIDFWRUVGULYLQJFRPPXQLW\DVVHPEO\7KHTXDQWL¿FDWLRQRIWKHZKROH DVVHPEODJHDQGLWVYDULDWLRQLQVL]HVSHFWUDFDQEHGRQHGLUHFWO\E\HSLÀXRUHVFHQFHPLFURVFRS\DQG WRDOHVVH[WHQWE\ÀRZF\WRPHWU\+RZHYHUIRUDFTXLULQJVLPLODUGDWDIRUSDUWLFXODUWD[RQRPLF groups other tools were required. One of the tools that was initially used to provide relative DEXQGDQFHVRIVSHFL¿FJURXSVZLWKLQ WKH33(V FRPPXQLW\ZDV WKHDQDO\VLV RISK\WRSODQNWRQ pigments by +3/&KLJKSHUIRUPDQFHOLTXLGFKURPDWRJUDSK\+3/&DQDO\VLVRIJURXSVSHFL¿F marker pigments has been widely applied in biological oceanography (Roy et al. 2011) to describe the structure and dynamics of the PPEs (Letelier et al., 1993; Andersen et al., 1996; Bidigare and Ondrusek, 1996; Latasa and Bidigare, 1998; Mojica et al., 2015). When coming to the molecular ¿HOGVHYHUDODSSURDFKHVZHUHGHYHORSHGWREHDEOHWRSURFHVVWKHQXPEHURIVDPSOHVW\SLFDOO\ associated with oceanographic cruises (cloning and libraries were very powerful for taxonomic % of clones Oligo Meso Eutro Figure 9. Overall composition of the photosynthetic picoeukaryotes community IURPD6RXWK3DFL¿FWUDQVHFW Composition is expressed as percent of clones. This V\QWKHWLF¿JXUHLVEDVHGRQDFRPSLODWLRQRIIRXUDSSURDFKHV6SODVWLGSULPHU VHWV 2;<)2;<5 DQG 3/$)2;<5 RQ VRUWHG PDWHULDO 6 SODVWLGSULPHUVHW3/$)2;<5RQ¿OWHUHGPDWHULDOWKURXJKPLFURQVDQG 18S nuclear primers on sorted material. Oligo, Meso and Eutro correspond to the WKUHHPDMRUUHJLRQVFRQVLGHUHGROLJRWURSKLFPHVRWURSKLFDQGHXWURSKLF0RGL¿HG from Shi et al., 2011. 24 General Introduction LGHQWL¿FDWLRQVEXWWRRWLPHFRQVXPLQJIRU¿HOGVWXGLHV'RWEORWK\EULGL]DWLRQDVVD\VFRQVLVWRQ quantifying the relative hybridization of membrane-bound DNA (typically PCR products of the WDUJHWJHQHXVLQJVSHFL¿FROLJRQXFOHRWLGHSUREHV7KXV33(FRPSRVLWLRQDQGYDULDELOLW\ZDV VWXGLHGZLWKGRWEORWVDQGSODVWLG6U'1$FODVVVSHFL¿FSUREHV)XOOHUet al., 2003, 2006b), allowing a fast analysis of many samples to assess distributional patterns of different PPEs classes over large spatial scales (Lepère et al., 2009; Kirkham et al., 2011a,b; Bouman et al., 2012; Kirkham et al.,0RUHUHFHQWO\KLJKWKURXJKSXWVHTXHQFLQJ+76WHFKQRORJLHVDQG Illumina) have provided a means of estimating species richness from deeply-sampled communities and for discovering novel species (Sogin et al., 2006; Amaral-Zettler et al., 2009; Logares et al., +76DOVRSURYLGHVSUR[LHVRIWKHUHODWLYHDEXQGDQFHRIVSHFL¿FWD[DLQDIDVWFKHDS and high-throughput scale and has been applied on broad studies of marine plankton from coastal (Massana et al., 2015) and open ocean (de Vargas et al., 2015) systems, including a particular analysis of the picoeukaryotic fraction. Although useful, HPLC pigment analysis, dot blot assays, and HTS surveys provide at best semiquantitative abundances, and to obtain cell abundances a true quantitative method such as FISH (Fluorescence In Situ Hybridization) is required (Pernthaler et al., 2001; Amann and Fuchs, 2008). 7KLVWHFKQLTXHDOORZVVLQJOHFHOOLGHQWL¿FDWLRQE\XVLQJJURXSVSHFL¿FSUREHVPRVWO\6U'1$ SUREHV+HUHDODEHOOHGSUREHSHQHWUDWHVLQWRWKH¿[HGPLFURELDOFHOOWKDWKDGEHHQLPPRELOL]HG LQDPHPEUDQHDQGELQGVWRWKHU51$LQWKHULERVRPHVZKHQ¿QGLQJWKHVDPHVHTXHQFHIRU K\EULGL]DWLRQWR¿QDOO\SURGXFHDÀXRUHVFHQWVLJQDO7KHODEHOOHGFHOOVFRXOGWKHUHIRUHEHREVHUYHG DQG TXDQWL¿HG XQGHU HSLÀXRUHVFHQFH PLFURVFRS\ WR REWDLQ DEXQGDQFHV RI VSHFL¿F JURXSV DQG rough morphological properties, at least the cell size (Figure 10). FISH has been applied both in coastal and open ocean sites to assess the abundance and distribution of well-known groups, such as prymnesiophytes (haptophytes), pelagophytes and chrysophytes (stramenopiles) (Jardillier et al., 2010) and mamiellophytes (chlorophytes) (Not et al.,1HYHUWKHOHVVFRQWUDU\WRVHPL quantitative approaches, manual counts of targeted cells is time-consuming and only few studies have applied FISH over large spatial scales (Not et al., 2008; Jardillier et al., 2010; Kirkham et al., E*UREet al., 2011). General Introduction 25 PHOTOSYNTHETIC PICOEUKARYOTES IN THE OCEANS: SPATIAL SCALES OF VARIABILITY The mentioned tools for evaluating PPEs community structure have been applied in surveys that have tracked the water column at different depths in oceanic regions with different trophic, temperature and salinity gradients. Combining the data obtained, the general consensus is that prymnesiophytes, chrysophytes and pelagophytes dominate in oligotrophic open ocean systems with minor contribution of chlorophytes, mostly mamiellophytes and new prasinophyte clades (Figure 9). On the other hand, coastal and nutrient rich systems contain the same PPE groups but in that areas mamiellophytes can dominate numerically, particularly in certain periods of the seasonal cycle that establishes in temperate regions (Not et al1HYHUWKHOHVV environmental parameters (Chl a, nitrate, phosphate, salinity, water column depth, latitude and temperature, among others) shaping PPEs community composition at global scale are still poorly characterized (Kirkham et al., 2013). A B Figure 10. (A) The principle of CARD-FISH FDWDO\VHG UHSRUWHG GHSRVLWLRQÀXRUHVFHQFH in situ hybridization). CARD-FISH combines CARD RI ÀXRUHVFHQWO\ ODEHOOHG W\UDPLGHV ZLWK VLQJOHFHOO LGHQWL¿FDWLRQ E\ ),6+ 7KH K\EULGL]DWLRQ LQYROYHV D single oligonucleotide that is covalently crosslinked to D KRUVHUDGLVK SHUR[LGDVH +53 ODEHO $PSOL¿FDWLRQ of the signal relative to that achieved with probes that DUH ODEHOOHG ZLWK D VLQJOH ÀXRURFKURPH LV EDVHG RQ the radicalization of multiple tyramide molecules by a single horseradish peroxidase. From Amann and Fuchs, 2008. (B) (SLÀXRUHVFHQFH PLFURVFRS\ LPDJH RI WKH CARD-FISH assay (green-labelled cells under blue ligth excitation) in Pelagomonas calceolata FFPS E\ XVLQJWKHSHODJRSK\WHFODVVVSHFL¿FSUREH3(/$ A B 32 Aims and outline of the thesis The mentioned thesis outline can be summarized under the umbrella of three major objectives and VHYHUDOVSHFL¿FRQHVDVIROORZV Objective 1: 7R FKDUDFWHUL]H WKH ¿QH YHUWLFDO GLVWULEXWLRQ RI PDMRU SK\WRSODQNWRQ JURXSV ZLWKLQWKH'&0GHYHORSLQJLQWHPSHUDWHDUHDVGXULQJVXPPHUVWUDWL¿FDWLRQ We hypothesize a vertical segregation of the investigated groups along the water column, particularly within the DCM, according to their different ecophysiological requirements in the light, nutrient and temperature gradients. In Chapter 1 ZH IRFXVHG RQ WKH PDMRU SKRWRV\QWKHWLF SLFRHXNDU\RWLF JURXSV 7KH VSHFL¿F objectives were: - To obtain reliable counts of targeted groups. - To evaluate their vertical distribution in abundance and size spectra. - To explore whether chlorophytes, prymnesiophytes, pelagophytes and chrysophytes are distributed differentially across the DCM. In Chapter 2, we expanded our study to other phytoplankton groups and more sampling stations to achieve the following purposes: - To obtain a comprehensive view of the phytoplankton composition within the DCM. - To explore the vertical segregation of PPEs as compared with Prochlorococcus, Synechococcus, GLDWRPVDQGGLQRÀDJHOODWHV Objective 2: To obtain global abundances of the prymnesiophyte–UCYN-A symbiosis A particular symbiosis has been recently described between prymnesiophyte species and the XQLFHOOXODU 1¿[LQJ F\DQREDFWHULXP 8&<1$ $W OHDVW WZR GLIIHUHQW 8&<1$ FODGHV H[LVW UCYN-A1 in symbiosis with an uncultured small prymnesiophyte and UCYN-A2 in symbiosis with the larger coccolitophore Braarudosphaera bigelowii. While previous molecular surveys show a widespread and overlapped distribution of the two clades, few microscopic observations of this symbiosis have been performed in the ocean. Aims and outline of the thesis 33 In chapter 3ZHTXDQWL¿HGWKLVSDUWLFXODUV\PELRVLVXQGHUWKHQH[WVSHFL¿FREMHFWLYHV 7RWDUJHWVSHFL¿FDOO\ERWKWKHSU\PQHVLRSK\WHKRVWDQGWKHF\DQREDFWHULDOV\PELRQWE\DGRXEOH CARD-FISH approach. 7RFRQ¿UPWKHEURDGGLVWULEXWLRQRIWKLVV\PELRVLV - To evaluate its ecological niche and distribution patterns. - To provide further evidence about the obligatory dependence of both symbiont and host. Objective 3: 7RVWXG\SHODJRSK\WHGLYHUVLW\DQGELRJHRJUDSK\DWVSHFLHVDQGLQWUDVSHFLHV OHYHOV XVLQJDJURXSVSHFL¿FDSSURDFK Pelagophytes are important components of marine photosynthetic picoeukaryotic assemblages and are widely distributed across the oceans. However, their diversity has been only evaluated in molecular surveys targeting all picoeukaryotes, suggesting that pelagophyte assemblages are formed by a single species, Pelagomonas calceolata. Here we wanted to expand the knowledge of WKHLUGLYHUVLW\XVLQJDJURXSVSHFL¿FDSSURDFKDQGKLJKWKURXJKSXWVHTXHQFLQJ The study of pelagophytes in chapter 4IROORZHGWKHQH[WVSHFL¿FREMHFWLYHV 7RGHVLJQJURXSVSHFL¿FSULPHUVFRYHULQJSDUWLDO6U'1$JHQHDQGWKHLQWHUQDOWUDQVFULEHG spacer 1 (ITS1). - To validate the prevalence and distribution of other pelagophyte genera aside from Pelagomonas. 7RHYDOXDWHWKHLQWUDVSHFL¿FYDULDELOLW\LQWZRPRVWLPSRUWDQWSHODJRSK\WHVSHFLHVPelagomonas calceolata and Aureococcus anophagefferens, using the ITS1 marker. Chapter 1 Chapter 1 37 Chapter 1 Vertical distribution of major photosynthetic picoeukaryotic JURXSVLQVWUDWL¿HGPDULQHZDWHUV Ana M. Cabello, Mikel Latasa, Irene Forn, Xosé Anxelu G. Morán and Ramon Massana. Summary Photosynthetic picoeukaryotes (PPEs) are fundamental contributors to oceanic primary production and form diverse communities dominated by prymnesiophytes, chlorophytes, pelagophytes, and chrysophytes. Here we studied the vertical distribution of these major groups in two offshore UHJLRQVRIWKHQRUWKHUQ,EHULDQ3HQLQVXODGXULQJVXPPHUVWUDWL¿FDWLRQ:HSHUIRUPHGD¿QHVFDOH YHUWLFDOVDPSOLQJHYHU\aPDFURVVWKH'&0DQGXVHGÀXRUHVFHQFHin situ hybridization (FISH) to determine the PPE composition and to explore the possible segregation of target groups in the light, nutrient and temperature gradients. Chlorophytes, pelagophytes and prymnesiophytes, in WKLVRUGHURIDEXQGDQFHDFFRXQWHGIRUWKHWRWDO33(VUHFRUGHGE\ÀRZF\WRPHWU\LQWKH$YLOpV canyon, and for more than half in the Galician Bank, while chrysophytes were undetected. Among these three groups, often the prymnesiophytes were dominant in biomass. In general, all groups were present throughout the water column with abundance peaks around the DCM, but their distributions differed: pelagophytes were located deeper than the other two groups, chlorophytes presented two peaks and prymnesiophytes exhibited surface abundances comparable to those at WKH'&07KLVVWXG\RIIHUV¿UVWLQGLFDWLRQVWKDWWKHYHUWLFDOGLVWULEXWLRQRIGLIIHUHQW33(JURXSV is heterogeneous within the DCM. Chapter 1 39 Introduction 0DULQHSKRWRV\QWKHWLFSLFRHXNDU\RWHV33(VGH¿QHGRSHUDWLRQDOO\DVFHOOVȝPDUHUHFRJQL]HG as major contributors to phytoplankton biomass :RUGHQet al., 2004; Cuvelier et al., 2010) and primary productivity in marine systems (Li, 1994, 1995; Agawin et al., 2000; Morán, 2007; Jardillier et al., 2010). This assemblage displays a tremendous diversity with members from all known algal groups and also uncultured lineages, as revealed by molecular surveys based on the 18S rRNA gene (Díez et al.,/ySH]*DUFtDet al.,0RRQYDQGHU6WDD\et al., 2001; Romari and Vaulot, 2004; Shi et al., 2009; Massana et al., 2011a) and the 16S rRNA gene sequence analysis (Rappé et al., 1998; Fuller et al., 2006a; McDonald et al., 2007; Lepère et al., 2009; Kirkham et al., 2011a; Shi et al., 2011). Molecular surveys have complemented previous reports based on culturing, pigment analysis, and ultrastructure, to reach an overall view of PPE diversity (Vaulot et al., 2008; Massana, 2011b). Due to the ecological relevance of PPEs, it is important to unveil its community structure over temporal and spatial scales and to identify the environmental factors driving community assembly. This effort requires the right tools to quantify the abundance of the different phytoplankton taxa in natural samples. Early studies were based in pigment analysis (Letelier et al.,$QGHUVHQet al., 1996; Bidigare and Ondrusek, 1996; Latasa and Bidigare, 1998; Suzuki et al., 2002), and these ZHUHODWHUFRPSOHPHQWHGZLWKGRWEORWK\EULGL]DWLRQDVVD\VEDVHGRQSODVWLG6U51$FODVV VSHFL¿FSUREHV(Fuller et al., 2006b; Lepère et al., 2009; Kirkham et al., 2011a,b; Bouman et al., 2012; Kirkham et al.,. However, this view based on pigments and rDNA gene frequencies FDQVWLOOEHGLIIHUHQWIURPWUXHFHOODEXQGDQFHVZKLFKFDQEHZHOOUHVROYHGE\&$5'),6+FRXQWV (Not et al., 2002, 2004, 2005, 2007; Biegala et al.,3LZRV]et al., 2015), a method that has been applied only in a few large spatial scale studies (Not et al., 2008; Jardillier et al., 2010; Kirkham et al., 2011b; Grob et al., 2011). Combining the data obtained from molecular surveys, pigment analysis, dot blot hybridization and FISH, the general consensus is that prymnesiophytes, chrysophytes and pelagophytes dominate in oligotrophic open ocean systems with minor FRQWULEXWLRQRIFKORURSK\WHVZKLFKEHFRPHGRPLQDQWLQFRDVWDODQGQXWULHQWULFKDUHDV One of the most pervasive gradients in the sea is the vertical structure of the water column, where phytoplankton is subjected to opposing resources: light that comes from above and nutrients often VXSSOLHGIURPEHORZ,QVWUDWL¿HGZDWHUVSK\WRSODQNWRQIUHTXHQWO\IRUPVDSHDNRISLJPHQWVDQG often of biomass) known as deep chlorophyll maximum (DCM), typically located towards the base of the pycnocline and strongly coupled to the nutricline (Cullen, 1982; Estrada et al., Sharples et al., 2001). Adaptation and competition for light and nutrients within this structure could explain the distinct vertical distributions of major phytoplankton groups such as cyanobacteria and eukaryotes (Latasa et al., 1992; Zubkov et al., 2000; Johnson et al., 2006; Hickman et al., 2009, 2010):LWKUHJDUGWRSDUWLFXODUWD[DZLWKLQWKH33(VRQO\DIHZVWXGLHVKDYHWDUJHWHGWKHLU relationship with the DCM, and always with a rough (tens of meters) vertical resolution. Photo-picoeukaryotes community structure in the DCM 40 In this work we studied in detail the vertical distribution of major PPEs groups during summer VWUDWL¿FDWLRQ LQ WKH WHPSHUDWH 1($WODQWLF EDVHG RQ &$5'),6+ JURXS VSHFL¿F SUREHV :H SHUIRUPHG D ¿QH VFDOH VDPSOLQJ ZLWKLQ WKH '&0 ! GHSWKV HYHU\ ¾2 m) to evaluate the variability of the PPE composition within this structure, and to explore whether chlorophytes, prymnesiophytes, pelagophytes and chrysophytes are distributed differentially. Because of their different ecophysiological features (pigment composition, nutrient requirements and temperature optima) (Roy et al., 2011; Kulk et al., 2012) we hypothesize a segregation of these groups along the water column, and especially at the DCM according to the strong gradients of light, nutrients and temperature. Our results indicate that PPE groups were not evenly distributed through the water column, in support of this hypothesis. Results Microscopic observations of target groups In this work we have applied a set of published FISH probes to obtain cell abundances of chlorophytes, prymnesiophytes, pelagophytes and chrysophytes at four stations in the temperate NE Atlantic in summer. The FISH signal obtained when targeting photosynthetic picoeukaryotes ZDVWULFN\7KHJUHHQÀXRUHVFHQWVLJQDORIWKH6U'1$SUREHZDVUHVWULFWHGWRDVPDOODUHD since the unlabeled chloroplast often occupied a substantial part of the cell volume. Nevertheless, detection of chlorophytes and prymnesiophytes was optimal, and probes CHLO02 and PRYM02 always labeled a clear population of cells. On the other hand, pelagophyte cells appeared weakly labeled with probe PELA01, specially in samples from surface or below the DCM, and positive FHOOVZHUHQRW HDVLO\GLVWLQJXLVKHGIURPRWKHU GLPÀXRUHVFHQWSDUWLFOHV7KHVHXQVSHFL¿FLWLHV were not due to natural peroxidases and were not removed by increasing the blocking reagent LQWKHK\EULGL]DWLRQEXIIHU7RKHOSGLVWLQJXLVKLQJSHODJRSK\WHFHOOVIURPQRQWDUJHWSDUWLFOHV DQGEDFNJURXQGÀXRUHVFHQFHZHGHYHORSHGDGRXEOHK\EULGL]DWLRQDVVD\DJDLQVWERWK6DQG 16S rRNA (Fig. 1). In a P. calceolata (pelagophyte) culture this resulted in cells with green labeled cytoplasm and red labeled plastid under blue and green light excitations, respectively. In environmental samples this procedure allowed a straightforward detection of target cells, even when both signals were faint. Surprisingly, the last targeted group, the chrysophytes, was never detected, so this group seemed absent from the four stations in this particular sampling period. 'LVWULEXWLRQSUR¿OHVRISKRWRV\QWKHWLFSLFRHXNDU\RWHV33( 7KHIRXUVHOHFWHGVWDWLRQV)LJDGLVSOD\HGDVWUDWL¿HGZDWHUFROXPQZLWKVXUIDFHWHPSHUDWXUHV EHWZHHQ&WKDWGHFUHDVHGXQWLOVWDELOL]DWLRQDW&EHORZP)LJE6DOLQLW\ Chapter 1 41 5 µm (A) (B) was higher in the Galicia Bank than in the Avilés canyon region. In the Avilés canyon region the thermocline started near the surface and had the strongest gradient between 40 and 60 m. In the Galicia region the thermocline was found between 40 and 70 m, with a clear surface mixed layer. )OXRUHVFHQFHSUR¿OHVUHYHDOHGYHU\FOHDU'&0SHDNVSODFHGWRZDUGWKHEDVH$YLOpVVWDWLRQVRU in the middle of the thermocline (Galicia stations), in close association with the nutricline (Fig. 61XWULHQWSUR¿OHVZHUHVLPLODULQERWKDUHDV7KHXSSHUZDWHUFROXPQZDVQXWULHQWGHSOHWHG down to the DCM depth. From here, nutrient concentrations increased with depth and maximum YDOXHVIRUQLWUDWHSKRVSKDWHDQGVLOLFDWHZHUHDQG0UHVSHFWLYHO\ (average of the deepest samples of the 4 stations). As shown in the graphs (Figs 2b), the selected sampling depths coveredLQGHWDLOWKHÀXRUHVFHQFHSUR¿OHVRIWKH'&0V The distribution of total 33(VFHOOVREWDLQHGE\ÀRZF\WRPHWU\IROORZHGWKHvertical pattern of the ÀXRUHVFHQFHSUR¿OHZLWKPD[LPDOFRXQWVFRLQFLGHQWZLWKWKH'&0SHDN)LJD$EXQGDQFH YDOXHVZHUHVLPLODUDWERWKUHJLRQVUDQJLQJEHWZHHQFHOOVPODWWKHVXUIDFHDQG 8000 cells mlDWWKH'&0GHSWK7KHYHUWLFDOGLVWULEXWLRQRIVSHFL¿F33(VJURXSVZDVREWDLQHG Figure 1.(SLÀXRUHVFHQFHPLFURVFRS\LPDJHVRIWKHGRXEOHK\EULGL]DWLRQDVVD\LQSHODJRSK\WHVIURPHQYLURQPHQWDO VDPSOHV/HIWSDQHOVFRUUHVSRQGWR'$3,VLJQDOEOXHODEHOHGQXFOHXVULJKWSDQHOVFRUUHVSRQGWRWKHFRPELQHG VLJQDORIWKH3(/$SUREHJUHHQODEHOHGF\WRSODVPXQGHUEOXHOLJKWH[FLWDWLRQDQGWKH3(/$SUREHUHG labeled plastid under green light excitation). (A)3HODJRSK\WHFHOOVRIW\SLFDOVL]HaP(B) A larger pelagophyte cell displaying the red plastid signal localized in two separated dots. Photo-picoeukaryotes community structure in the DCM 48 Discussion 7D[RQRPLFFRPSRVLWLRQRIWKH'&0FRPPXQLW\DWWKHWZRVWXG\VLWHV Sampling at the Avilés canyon and Galicia Bank regions showed the common DCM structure GHYHORSHGGXULQJWKHVXPPHUVWUDWL¿FDWLRQLQWHPSHUDWHDUHDVIROORZLQJWKH³7\SLFDO7URSLFDO 6WUXFWXUH´ GH¿QHG E\ +HUEODQG DQG9RLWXULH] 9HUWLFDO SUR¿OHV LQ ERWK RFHDQRJUDSKLF regions showed similar ranges of temperature and nutrient concentrations, whereas the salinity was higher in the Galicia Bank than in the Avilés region, as expected from the water masses composing these regions (Botas et al.,*RQ]iOH]4XLUyVet al., 2004). Differences in the picophytoplanktonic community were also detected, both at the cyanobacterial level (Synechococcus was present in both regions and Prochlorococcus only in Galicia, Latasa et al., Chapter 2), and at the PPEs composition. Moreover, in the Avilés stations, chlorophytes, SHODJRSK\WHV DQG SU\PQHVLRSK\WHV FRPSOHPHQWHG WKH 33( DVVHPEODJHV PHDVXUHG E\ ÀRZ cytometry, while these three groups accounted for about 50% of PPE cells in Galicia stations. In other studies, both based on FISH and dot blot hybridization, it is common not to cover the total PPE abundance with the assayed probes (Kirkham et al., 2011b; Grob et al., 2011; Piwosz et al.,, so the situation detected in Avilés is more the exception than the rule, and the question remaining is which other groups are completing the PPE assemblage in the Galicia stations. It is remarkable the fact that we did not detect chrysophytes, known to be important within PPEs LQWHPSHUDWHDQGWURSLFDO$WODQWLFDQG3DFL¿FZDWHUVEDVHGRQ),6+aRIFHOOV(Jardillier et al., 2010; Grob et al., 2011; Hartmann et al., and dot blot data (~40% of the signal) (Lepère et al., 2009; Kirkham et al., 2011b). Curiously enough, the abundance of chrysophytes in the Avilés region in a station closer to the coast (bottom depth of 110 m) and taken one month later was ~500 cells ml, being most cellsȝPLQVL]HGDWDQRWVKRZQ$OVRZHQRWLFHG that the probe &+5<62ZDVQRWODEHOLQJVRPHRIRXUFKU\VRSK\WHFXOWXUHVVRLWFDQQRW be discarded the possibility that we were missing some chrysophyte species in our survey. &U\SWRSK\WHVZHUHGHWHFWHGE\ÀRZF\WRPHWU\LQRXUVDPSOHVEXWDOZD\VDWORZDEXQGDQFHV less than 1% of PPEs cells (data not shown). Other PPE cells in Galicia samples might include dictyochophytes, bolidophytes, pinguiophytes, eustigmatophytes, or chlorarachniophytes. These groups are considered minor components of the community based on dot blot surveys (Kirkham et al., although dictyochophytes and bolidophytes in particular are often found in clone libraries from the picoplankton size fraction (Kirkham et al., 2011a,b; Massana, 2011a; Shi et al., 2011). Our results agree with previous FISH data in the temperate North Atlantic, which show the PPE FRPPXQLW\ GRPLQDWHG E\ FKORURSK\WHV  RI WKH FHOOV IROORZHG E\ SU\PQHVLRSK\WHV Chapter 1 49 pelagophytes, and minor components like cryptophytes and bolidophytes (Not et al., 2002, 2007; Biegala et al.,  :LWKLQ FKORURSK\WHV 0DPLHOORSK\FHDH VSHFLHV SDUWLFXODUO\ ZLWKLQ WKH genus MicromonasFRPPRQO\GRPLQDWHWKH33(FRPPXQLW\LQQXWULHQWULFKRIIVKRUHDQGFRDVWDO waters (Not et al., 2004, 2005). In our samples chlorophytes were mainly composed by cells <2 +m that likely corresponded to Mamiellophyceae, whereas pelagophytes and prymnesiophytes were VOLJKWO\ODUJHU,QWHUHVWLQJO\WRWDO),6+FRXQWVRIFKORURSK\WHVZHUHVLJQL¿FDQWO\FRUUHODWHGWRWKH VPDOOHURIWKHWZR33(SRSXODWLRQVREVHUYHGE\ÀRZF\WRPHWU\VHH([SHULPHQWDOSURFHGXUHV (Fig. 7). The slope of the regression close to the 1:1 line indicatedWKDWWKH VRFDOOHGµVPDOO¶ picoeukaryotes &DOYR'tD]et al., 2008) were made up to a large extent by chlorophytes. Since pelagophytes and prymnesiophytes presented similar abundances in both regions, the decrease of chlorophytes and the presence of other groups in Galicia stations were responsible for a shift in WKH33(FRPPXQLW\FRPSRVLWLRQ,QIDFWWKHSHUFHQWDJHRIµVPDOO¶SLFRHXNDU\RWHVH[SODLQHGE\ FKORURSK\WHVZDVPXFKORZHULQ*DOLFLDWKDQLQ$YLOpV)LJ,QDQRIIVKRUHHVWXDULQHWUDQVHFW in the English Channel, a similar community shift was observed regarding chlorophytes, which doubled the abundance from offshore to the coast, while pelagophytes maintained their abundances (Biegala et al.,7KHLQFUHDVHRIFKORURSK\WHVLQ$YLOpVVWDWLRQVZRXOGUHÀHFWWKHLQÀXHQFHRI coastal waters explained by the hydrodynamics of the Avilés canyon, known to produce upwelling DQGGHÀHFWLRQRIFRVWDOZDWHUVWRZDUGVRIIVKRUHDUHDV*RQ]iOH]4XLUyVet al., 2004). 'small' picoeukaryotes (cells ml-1) 0 2000 4000 6000 8000 chlorophytes (cells ml-1) 0 2000 4000 6000 8000 st A st B st C st D y = 0.88x - 649.6 R2 = 0.63 Figure 7. 5HODWLRQVKLSRIFKORURSK\WH),6+FRXQWVDQGWKHSRSXODWLRQRIµVPDOO¶33(VW\SLFDOO\PFRXQWV GHULYHGIURPÀRZF\WRPHWU\'DWDIURPHDFKVWDWLRQDUHUHSUHVHQWHGZLWKDGLIIHUHQWV\PERO7KHUHJUHVVLRQOLQH (solid line; P < 0.05) and the 1:1 line (dashed line) are plotted. Photo-picoeukaryotes community structure in the DCM 50 Here we determined also the size distribution of the three algal groups, and the breadth of the size spectra was consistent with their taxonomic diversity. Thus, mamiellophytes, likely the major contributors to chlorophytes, and pelagophytes, both having the majority of cells in a single size class, are known to be less diverse than prymnesiophytes based on rDNA (Liu et al., 2009; Pernice et al.,. 7KH¿UVWWZRJURXSVDUHUHVWULFWHGWRWKHSLFRHXNDU\RWLFVL]HIUDFWLRQVFHOOVP while prymnesiophytes are also well represented in the nanoplankton, including cells larger than 5 P/LXet al., 2009). As a result, prymnesiophytes do not need to be the most abundant group to become important contributors to phytoplankton biomass (Liu et al., 2009; Cuvelier et al., 2010). 9HUWLFDOGLVWULEXWLRQSDWWHUQV 2XULQWHQVLYHVDPSOLQJLQDERXWPLQWHUYDOVDFURVVWKHÀXRUHVFHQFHJUDGLHQWRIWKH'&0OD\HU allowed us to determine in detail the vertical distributions of the target groups and to observe that they presented decoupled abundance peaks. Pelagophytes were located deeper than the other two groups, chlorophytes presented two peaks (well marked in Galicia) and prymnesiophytes exhibited surface abundances comparable to those at the DCM. Although their distributions presented the differences just mentioned above, all groups occurred throughout the water column (not restricted to particular layers), usually with subsurface abundance maxima around the DCM peak. This overall regular pattern could be, in part,EHFDXVHZHXVHG'LYLVLRQUDQN&+/2DQG35<0 RU &ODVVUDQN 3(/$ SUREHV ZKLFK FDQ WDUJHW VSHFLHV ZLWK GLIIHUHQW HFRSK\VLRORJLFDO requirements. The high prymnesiophyte abundances in surface waters suggested that surface species might be different than the ones occupying the DCM. As an example of this case, we GHWHFWHGLQWKHVHSUR¿OHVDSU\PQHVLRSK\WHVSHFLHVKDUERULQJDF\DQREDFWHULDOV\PELRQWWKDWZDV UHVWULFWHGWRVXUIDFHZDWHUVZKHUHLWDFFRXQWHGIRURISU\PQHVLRSK\WHFHOOVDQGZDV absent from the DCM (Cabello et al., 2015). This provides evidence that our level of taxonomic resolution likely masked vertical gradients. Another important observation was the presence of two chlorophyte peaks, well marked in the Galicia region. Micromonas and Ostreococcus species DUHNQRZQWRFRPSULVHVHYHUDOSK\ORJHQHWLFFODGHVDQGVRPHDUHUHODWHGWRORZOLJKWRUKLJK light adapted ecotypes, with different optima for irradiance and also for nutrients, temperature and salinity (Rodríguez et al., 2005; Foulon et al., 2008). Although niche partitioning in depth of these ecotypes remains unclear'HPLU+LOWRQet al., 2011), the observed chlorophyte peaks in our SUR¿OHVFRXOGEHWKHUHVXOWRIVHOHFWHGHFRW\SHVDGDSWHGWRGLIIHUHQWQXWULHQWDQGOLJKWUHJLPHV,WLV noticeable that the shallowest peak was placed at the base of the mixed layer and the deepest peak within the nutricline, coinciding with settings that characterize the distribution of Ostreococcus HFRW\SHV'HPLU+LOWRQet al., 2011). 2QWKHRWKHUKDQGWKHFODVV3HODJRSK\FHDHKDVH[SHULHQFHGORZHYROXWLRQDU\GLYHUVL¿FDWLRQEDVHG on the 18S rDNA (Pernice et al.,DQGWKHYDVWPDMRULW\RIVHTXHQFHVIURPPROHFXODUVXUYH\V Chapter 1 51 DI¿OLDWHWRPelagomonas calceolata (Shi et al., :RUGHQet al., 2012). Thus, the PELA01 probe is probably targeting mostly the P. calceolata population, and this is consistent with the observation that pelagophytes exhibited a more conserved distributional trend than chlorophytes and prymnesiophytes. Pelagophytes accumulated half of their population deeper than the other JURXSVDQGGLVSOD\HGJUHDWHUFRQWULEXWLRQVZLWKLQWKHGRZQVORSHRIWKH'&0JUDGLHQWLQGLFDWLQJ a preference to occupy deeper layers. The increased relevance of pelagophytes within the DCM UHODWLYHWRVXUIDFHZDWHUVZDVSUHYLRXVO\REVHUYHGLQSLJPHQWEDVHGVXUYH\VZKLFKSURSRVHG an adaptation of pelagophytes to low light conditions and perhaps high nutrient requirements (Claustre and Marty, 1995; Barlow et al., 1997; Marty et al., 2008). Pelagomonas calceolata is FRQVLGHUHGDORZOLJKWDGDSWHGVSHFLHV7LPPHUPDQVet al., 2005; Dimier et al., 2009) and a recent PROHFXODUVXUYH\VKRZHGKLJKH[SUHVVLRQOHYHOVRIQLWURJHQDVVLPLODWLRQJHQHVLQSHODJRSK\WHV indicating that this group was an important contributor to nitrate assimilation within the DCM (Dupont et al., 2015). &RQFOXGLQJUHPDUNV :HZHUHDEOHWRHYDOXDWHIRUWKH¿UVWWLPHWKHYDULDELOLW\RIWKH33(FRPPXQLW\VWUXFWXUHLQ WHUPVRIFRPSRVLWLRQFHOOVL]HDQGELRPDVVZLWKLQW\SLFDO'&0VRIWHPSHUDWHUHJLRQV2XU¿QH VDPSOLQJDSSURDFKSURYLGHGFRQ¿GHQFHWRHVWDEOLVKWKHYHUWLFDOVWUXFWXULQJRIWKHKDELWDWVRIWKH three groups, which not always revealed smooth distributions. Although groups generally peaked in the vicinity of the DCM depth, the abundance maxima of each was vertically separated within this layer. At the level of taxonomic resolution resolved by our probes, ZHFRQ¿UPHGWKHSUHIHUHQFH of pelagophytes to occupy the deepest layers, the intriguing presence of two chlorophyte peaks and the high abundance of prymnesiophytes within surface relative to DCM waters. This study has shown that the DCM should be considered a heterogeneous structure that requires more than a single sample to obtain a representative picture of its phytoplanktonic composition. Experimental procedures 6WXG\DUHDVDQGVDPSOLQJSURFHGXUHV 7KHRFHDQRJUDSKLFFUXLVH,1'(0$5(6WRRNSODFHGXULQJVXPPHU-XO\ August) on board the R/V Thalassa (IFREMER / IEO) in two regions near the Iberian Peninsula, the Avilés canyon (Cantabrian Sea) and the Galicia Bank (NE Atlantic). Two offshore stations ERWWRPGHSWK!PZHUHVHOHFWHGZLWKLQHDFKVDPSOLQJUHJLRQ)LJDStations A, B, &DQG'FRUUHVSRQGHGWRFDVWVDQGUHVSHFWLYHO\At each station, vertical casts ZHUHSHUIRUPHGDIWHUVXQVHWZLWKD&7'SUR¿OHUUHFRUGLQJWHPSHUDWXUHDQGVDOLQLW\¿WWHGZLWK Photo-picoeukaryotes community structure in the DCM 52 DÀXRURPHWHU:(7ODEV(&2$)/DQGDERWWOH1LVNLQURVHWWH:HFDUULHGRXWDQLQWHQVLYH VDPSOLQJZLWKLQWKH'&02QWKHEDVLVRIWKHUHDOWLPHÀXRUHVFHQFHSUR¿OHUHYHDOHGGXULQJWKH downcast, we chose the depth just below the DCM where to start the sampling, and during the XSFDVW1LVNLQERWWOHVZHUHFORVHGHYHU\VHFRQGV$YLOpVFDQ\RQRUVHFRQGV*DOLFLD Bank) at a constant ascent rate of 0.25 m s from the chosen initial depth to just above the DCM. 7KLVJDYHXVD¿QHYHUWLFDOVDPSOLQJUHVROXWLRQRIWRP7KHODVWERWWOHVZHUHFORVHGZLWKLQ the mixed layer and at surface. From the 22 bottles continuously sampled, we chose 11 or 12 to ¿QHO\UHSUHVHQWWKHÀXRUHVFHQFHJUDGLHQWRIWKH'&0. )ORZF\WRPHWULFFRXQWVRISKRWRV\QWKHWLFSLFRHXNDU\RWHV33( PPEs cells were counted from samples (1.8 ml) preserved on board with 1% paraformaldehyde SOXVJOXWDUDOGHK\GHÀDVKIUR]HQLQOLTXLGQLWURJHQDQGNHSWDWí&XQWLODQDO\VLVLQ WKHODERUDWRU\ZLWKD)$&6&DOLEXUÀRZF\WRPHWHU%HFWRQ'LFNLQVRQHTXLSSHGZLWKDODVHU HPLWWLQJDWQP7ZRGLIIHUHQWO\VL]HGJURXSVRI33(VUHIHUUHGWRDVµVPDOO¶DQGµODUJH¶ ZHUHGLVFULPLQDWHGIURPF\DQREDFWHULDEDVHGRQWKHLURUDQJH)/QPDQGUHG)/! QPÀXRUHVFHQFHDQGOLJKWVFDWWHUVLJQDOV&DOYR'tD]DQG0RUiQ)RUHVWLPDWLQJFHOO DEXQGDQFHVFDOLEUDWLRQRIWKHF\WRPHWHUÀRZUDWHZDVSHUIRUPHGGDLO\DQGDVROXWLRQRIȝP ÀXRUHVFHQWODWH[EHDGVUHI)0ROHFXODU3UREHVZDVDGGHGDVDQLQWHUQDOVWDQGDUG&DOYR 'tD]DQG0RUiQ&HOOVXSWRFDPLQVL]HZHUHURXWLQHO\GHWHFWHG&RXQWVRIµVPDOO¶DQG µODUJH¶HXNDU\RWLFFHOOVZHUHVXPPHGXSWRREWDLQWRWDO33(VFRXQWV &$5'),6+FRXQWV&$WDO\]HG5HSRUWHU'HSRVLWLRQ)OXRUHVFHQW,Q6LWX+\EULGL]DWLRQ )RUZKROHFHOO&$5'),6+POVDPSOHVZHUHSUH¿OWHUHGE\ȝP¿[HGZLWKIRUPDOGHK\GH ¿QDOFRQFHQWUDWLRQ¿OWHUHGRQPSRUHVL]H1XFOHRSRUH¿OWHUVPPGLDPHWHUDQG NHSWIUR]HQ*URXSVSHFL¿FROLJRQXFOHRWLGHSUREHVZHUHDSSOLHGWRWDUJHWFKORURSK\WHV&+/2 SU\PQHVLRSK\WHV 35<0 FKU\VRSK\WHV &+5<62 DQG SHODJRSK\WHV 3(/$ DQG 3(/$'HVLJQDQGWHVWLQJRIWKH6U'1$SUREHV&+/235<0DQG3(/$ZDV described before (Simon et al., 2000; Not et al., 3UREHV&+5<62DQG3(/$ targeting the plastid 16S rDNA, were described for dot blot hybridizations (Fuller et al., 2006b) DQGPRGL¿HGIRU&$5'),6+E\-DUGLOOLHUet al. (2010) and in this work, respectively. Probes were SXUFKDVHGZLWKD¶DPLQROLQN&IURP7KHUPR)LVKHU6FLHQWL¿FDQGODEHOHGZLWKKRUVHUDGLVK perodidase, HRP (Roche Diagnostic Boehringer) as described before (Urdea et al., 1988; Amann et al., 1992))LOWHUVIRU&$5'),6+ZHUHHPEHGGHGLQZYORZJHOOLQJSRLQWDJDURVH WRPLQLPL]HFHOOORVV7KHK\EULGL]DWLRQZDVFDUULHGRXWE\FRYHULQJ¿OWHUSLHFHVZLWKORI K\EULGL]DWLRQEXIIHUGHLRQL]HGIRUPDPLGH>H[FHSWIRU&+5<62DQG3(/$WKDW Chapter 1 53 ZDV@01D&OP07ULV+&O>S+@VRGLXPGRGHF\OVXOIDWH>6'6@DQG mg ml%ORFNLQJUHDJHQW>5RFKH'LDJQRVWLF%RHKULQJHU@FRQWDLQLQJORI+53ODEHOHGSUREH VWRFNDWQJO–1DQGLQFXEDWLQJDW&RYHUQLJKW$IWHUWZRVWHSVRIPLQDW&LQD ZDVKLQJEXIIHUP01D&O>RUP0ZKHQK\EULGL]LQJZLWKIRUPDPLGH@P0('7$ 6'6P07ULV+&O>S+@WKH¿OWHUVZHUHHTXLOLEUDWHGLQ3%6IRUPLQDWURRP WHPSHUDWXUH577\UDPLGHVLJQDODPSOL¿FDWLRQZDVGRQHIRUPLQDW57LQWKHGDUNLQDVROXWLRQ containing 1x PBS, 2 M NaCl, 1 mg ml blocking reagent, 100 mg ml dextran sulfate, 0.0015% H2O2DQGJPO$OH[DRUJPO$OH[DODEHOHGW\UDPLGH)LOWHUVZHUHWUDQVIHUUHGWR 3%6EXIIHUIRUPLQULQVHGZLWKGLVWLOOHGZDWHUDQGDLUGULHG3UHSDUDWLRQVZHUHFRXQWHUVWDLQHG ZLWK¶GLDPLGLQRSKHQ\OLQGROH'$3,DWJPO–1, mounted in antifading reagent (77% JO\FHURO9(&7$6+,(/'DQG[3%6DQGNHSWIUR]HQXQWLOPLFURVFRS\1RSUREH controls were carried out at surface and DCM in all stations to verify the absence of endogenous peroxidases in these samples. Hybridization conditions for probe 3(/$ were optimized using different formamide concentrations in the buffer. At 20% formamide, bright FISH signals were obtained in pelagophyte cultures used as positive controls (Pelagomonas calceolata [CCMP1214], Aureoumbra lagunensis >&&03@DQGAureococcus anophagefferens [CCMP1706]) while no hybridization signal was seen in other marine algal cultures used as negative controls (Pinguiococcus pyrenoidosus [CCMP1144] from Pinguiophyceae, Rhizocromulina sp. [&&03@IURP 'LFW\RFKRSK\FHDH and Ochromonas sp. [CCMP584 and RCC21] from Chrysophyceae). 'RXEOH&$5'),6+IRUSHODJRSK\WHV 3UREH3(/$ZDV LQLWLDOO\ WHVWHGLQHQYLURQPHQWDO VDPSOHV IURPWKH1:0HGLWHUUDQHDQDW WKH'&0GHSWKDQGEHORZUHVXOWLQJLQXQVSHFL¿FVLJQDOVDQGIDLQWÀXRUHVFHQFHRIWDUJHWFHOOV 7R VROYHWKLVZH GLGDGRXEOH&$5'),6+IRU SHODJRSK\WHVE\FRPELQLQJSUREHV3(/$ DJDLQVW HXNDU\RWLF F\WRSODVP DQG 3(/$ DJDLQVW FKORURSODVWV 3UREH 3(/$ ZDV tested on pelagophytes cultures and cultures from other marine algal classes and bright and clear ÀXRUHVFHQFHVLJQDOORFDOL]HGLQWKHFKORURSODVWZDVREWDLQHGRQO\ZLWKWKHSHODJRSK\WHFXOWXUHV Pelagophytes in INDEMARES samples were detected by a double hybridization procedure, using SUREHV 3(/$ DPSOL¿HG ZLWK$OH[D W\UDPLGH DQG 3(/$ DPSOL¿HG ZLWK$OH[D W\UDPLGH7KHGRXEOHK\EULGL]DWLRQZDVSHUIRUPHGLQVHTXHQFHDVSURSRVHGLQ3HUQWKDOHUet al.IRU³0XOWLFRORU&$5'),6+´$IWHUWKH¿UVWK\EULGL]DWLRQWKH3(/$SHUR[LGDVHV were inactivated with 0.01M HCl for 10 minutes at RT in the dark. Filter sections were then rinsed WZLFHZLWK0LOOL4ZDWHUDQGDLUGULHGEHIRUHVWDUWLQJWKHVHFRQGK\EULGL]DWLRQ:HWULHGGLIIHUHQW FRPELQDWLRQVRISUREHFRORUDQGDSSOLFDWLRQRUGHULQPelagomonas calceolata and the proposed combination nicely provided separated colored signals at different parts of the cell. Photo-picoeukaryotes community structure in the DCM 54 &RXQWLQJQDWXUDOFHOOV Filters were observed E\ HSLÀXRUHVFHQFH PLFURVFRS\ 2O\PSXV %; DW [ DW GLIIHUHQW excitations: UV (DAPI signal), blue light (Alexa 488 signal, green emission) or green light (Alexa VLJQDOUHGHPLVVLRQ&RXQWVRIK\EULGL]HGFHOOVZHUHGRQHIURPUDQGRPO\FKRVHQ¿HOGV [PRUZKHQFHOOGHQVLWLHVZHUHORZHUWKDQaFHOOVSHU¿HOGIURPWZRWUDQVHFWVa PP[PDFURVVWKH¿OWHUVHFWLRQ'XHWRWKHODUJHQXPEHURIVDPSOHVDQGWKHWLPHQHHGHG IRUHDFKFRXQWZHGLGQRWFRXQWUHSOLFDWHVIRUHDFKVDPSOH,QVWHDGZHFDOFXODWHGWKHFRHI¿FLHQW of variation (CV) in two samples displaying high (~1700 cells mlDQGORZaFHOOVPO) DEXQGDQFHRISHODJRSK\WHV7KUHH¿OWHUVHFWLRQVZHUHK\EULGL]HGLQWZRUHSOLFDWH¿OWHUVDWRWDO of six hybridizations per sample), yielding CV ranging from 24% (low abundance) to 18% (high DEXQGDQFH+\EULGL]HGFHOOVZHUHFODVVL¿HGLQWR¿YHVL]HFODVVHVDQG!P E\YLVXDOPHDVXUHPHQWVXVLQJDQRFXODUPLFURPHWHUPLQFUHPHQWV7KHW\SLFDOELRYROXPH RIHDFKVL]HFODVVZDVWDNHQDVWKHDYHUDJHRIWKHELRYROXPHRIWKHWZRVL]HOLPLWVDVVXPLQJD sphere. Biovolumes were converted to carbon biomass following the formula: pgC cell = 0.261* YRO>LQP])0.860 0HQGHQ'HXHUDQG/HVVDUG. $FNQRZOHGJHPHQWV Financial support has been provided by the Spanish Ministry of Economy and Competitivity WKURXJK SURMHFW )$0262 &70&0$5 WR 0/ 0(),672 &70 3WR50DQG0,&52%)32&($1(8WR&3$$0&DEHOORZDV UHFLSLHQWRID6SDQLVK)3,JUDQW%(6:HWKDQN)6iQFKH]IRULQYLWLQJXVWR participate in the INDEMARES 0710 cruises and the chief scientists, technicians, researchers and FUHZIRUWKHLUFROODERUDWLRQ:HWKDQN*6DOD]DUDQG)0&RUQHMR&DVWLOORIRUGDWDWUHDWPHQW advice. Chapter 1 55 Depth (m) 0 20 40 60 80 temperature (ºC) 10 14 18 22 NO30246 0,0 0,1 0,2 0,3 0,4 0,5 0,6 0,0 0,5 1,0 1,5 2,0 2,5 3,0 10 14 18 22 fluorescence ru 0246810 Depth (m) 0 20 40 60 80 0246810 (µM) PO43- (µM) Si(OH)4(µM) fluorescence ru temperature (ºC) NO30246 0,0 0,1 0,2 0,3 0,4 0,5 0,6 0,0 0,5 1,0 1,5 2,0 2,5 3,0 (µM) PO43- (µM) Si(OH)4(µM) St A St B St C St D Fig. S1. Vertical profiles of inorganic nutrients (silicate, phosphate and nitrate) at each station, together with temperature and fluorescence profiles. Supplementary material Figure S1. 9HUWLFDOSUR¿OHVRILQRUJDQLFQXWULHQWVVLOLFDWHSKRVSKDWHDQGQLWUDWHDWHDFKVWDWLRQ WRJHWKHUZLWKWHPSHUDWXUHDQGÀXRUHVFHQFHSUR¿OHV Photo-picoeukaryotes community structure in the DCM 56 Figure S2. &RQWULEXWLRQRIFHOOVL]HFODVVHVRIHDFKJURXSDORQJWKHYHUWLFDOSUR¿OHV prymnesiophytes depth (m) 0 10 20 30 40 50 60 70 Contibution of size classes (%) 020406080 100 depth (m) 0 10 20 30 40 50 60 70 80 < 2 µm 2-3 µm 3-4 µm 4-5 µm > 5 µm 020406080 100 Contibution of size classes (%) St A St B St C St D Fig. S2. Contribution of cell size classes of each group along the vertical profiles. Chapter 1 57 depth (m) 0 10 20 30 40 50 60 70 Contribution of size classes (%) 020406080 100 depth (m) 0 10 20 30 40 50 60 70 80 020406080 100 Contribution of size classes (%) pelagophytes St A St B St C St D Fig. S2. cont. < 2 µm 2-3 µm 3-4 µm 4-5 µm > 5 µm Figure S2. cont. Photo-picoeukaryotes community structure in the DCM 64 3LZRV].6SLFK.&DáNLHZLF]-:H\GPDQQ$.XELV]\Q$0DQG:LNWRU-0 'LVWULEXWLRQRIVPDOOSK\WRÀDJHOODWHVDORQJDQ$UFWLFIMRUGWUDQVHFWEnviron Microbiol 17:  2406. Rappé, M.S., Suzuki, M.T., Vergin, K.L., and Giovannoni, S.J. (1998) Phylogenetic diversity of XOWUDSODQNWRQSODVWLGVPDOOVXEXQLWU51$JHQHVUHFRYHUHGLQHQYLURQPHQWDOQXFOHLFDFLGVDPSOHV IURPWKH3DFL¿FDQG$WODQWLFFRDVWVRIWKH8QLWHG6WDWHVAppl Environ Microbiol 64± Rodríguez, F., Derelle, E., Guillou, L., Le Gall, F., Vaulot, D., and Moreau, H. (2005) Ecotype diversity in the marine picoeukaryote Ostreococcus (Chlorophyta, Prasinophyceae). Environ Microbiol 7± Romari, K. and Vaulot, D. (2004) Composition and temporal variability of picoeukaryote communities at a coastal site of the English Channel from 18S rDNA sequences. Limnol Oceanogr 49: 784–798. Roy, S., Llewellyn, C.A., Egeland, E.S., and Johnsen, G. (2011) Phytoplankton pigments: Characterization, chemotaxonomy and applications in oceanography. Cambridge University Press: Cambridge. Sharples, J., Moore, C.M., Rippeth, T.P., Holligan, P.M., Hydes, D.J., Fisher, N.R., and Simpson, J.H. (2001) Phytoplankton distribution and survival in the thermocline. Limnol Oceanogr 46: 486–496. Shi, X.L., Marie, D., Jardillier, L., Scanlan, D.J., and Vaulot, D. (2009) Groups without cultured UHSUHVHQWDWLYHV GRPLQDWH HXNDU\RWLF SLFRSK\WRSODQNWRQ LQ WKH ROLJRWURSKLF 6RXWK (DVW 3DFL¿F Ocean. PLoS One 4: e7657. Shi, X.L., Lepère, C., Scanlan, D.J., and Vaulot, D. (2011) Plastid 16S rRNA gene diversity among HXNDU\RWLFSLFRSK\WRSODQNWRQVRUWHGE\ÀRZF\WRPHWU\IURPWKH6RXWK3DFL¿F2FHDQPLoS One 6: e18979. Simon, N., Campbell, L., Ornolfsdottir, E., Groben, R., Guillou, L., Lange, M., and Medlin, /.2OLJRQXFOHRWLGHSUREHVIRUWKHLGHQWL¿FDWLRQRIWKUHHDOJDOJURXSVE\GRWEORWDQG ÀXRUHVFHQWZKROHFHOOK\EULGL]DWLRQJ Euk Microbiol 47: 76–84. Suzuki, K., Minami, C., Liu, H., and Saino, T. (2002) Temporal and spatial patterns of FKHPRWD[RQRPLF DOJDO SLJPHQWV LQ WKH VXEDUFWLF 3DFL¿F DQG WKH %HULQJ 6HD GXULQJ WKH HDUO\ summer of 1999. Deep Sea Res II 49: 5685 –5704. 7LPPHUPDQV.5YDQGHU:DJW%9HOGKXLV0-:0DDWPDQ$DQGGH%DDU+-: 3K\VLRORJLFDOUHVSRQVHVRIWKUHHVSHFLHVRIPDULQHSLFRSK\WRSODQNWRQWRDPPRQLXPSKRVSKDWH iron and light limitation. J Sea Res 53: 109–120. Chapter 1 65 8UGHD 06 :DUQHU %' 5XQQLQJ -$ 6WHPSLHQ 0 &O\QH - DQG +RUQ 7 $ FRPSDULVRQRIQRQUDGLRLVRWRSLFK\EULGL]DWLRQDVVD\PHWKRGVXVLQJÀXRUHVFHQWFKHPLOXPLQHVFHQW and enzyme labeled synthetic oligodeoxyribonucleotide probes. Nucleic Acids Res 16± 9DXORW'(LNUHP:9LSUH\0DQG0RUHDX+7KHGLYHUVLW\RIVPDOOHXNDU\RWLF SK\WRSODQNWRQPLQPDULQHHFRV\VWHPVFEMS Microbiol Rev 32: 795–820. :RUGHQ$=1RODQ-.DQG3DOHQLN%$VVHVVLQJWKHG\QDPLFVDQGHFRORJ\RIPDULQH picophytoplankton: the importance of the eukaryotic component. Limnol Oceanogr 49: 168–179. :RUGHQ$=-DQRXVNRYHF-0F5RVH'(QJPDQ$:HOVK500DOIDWWL6et al. (2012) Global distribution of a wild alga revealed by targeted metagenomics. Curr Biol 22: R675–677. Zubkov, M. V., Sleigh, M.A., Burkill, P.H., and Leakey, R.J.G. (2000) Picoplankton community structure on the Atlantic Meridional Transect: a comparison between seasons. Prog Oceanogr 45: ± Chapter 2 Chapter 2 69 Chapter 2 Distribution of phytoplankton groups within the deep chlorophyll maximum Mikel Latasa, Ana M. Cabello, Xosé Anxelu G. Morán, Ramon Massana and Renate Scharek. 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Sample location, parameters analyzed and main characteristics of the DCM layer. Cast Coordinates (W, N) Date Parameters analyzed Depth (m) Chl a max (mg m-3) Width (m) Asymmetry 48 06.2745, 43.7565 29/07 FCM, HPLC 32 1.59 11 -0.048 52 06.2693, 44.0028 30/07 FCM, HPLC, Nutrients, FISH, Microscopy 54 0.628 39 -0.109 55 06.3852, 43.7555 31/07 FCM, HPLC, Nutrients 22 1.87 17 0.252 66 06.2718, 44.1720 02/08 FCM, HPLC 59 0.411 65 0.390 73 05.6977, 44.0425 04/08 FCM, HPLC, Nutrients, FISH, Microscopy 42 1.22 13 0.886 75 11.2572, 43.1665 07/08 FCM, HPLC 54 0.417 32 -0.020 80 12.3895, 43.1682 08/08 FCM, HPLC, Nutrients, FISH, Microscopy 56 0.449 36 0.218 83 11.7078, 42.9993 09/08 FCM, HPLC, Nutrients, FISH 52 0.507 27 -0.053 88 12.1622, 42.8353 10/08 FCM, HPLC 65 0.616 26 0.082 92 11.4838, 42.8332 11/08 FCM, HPLC 38 0.487 24 0.287 101 12.1630, 42.6675 13/08 FCM, HPLC, Nutrients 58 0.474 30 0.068 Variables analyzed )RUQXWULHQWDQDO\VLVP/RIXQ¿OWHUHGVDPSOHZHUHIUR]HQDQGNHSWDW&LPPHGLDWHO\DIWHU VDPSOLQJ,QRUJDQLFQXWULHQWVZHUHDQDO\]HGE\VWDQGDUGQXWULHQWWHFKQLTXHVZLWKD6.$/$56DQ 3OXV$XWRDQDO\]HU'HWHFWLRQOLPLWVIRUWKHGLIIHUHQWPROHFXOHVZHUHLQPPROP12), 1232DQG6L2). )RUÀRZF\WRPHWU\DQDO\VLVP/RIVDPSOHZHUH¿[HGZLWKSDUDIRUPDOGHK\GHSOXV JOXWDUDOGHK\GHÀDVKIUR]HQLQOLTXLGQLWURJHQDQGNHSWDWí&7KHDQDO\VLVZDVSHUIRUPHG ZLWKD)$&6&DOLEXUÀRZF\WRPHWHU%'%LRVFLHQFHVHTXLSSHGZLWKDODVHUHPLWWLQJDWQP 7KHÀRZUDWHZDVFDOLEUDWHGGDLO\E\ZHLJKWWRHVWLPDWHFHOOFRQFHQWUDWLRQV$VROXWLRQRIȝP ÀXRUHVFHQWODWH[EHDGVUHI)0ROHFXODU3UREHVZDVDGGHGDVDQLQWHUQDOVWDQGDUGVRWKDW DOOFHOOXODUYDULDEOHVZHUHUHODWHGWRÀXRUHVFHQWEHDGVYDOXHV Prochlorococcus and Synechococcus ÀRZF\WRPHWU\FHOOFRXQWVZHUHFRQYHUWHGWRFDUERQ&DIWHUHVWLPDWLQJWKHLUFHOOYROXPHIURPWKH UHODWLYHVLGHVFDWWHUVLJQDODVGHVFULEHGLQ&DOYR'tD]DQG0RUiQDQGXVLQJWKHDYHUDJH YDOXHRIIJ&P:RUGHQet al. Phytoplankton groups within the DCM 80 Figure 4.9HUWLFDOSUR¿OHVRISLJPHQWV¿OOHGFLUFOHVERWWRP[D[LVQJ/DQGFHOOFRXQWVHPSW\FLUFOHVDQGWULDQJOHVWRS[D[LVFHOOP/PHDVXUHGE\ÀRZF\WRPHWU\ ),6+ DQG OLJKW PLFURVFRS\ 3HU SHULGLQLQ )XFR IXFR[DQWKLQ ¶KH[ ¶KH[DQR\OR[\IXFR[DQWKLQ ¶EXW ¶EXWDQR\OR[\IXFR[DQWKLQ 9LRO YLROD[DQWKLQ =HD ]HD[DQWKLQ'9DGLYLQ\OFKORURSK\OOa, Chl aFKORURSK\OOa6\QSynechococcus'LQRGLQRÀDJHOODWHV'LDWGLDWRPV&RFFRFRFFROLWKRSKRULGV3U\PVPDOOP SU\PQHVLRSK\WHV3HODJSHODJRSK\WHV&KORURFKORURSK\WHV3URProchlorococcus.1RWHWKHFKDQJHVLQVFDOHV)OXRUHVFHQFHSUR¿OHVDUHSUHVHQWHGDVJUH\DUHDVIRUUHIHUHQFH ZLWKRXWVFDOHWRVKRZWKHVKDSHRIWKHSUR¿OH,QWKH¶KH[FRFFRVDQG3U\PSORWVWKHRIIVHWWRS[D[LVFRUUHVSRQGVWRFHOOFRQFHQWUDWLRQVRIVPDOOSU\PQHVLRSK\WHV Chapter 2 81 Figure 4. 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Parameters of photoacclimation for pigment cell-1 and pigment C-1 versus percent irradiance. See text for equation. P max P min k R2 Mean±S.D. DVChl a per Pro fg cell-1 mg g-1 4.2734 26.93 0.3264 12.51 1.077 0.2745 0.87 0.47 1.50±1.14 21.3±6.05 Zeax per Syn fg cell-1 mg g-1 0.7118 --- 0.4856 --- 0.4018 --- 0.11 --- 0.631±0.166 4.90±1.30 Viol per chloro fg cell-1 mg g-1 --- --- --- --- --- --- --- --- 1.38±0.84 1.81±1.02 19'but-fuco per pelago fg cell-1 mg g-1 119.98 68.22 62.77 30.17 0.878 0.892 0.37 0.49 82.3±24.2 43.0±13.9 19'hex-fuco per prym fg cell-1 mg g-1 301.6 85.28 67.1 15.31 0.144 0.4215 0.32 0.87 193.8±78.9 48.4±22.8 Per per dino fg cell-1 462.17 50.57 0.2153 0.45 333±139 Fuco per diat fg cell-1 2809.6 827.6 0.6713 0.71 1659±643 *Violaxanthin per chlorophyte versus irradiance failed to adjust. Zeaxanthin per C-1 in Synechococcus failed to adjust. Changes in pigment per C content were not calculated for dinoflagellates and diatoms. 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I 50± Phytoplankton groups within the DCM 98 9HOGKXLV0DQG.UDD\*9HUWLFDOGLVWULEXWLRQDQGSLJPHQWFRPSRVLWLRQRIDSLFRSODQ NWRQLFSURFKORURSK\WHLQWKHVXEWURSLFDO1RUWK$WODQWLFDFRPELQHGVWXG\RI+3/&DQDO\VLVRI SLJPHQWVDQGÀRZF\WRPHWU\Mar. Ecol. Prog. Ser. 68± 9HQULFN(/7KHYHUWLFDOGLVWULEXWLRQRIFKORURSK\OODQGSK\WRSODQNWRQVSHFLHVLQWKH 1RUWK3DFL¿FFHQWUDOHQYLURQPHQWJ. Plankton Res. 10± 9HQULFN(/3K\WRSODQNWRQVSHFLHVVWUXFWXUHLQWKHFHQWUDO1RUWK3DFL¿F YDULDELOLW\DQGSHUVLVWHQFHJ. Plankton Res. 21± :DLWH$)LVKHU$7KRPSVRQ3DDQG+DUULVRQ3-6LQNLQJUDWHYHUVXVFHOOYROXPH UHODWLRQVKLSVLOOXPLQDWHVLQNLQJUDWHFRQWUROPHFKDQLVPVLQPDULQHGLDWRPVMar. Ecol. Prog. Ser. 157± :RUGHQ$=1RODQ--.DQG3DOHQLN%$VVHVVLQJWKHG\QDPLFVDQGHFRORJ\RIPDULQH SLFRSK\WRSODQNWRQ7KHLPSRUWDQFHRIWKHHXNDU\RWLFFRPSRQHQWLimnol. Ocean. 49± :ULJKW6:DQG0DQWRXUD5)&*XLGHOLQHVIRUFROOHFWLRQDQGSLJPHQWDQDO\VLVRI¿HOG VDPSOHV,Q-HIIUH\6:0DQWRXUD5)&DQG:ULJKW6:HGVPhytoplankton pigments in oceanographhy: guidelines to modern methods81(6&23DULVSS± =DSDWD05RGULJXH])DQG*DUULGR-/6HSDUDWLRQRIFKORURSK\OOVDQGFDURWHQRLGV IURPPDULQHSK\WRSODQNWRQDQHZ+3/&PHWKRGXVLQJDUHYHUVHGSKDVH&FROXPQDQGS\ULGLQH FRQWDLQLQJPRELOHSKDVHVMar. Ecol. Prog. Ser. 195± =XENRY09DQG7DUUDQ*$+LJKEDFWHULYRU\E\WKHVPDOOHVWSK\WRSODQNWRQLQWKH 1RUWK$WODQWLF2FHDQNature 455± Chapter 3 Chapter 3 101 Chapter 3 Global distribution and vertical patterns of a prymnesiophytecyanobacteria obligate symbiosis Summary A marine symbiosis has been recently discovered between prymnesiophyte species and the unicellular diazotrophic cyanobacterium UCYN-A. At least 2 different UCYN-A phylotypes exist, the clade UCYN-A1 in symbiosis with an uncultured small prymnesiophyte and the clade UCYN-A2 in symbiosis with the larger Braarudosphaera bigelowii. We targeted the prymnesiophyte–UCYN-A1 symbiosis by double CARD-FISH (CAtalyzed Reporter DepositionFluorescence In Situ Hybridization) and analyzed its abundance in surface samples from the 0$/$63,1$FLUFXPQDYLJDWLRQH[SHGLWLRQ2XUXVHRIDVSHFL¿FSUREHIRUWKHSU\PQHVLRSK\WH partner allowed us to verify that this algal species virtually always carried the UCYN-A symbiont, indicating that the association was also obligate for the host. The prymnesiophyte–UCYN-A1 symbiosis was detected in all ocean basins, displaying a patchy distribution with abundances (up to 500 cells ml-1WKDWFRXOGYDU\RUGHUVRIPDJQLWXGH$GGLWLRQDOYHUWLFDOSUR¿OHVWDNHQDWWKH1( Atlantic showed that this symbiosis occupied the upper water column and disappeared towards WKH'HHS&KORURSK\OO0D[LPXPZKHUHWKHELRPDVVRIWKHSU\PQHVLRSK\WHDVVHPEODJHSHDNHG Moreover, sequences of both prymnesiophyte partners were searched within a large 18S rDNA metabarcoding dataset from the Tara-Oceans expedition around the world. This sequence-based analysis supported the patchy distribution of the UCYN-A1 host observed by CARD-FISH, and highlighted an unexpected homogeneous distribution (at low relative abundance) of B. bigelowii in the open ocean. Our results demonstrate that partners are always in symbiosis in nature and show contrasted ecological patterns of the two related lineages. Ana M. Cabello, Francisco M. Cornejo-Castillo, Nicolas Raho, Dolors Blasco, Montserrat Vidal, Stéphane Audic, Colomban de Vargas, Mikel Latasa, Silvia G. Acinas and Ramon Massana (2015). Global distribution and vertical patterns of a prymnesiophyte-cyanobacteria obligate symbiosis. ISME J. Doi: 10.1038/ismej.2015.147 Chapter 3 103 Introduction 6\PELRVLVEHWZHHQF\DQREDFWHULDDQGHXNDU\RWLFRUJDQLVPVLVDZLGHVSUHDGSKHQRPHQRQUHSRUWHG both in land and in aquatic systems (Rai et al., 2002). In the marine environment, cyanobacteria appear associated with multicellular organisms, such as ascidians and sponges, and with singleFHOOHG RUJDQLVPV VXFK DV GLDWRPV GLQRÀDJHOODWHV UDGLRODULDQV DQG WLQWLQQLGV &DUSHQWHU DQG Foster, 2002; Foster et al.  7KHVH ¿QGLQJV GHULYH IURP PLFURVFRSLF REVHUYDWLRQV DQG RIWHQWKHPXWXDOEHQH¿WEHWZHHQWKHKRVWDQGWKHV\PELRQWLVSRRUO\XQGHUVWRRG*HQHUDOO\LWLV assumed that the cyanobacteria provides organic carbon to the host through photosynthesis, while the host provides a stable and secure environment. Diazotrophic cyanobacteria symbionts can in addition provide nitrogen-derived metabolites through N2¿[DWLRQDVKDVEHHQGHPRQVWUDWHGLQ some diatom species (Foster et al., 2011). A related and particular symbiosis has been recently described between two uncultured SLFRSODQNWHUVDVPDOOȝPSU\PQHVLRSK\WHDQGWKHXQLFHOOXODUGLD]RWURSKLFF\DQREDFWHULD UCYN-A (Thompson et al.  *HQRPLFV UHYHDOHG WKDW 8&<1$ ODFNHG NH\ PHWDEROLF pathways commonly shared by diazotrophic cyanobacteria, such as oxygenic photosynthesis and the Calvin cycle, suggesting it was an obligate symbiont (Zehr et al., 2008; Tripp et al., 2010). Indeed, QDQR6,06GHPRQVWUDWHGWKDWWKHV\PELRQWJDYH¿[HGQLWURJHQWRWKHKRVWDQGREWDLQHGRUJDQLF carbon in return (Thompson et al.7KH6U'1$SU\PQHVLRSK\WHVHTXHQFHLGHQWL¿HG by single cell analysis (Thompson et al., 2012), was identical to an environmental sequence from WKH6RXWK3DFL¿F6KLet al., 2009) and related (98.2%) to Braarudosphaera bigelowii, a coastal QDQRSODQNWRQLFFRFFROLWKRSKRUH+DJLQRet al., 2009). Later, endosymbiotic UCYN-A was also discovered in B. bigelowii E\7(0 REVHUYDWLRQV+DJLQR et al.  3K\ORJHQHWLFDQDO\VHV using the UCYN-A nitrogenase (nifH) gene revealed at least three distinct clades, UCYN-A1, $DQG$7KRPSVRQet al.DQGKLJKOLJKWHGVSHFL¿FLW\EHWZHHQKRVWDQGV\PELRQW pairings, suggesting co-evolution between symbionts and hosts (Bombar et al., 2014; Thompson et al., 2014). Thus, the clade UCYN-A1 was associated with an open ocean small prymnesiophyte (hereafter UCYN-A1 host), and the clade UCYN-A2 was associated with the coastal and larger B. bigelowiiKHUHDIWHU8&<1$KRVWZKLOVWQRKRVWKDVEHHQ\HWSURSRVHGIRU8&<1$ (QYLURQPHQWDOVXUYH\VRInifH genes indicated a rather global distribution of this symbiosis in the oceans (Moisander et al., 2010). Further reports showed that the distinct UCYN-A clades were widespread and often coexisted (Thompson et al., 2014). Nevertheless, microscopic observations of the symbiosis or sequencing data of the prymnesiophytes verifying this widespread distribution are still scarce. Recently, a double CARD-FISH (CAtalyzed Reporter Deposition-Fluorescence In Situ Hybridization) approach targeting UCYN-A and prymnesiophyte cells, allowed visualization DQGTXDQWL¿FDWLRQRIWKLVDVVRFLDWLRQLQWKH1RUWK$WODQWLF.UXSNHet al., 2014a,b). Later, the VDPHGRXEOH&$5'),6+WHFKQLTXHEXWVSHFL¿FDOO\WDUJHWLQJWKHWZRGLIIHUHQWSU\PQHVLRSK\WH Distribution of a prymnesiophyte–UCYN-A symbiosis 104 host phylotypes made it possible to distinguish both types of associations and to observe their cooccurrence in the subtropical South Atlantic (Cornejo-Castillo et al., submitted). The emerging picture derived from the above studies was that the prymnesiophyte–UCYN-A association showed a tendency for being more abundant in oligotrophic warm waters, but the environmental drivers explaining its distribution are still poorly understood. In this sense, quantifying the symbiosis by both CARD-FISH and 18S rDNA metabarcoding in a large set of samples covering a wide geographic range help evaluating accurately its distribution in marine ecosystems. ,QWKLVZRUNZHVWXGLHGWKHGLVWULEXWLRQRI8&<1$DQG8&<1$KRVWVLQVDPSOHVUHSUHVHQWDWLYH of the World’s major oceans. First, we targeted the prymnesiophyte–UCYN-A1 symbiosis by GRXEOH &$5'),6+ XVLQJ WKH VSHFL¿F SUREH IRU WKH 8&<1$ KRVW &RUQHMR&DVWLOOR et al., VXEPLWWHGDQGWKHJHQHUDOSUREH8&<1$WDUJHWLQJDOOFODGHVRI8&<1$.UXSNHet al., 7KLV),6+VHWXSZDVXVHGWRTXDQWLI\FHOODEXQGDQFHVLQVXUIDFHZDWHUVWUDFNHGGXULQJ WKH0$/$63,1$H[SHGLWLRQWRDQDO\]HYHUWLFDOGLVWULEXWLRQVLQWKH1($WODQWLFQHDUWKH,EHULDQ Peninsula, and to evaluate whether the host can appear without the cyanobacteria. Second, we analyzed the global distribution of both hosts within an 18S rDNA metabarcoding dataset from the Tara-Oceans expedition sampled at surface and DCM depths (de Vargas et al., 2015). The aim RIWKLVZRUNZDVWRFRQ¿UPWKHEURDGGLVWULEXWLRQRIWKHSU\PQHVLRSK\WH±8&<1$DVVRFLDWLRQ evaluate its putative ecological niche and provide further evidence about the obligatory dependence of both symbiont and host. Materials and methods Oceanographic cruises We analyzed data from three independent cruises. The MALASPINA circumnavigation expedition WRRNSODFHIURP'HFHPEHUWR-XO\RQERDUGWKH59HespéridesDQGWUDFNHGVXEWURSLFDO DQG WURSLFDO ODWLWXGHV RI WKH$WODQWLF ,QGLDQ DQG 3DFL¿F RFHDQV VHH )LJXUH 6D IRU D PDS RI stations). The Tara2FHDQVH[SHGLWLRQ.DUVHQWLet al.WRRNSODFHIURP6HSWHPEHUWR March 2012 on board the TaraVFKRRQHUDQGHQFRPSDVVHGDGLIIHUHQWWUDFNRQWKHVDPHRFHDQV SOXVWKH0HGLWHUUDQHDQ6HDDQGVXEDQWDUFWLFZDWHUV)LJXUH6E7KH,1'(0$5(6FUXLVH WRRNSODFHLQVXPPHU-XO\$XJXVWRQERDUGWKH59Thalassa ,)5(0(5,(2 LQWKH1($WODQWLFQHDUWKH,EHULDQSHQLQVXOD)LJXUH6D)RU0$/$63,1$DQG,1'(0$5(6 sampling and acquisition of hydrological variables are detailed below. Seawater sampling, DNA extract collection and 18S rDNA sequencing for the Tara-Oceans expedition is described in detail elsewhere (de Vargas et al., 2015). Chapter 3 105 Sampling procedures during MALASPINA and INDEMARES cruises 'XULQJ WKH 0$/$63,1$ FUXLVH VXUIDFH VDPSOHV a P IRU &$5'),6+ DQG FKORURSK\OO D DQDO\VLVZHUHFROOHFWHGZLWKD/1LVNLQERWWOHDWDURXQGQRRQLQVWDWLRQVPLQLPXPERWWRP depth ~2 000 m). Conductivity-Temperature-Depth (CTD) casts were performed from surface to PHVRSHODJLFGHSWKVZLWKD6HDELUG3OXVSUREHPRXQWHGRQDERWWOH1LVNLQURVHWWHDQGZDWHU ZDVWDNHQDWVHYHUDOGHSWKVIRUQXWULHQWDQDO\VLV,QWKH,1'(0$5(6FUXLVHYHUWLFDOSUR¿OHV ZHUHWDNHQDWRIIVKRUHVWDWLRQVLQWKH$YLOéVFDQ\RQVWDWLRQV$%DQGWKH*DOLFLDQ%DQNVWDWLRQV &'&DVWVZHUHSHUIRUPHGDIWHUVXQVHWZLWKD&7'SUR¿OHU¿WWHGZLWKD)OXRURPHWHU:(7ODEV (&2$)/DQGDERWWOH1LVNLQURVHWWH%DVHGRQWKHGRZQFDVWÀXRUHVFHQFHSUR¿OHVHDZDWHU was collected at 7-8 depths from below the Deep Chlorophyll Maximum (DCM) to surface (~5 m), with higher frequency above the DCM. For chlorophyll a determination, aliquots of 250 ml 0$/$63,1$ RU  / ,1'(0$5(6 ZHUH ¿OWHUHG WKURXJK :KDWPDQ *)) ¿OWHUV  PP diameter) and stored at -20ºC until extraction on board or in the lab, respectively. Samples for LQRUJDQLF QXWULHQWVZHUH GUDZQLQWR SRO\HWK\OHQHYLDOV DQGNHSW IUR]HQXQWLO DQDO\VLV)RUWKH &$5'),6+DVVD\PODOLTXRWVIRU0$/$63,1$RUPODOLTXRWVIRU,1'(0$5(6ZHUH ¿[HGZLWKIRUPDOGHK\GHRU¿QDOFRQFHQWUDWLRQUHVSHFWLYHO\¿OWHUHGRQP SRUHVL]H1XFOHRSRUH¿OWHUVPPGLDPHWHUDQGNHSWIUR]HQXQWLOSURFHVVHG Chlorophyll a and inorganic nutrient measurements 'XULQJ0$/$63,1$SLJPHQWVZHUHH[WUDFWHGE\SODFLQJWKH¿OWHUVLQPORIDFHWRQH DW&IRUKDQGGHWHUPLQLQJWKHÀXRUHVFHQFHRIWKHH[WUDFWLQD7XUQHU'HVLJQVÀXRURPHWHU <HQWVFKDQG0HQ]HO$&KORURSK\OODVWDQGDUG6LJPD$OGULFKZDVXVHGWRFDOLEUDWHWKH ÀXRURPHWHUDQGQRSKDHRSK\WLQFRUUHFWLRQZDVDSSOLHG)RU,1'(0$5(6VDPSOHVSLJPHQWV ZHUHH[WUDFWHGZLWKDFHWRQHVRQLFDWHGNHSWDW&IRUKDQGFOHDUHGE\¿OWUDWLRQWKURXJK *))¿OWHUV7RWDOFKORURSK\OODZDVGHWHUPLQHGE\+3/&IROORZLQJWKHSURFHGXUHGHVFULEHGE\ Latasa (2014). In MALASPINA samples, nitrate (NO -) concentration was measured spectrophotometrically ZLWKD 6NDODUDXWRDQDO\]HU 6NDODU6$1SOXV IROORZLQJVWDQGDUG SURFHGXUHV*UDVVKRIIet al., 1999; Moreno-Ostos, 2012), and phosphate (PO4 ) concentration was measured manually with a 3HUNLQ(OPHUVSHFWURSKRWRPHWHU)RU,1'(0$5(6ERWKQXWULHQWVZHUHPHDVXUHGZLWKD6NDODU DXWRDQDO\]HU'HWHFWLRQOLPLWVZHUHȝ0IRU12 -DQGȝ0IRU324 13UDWLRVZHUH FDOFXODWHGIURPWKHUDWLREHWZHHQVLJQL¿FDQWVORSHYDOXHVP < 0.05) of nitrate and phosphate across the nutricline. Dissolved organic nitrogen and phosphorus (DON and DOP) were estimated as the difference between total (TN and TP) and inorganic values. TN and TP concentrations ZHUHGHWHUPLQHGE\QLWUDWHDQGSKRVSKDWHPHDVXUHVGRQHDIWHUDONDOLQHDQGDFLGLFSHUVXOSKDWH R[LGDWLRQUHVSHFWLYHO\*UDVVKRIIet al., 1999; Moreno-Ostos, 2012). Distribution of a prymnesiophyte–UCYN-A symbiosis 112 Figure 4 Contribution of prymnesiophytes to total community reads (a) and contribution of UCYN-A1 host (b) and UCYN-A2 host (c) to total prymnesiophyte reads at surface and DCM depths of Tara-Oceans stations (V9 rDNA metabarcoding data). DCM depth was plotted in panel (c). Only stations having both surface and DCM samples are shown (29 out of 40). In the 11 stations not shown here, the percentage of prymnesiophytes to total community reads DWVXUIDFHZDVVLPLODURQDYHUDJH Prymnesiophytes 4791618222325303234363841425264656672767885 100 102 111 122 123 124 Reads contribution (%) 0 5 10 15 20 25 UCYN-A1 host 4791618222325303234363841425264656672767885 100 102 111 122 123 124 Reads contribution (%) 0 1 2 3 4 5 6 7 25 30 35 UCYN-A2 host Station (Tara-Oceans) 4791618222325303234363841425264656672767885 100 102 111 122 123 124 Reads contribution (%) 0 1 2 3 4 5 6 7 8 9 DCM depth (m) 0 20 40 60 80 100 120 140 160 surface DCM DCM depth a b c in samples inside and outside a given range of the environmental variables (Table 1). Abundances ZHUHVLJQL¿FDQWO\KLJKHULQVDPSOHVZLWKWHPSHUDWXUHVORZHUWKDQ&DQGSKRVSKDWHORZHUWKDQ ȝ01RGLIIHUHQFHVLQDEXQGDQFHVZHUHIRXQGEHWZHHQVDPSOHVZLWKQLWUDWHFRQFHQWUDWLRQV below and above the median value observed in all samples (0.27 ȝ0RUEHWZHHQVDPSOHVZLWK 13UDWLRVEHORZRUDERYH0RUHRYHUV\PELRVLVDEXQGDQFHVZHUHQRWVLJQL¿FDQWO\GLIIHUHQW LQROLJRPHVRRUHXWURSKLFZDWHUVDOWKRXJKWKH\ZHUHVLJQL¿FDQWO\ORZHULQXOWUDROLJRWURSKLF stations. Chapter 3 113 Figure 5 Cell counts of the prymnesiophyte–UCYN-A1 symbiosis versus (a) total chlorophyll, (b) temperature, (c) nitrate, (d) phosphate, (e13UDWLRf) DOP and (g) DON measured in surface waters during the MALASPINA (white GRWVDQG,1'(0$5(6EODFNGRWVFUXLVHV5HJUHVVLRQOLQHVDUHSORWWHGLQFDVHVGLVSOD\LQJDVLJQL¿FDQWUHODWLRQVKLS (P < 0.05). Vertical distribution pattern in the water column The vertical distribution of the prymnesiophyte–UCYN-A1 symbiosis was studied in detail in IRXUYHUWLFDOSUR¿OHVLQWKH1($WODQWLF)LJXUH$VWUDWL¿HGZDWHUFROXPQZDVREVHUYHGLQDOO FDVHVZLWKD'&0SHDNFRXSOHGWRWKHEHJLQQLQJRIWKHQXWULFOLQHDQGGHSWKVDERYHWKH'&0 KDYLQJORZQLWUDWHDQGSKRVSKDWHFRQFHQWUDWLRQVȝ012; 0.075 ȝ0324). At selected depths, symbiosis counts together with total prymnesiophyte counts were obtained by GRXEOHDQGVLQJOH&$5'),6+DVVD\VUHVSHFWLYHO\,QDOOSUR¿OHV8&<1$KRVWFHOOVRFFXSLHG the upper water column and decreased towards the DCM, where they disappeared. The decrease in V\PELRVLVDEXQGDQFHVDSSHDUHGPRUHFRXSOHGWRWKHEHJLQQLQJRIWKHÀXRUHVFHQFHJUDGLHQWWKDQ to the beginning of the nutricline. On the contrary, the other prymnesiophyte cells, not involved LQV\PELRVLVSHDNHGDWWKH'&0DQGZHUHSUHVHQWWKURXJKRXWWKHSKRWLFZDWHUFROXPQ6WDWLRQ %ODFNHGWKHVXUIDFHVDPSOHVEXWLWVYHUWLFDOSDWWHUQZDVFRQVLVWHQWZLWKWKHRWKHUWKUHHSUR¿OHV The abundance and contribution of the UCYN-A1 host varied between the two regions sampled. +LJKHVWDEXQGDQFHVZHUHVHHQLQWKHXSSHUZDWHUFROXPQRIVWDWLRQ$FHOOVPO-1 on average) in the Avilés canyon region, where they represented on average 70% of the prymnesiophyte cells XSWRDWP,QWKH*DOLFLDQ%DQNUHJLRQ8&<1$KRVWDEXQGDQFHVWRFHOOVPO-1 on average) had a lower contribution to total prymnesiophytes (8-16% on average). Malaspina cruise Indemares cruise Chla (mg m-3) 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 Log abundances (cells ml-1) 0.0 0.5 1.0 1.5 2.0 2.5 3.0 Temperature (ºC) 18 20 22 24 26 28 30 Phosphate (μM) 0.00 0.05 0.10 0.15 0.20 0.25 Nitrate (μM) 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 DOP (μM) 0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.35 DON (μM)N/P ratio 0.0 0.5 1.0 1.5 2.0 2.5 3.0 5 10 15 20 25 30 35 2 4 6 8 10 12 a b c d e f g y = -0.114x + 4.157 R2 = 0.168 y = -4.086x + 1.857 R2 = 0.171 Log abundances (cells ml-1) Distribution of a prymnesiophyte–UCYN-A symbiosis 114 Table 1: Averaged abundances of symbiosis inside and outside of a given range of values for temperature, phosphate, QLWUDWH13UDWLRDQGFKORURSK\OOa. Thresholds for temperature and inorganic nutrients were around the median value LQWKH0$/$63,1$DQG,1'(0$5(6VXUIDFHVDPSOHVLQRUGHUWRREWDLQDQHTXDOQXPEHURIVDPSOHVLQHDFK category. Chlorophyll a UDQJHVZHUHHVWDEOLVKHGDFFRUGLQJWRWKHWURSKLFZDWHUFODVVL¿FDWLRQRI6KXVKNLQDet al., 1997. LQGLFDWHVZKHQDEXQGDQFHVYDOXHVZHUHVLJQL¿FDQWO\GLIIHUHQW(P <0.05; Mann-Whitney test) inside and outside the corresponding range. abundances (cells ml-1) number of samples in range out range in range out range ---------------- ---------------- ---------- ---------- Variable Range Average SD Average SD n n Temperature < 25C (*) 21 120 7 25 33 29 PO4 3- < 0.08 M (*) 61 118 6 14 30 26 NO3 - < 0.27 M 57 119 24 67 28 29 N/P ratio < 16 72 135 13 22 26 18 TChla (1) < 0.06 mg m-3 (*) 3 17 43 18 9 52 TChla (2) 0.06-0.1 mg m-3 64 20 27 18 16 45 TChla (3) 0.1-0.3 mg m-3 35 19 39 18 30 31 TChla (4) > 0.3 mg m-3 28 11 38 19 6 55 (1) Ultraoligotrophic, (2) Oligotrophic, (3) Mesotrophic, (4) Eutrophic The vertical distribution of the symbiosis could also be inferred by the contribution of UCYN-A host reads in surface versus DCM samples of the Tara-Oceans cruise. Considering only the stations with high UCYN-A1 host abundance in surface samples (stations 76, 78 in the South Atlantic; VWDWLRQLQWKH*LEUDOWDU6WUDLWDQGVWDWLRQLQWKH5HG6HDWKHFRQWULEXWLRQDWWKH'&0ZDV about 150 times lower (Figure 4b). In the other stations where UCYN-A1 host was scarce at surface, it was equally scarce at the DCM. By contrast, UCYN-A2 host was detected at similar DEXQGDQFHLQVXUIDFHDQG'&0VDPSOHVLQDOOVWDWLRQVZKHUHLWDSSHDUHGDYHUDJHGVXUIDFH'&0 UDWLRRI)LJXUHF7KHFRQWULEXWLRQRI8&<1$KRVWZDVRQO\VLJQL¿FDQWO\ORZHU at the DCM (as compared with surface) in stations where the DCM was deeper than 60 m (MannWhitney Test; P = 0.001, n= 29), a difference not seen for total prymnesiophytes (Figure 4a). Chapter 3 115 Figure 6 'HSWKSUR¿OHVRIK\GURORJLFDOYDULDEOHVULJKWDQGGLVWULEXWLRQLQGHSWKRIWKHSU\PQHVLRSK\WH±8&<1$ V\PELRVLV EODFN GRWV DQG WKH UHVW RI WKH SU\PQHVLRSK\WH FRPPXQLW\ ZKLWH GRWV OHIW IRU WKH ,1'(0$5(6 VWDWLRQVLQWKH1($WODQWLF6WDWLRQV$DQG%FRUUHVSRQGWRWKH$YLOpVFDQ\RQUHJLRQVWDWLRQV&DQG'FRUUHVSRQGWR WKH*DOLFLDQ%DQNUHJLRQ7KHJUH\VKDGHGDUHDUHSUHVHQWVWKHÀXRUHVFHQFHSUR¿OHUHFRUGHGDWHDFKVWDWLRQ(UURUEDUV represent standard errors. st. A Depth (m) 0 20 40 60 abundance (cells ml-1) 0 200 400 600 800 UCYN-A1 host other prymnesiophytes st. B Depth (m) 0 20 40 60 0 100 200 300 400 st. C Depth (m) 0 20 40 60 0 300 600 900 1200 st. D Fluorescence ru. 0246810 Depth (m) 0 20 40 60 0 300 600 900 1200 01234567 PO43- (μM) 0.00.1 0.20.3 0.40.5 0.6 Temp Temperature (ºC) 10 12 14 16 18 20 22 NO3- (μM) NO3PO43- Distribution of a prymnesiophyte–UCYN-A symbiosis 116 Discussion Characterization of the partnership Microscopic observations revealed a population size spectra and phenotype consistent with SUHYLRXVUHSRUWVZLWKVPDOOSU\PQHVLRSK\WHKRVWFHOOVRIPW\SLFDOO\ZLWKRQHF\DQREDFWHULDO UCYN-A1 symbiont (Thompson et al..UXSNHet al., 2014a). On the other hand, the cell VL]HRIWKH8&<1$KRVWLQRSHQRFHDQVDPSOHVZDVPFOHDUO\VPDOOHUWKDQWKDWGHVFULEHG LQFRDVWDOVLWHVDERXWȝP7KRPSVRQet al., 2014; Hagino et al.7KLVLVLQDJUHHPHQW with a greater contribution of B. bigelowiiUHDGVLQWKHȝPVL]HIUDFWLRQDVFRPSDUHGZLWKWKH PIUDFWLRQLQWKHTara-Oceans metabarcoding data analysed here, as well as of UCYN-A2 reads in the same smaller size fraction in metagenomic samples of the South Atlantic (CornejoCastillo et al., submitted). The hypothesis about the obligatory dependence of UCYN-A with its host (Tripp et al., 2010; Thompson et al., 2012; Bombar et al., 2014) has been recently reinforced with the strong coupling REVHUYHGLQWKHWUDQVIHURIFDUERQDQGQLWURJHQPHWDEROLWHVEHWZHHQSDUWQHUFHOOV.UXSNHet al., 2014b). On the other hand, the possibility of a free-living population of the host could not be HYDOXDWHGVLQFHWKH),6+SUREHXVHGLQSUHYLRXVVWXGLHV.UXSNHet al., 2014a) targeted the whole SU\PQHVLRSK\WHDVVHPEODJHDQGQRWWKHVSHFL¿FKRVWSK\ORW\SHV2XUXVHRIDVSHFL¿FSUREHIRU the UCYN-A1 host allowed us to validate that this association was also obligatory for the host since i) we never observed a free living population of targeted prymnesiophytes in the absence of UCYN-A and ii) counts of free partner cells, when detected, were correlated, suggesting a disruption of the association due to sample manipulation (Thompson et al.,.UXSNHet al., 7RRXUNQRZOHGJHWKLVLVRQHRIWKHIHZFDVHVVKRZLQJDQREOLJDWRU\GHSHQGHQF\RID SK\WRSODQNWRQ VSHFLHV DQG LWV V\PELRQW 7KH KLJK GHJUHH RI VSHFL¿FLW\ EHWZHHQ SDUWQHUV DQG the reductive evolution of the UCYN-A genomes (Bombar et al., 2014) are features analogous to those observed in some freshwater diatom species, which harbor N2¿[LQJF\DQREDFWHULDDV endosymbionts that are inseparable from the host and transferred to daughter cells during host cell GLYLVLRQ1DND\DPDet al., 2014). The UCYN-A symbiont of B. bigelowii is also an endosymbiont as observed by transmission electron microscopy (Hagino et al.,DQGLWLVOLNHO\WKDWWKH prymnesiophyte–UCYN-A1 association presents analogous structural properties. In our study, about 97% of the hosts carried a single UCYN-A1 cell. This supports previous REVHUYDWLRQVIURPWKH1RUWK3DFL¿F7KRPSVRQet al.DQGWKH1RUWK$WODQWLF.UXSNHet al., D7KHUHIRUHZHFRQVLGHUWKDWWKH8&<1$KRVWUDWLRLVWKHPRVWSODXVLEOHDVVXPSWLRQ for estimating N host requirements and metabolite exchanges between partner cells and evaluating the contribution of this partnership to N2¿[DWLRQ,QDGGLWLRQZHVXJJHVWWKDWWKHSUHVHQFHRIWZR UCYN-A1 cells per host may derive from repartition of symbionts before host cell division. We PDGHSXQFWXDOFDSWXUHVRIKRVWFHOOVOLNHO\GLVSOD\LQJGLIIHUHQWVWDJHVRIFHOOGLYLVLRQ )LJXUH 6,WLVNQRZQIURP¿HOG-DFTXHWet al.DQGODERUDWRU\VWXGLHV-DFTXHWet al., 2001) Chapter 3 117 WKDWSKRWRV\QWKHWLFSLFRHXNDU\RWHVGLYLGHGXULQJDYHU\VKRUWWLPHZLQGRZMXVWEHIRUHRUGXULQJ WKHGDUNSHULRG:HGHWHFWHGWZRFHOOVSHUKRVWPRVWO\LQ,1'(0$5(6VDPSOHVZKLFKZHUH collected after sunset and thus the symbiotic population could be in active cell division. In the future, microscopic observations during diel cycles coupled with transcriptomic analyses would shed light on the division mechanisms within this symbiosis. A widespread distribution of the symbiosis in the marine ecosystem The distribution obtained based on CARD-FISH counts, together with the 18S rDNA metabarcoding from the circumglobal expedition Tara-Oceans, expands the geographic distribution of the symbiosis and presents contrasted ecological patterns of both types. The UCYN-A1 host appeared in few spots of high abundance and was undetected in ~56% of CARD-FISH surface samples. The distribution of this host based on V9 reads supported this patchy distribution. Although the metabarcoding method has a much lower detection limit than the CARD-FISH method, we could not detect the presence of UCYN-A1 host reads in a substantial number of stations (~62%). The 8&<1$KRVWZDVGHWHFWHGRQO\RFFDVLRQDOO\E\&$5'),6+OLNHO\EHFDXVHLWVDEXQGDQFHZDV below the detection threshold, consistent with reported cell abundances of B. bigelowii of up to 1 cells ml-1LQPDULQHV\VWHPV.RQQRet al., 2007). This limitation to obtain a reliable signal of the UCYN-A2 host was offset with the metabarcoding approach. Thus, the UCYN-A2 host showed an unexpected homogeneous distribution at low relative abundance in the open ocean, with targeted reads detected throughout Tara-Oceans samples. Our CARD-FISH observations expanded the geographic coverage of UCYN-A1 in symbiosis to the South Atlantic gyre and the southern Indian Ocean. In these regions, no records of the prymnesiophyte phylotypes or UCYN-A were documented previously, except occasional detections of UCYN-A nifH sequences (no clade distinction) in the Benguela upwelling system, in the Arabian Sea and near Madagascar (Mazard et al., 2004; Moisander et al., 2010; Sohm et al.,D0RUHRYHURXUPHWDEDUFRGLQJDQDO\VLVUHSUHVHQWHGWKH¿UVWGHWHFWLRQRIERWKKRVWVLQ WKH5HG6HDDQGZLGHQHGWKHSUHVHQFHRIWKH8&<1$KRVWLQWKH6RXWK3DFL¿FWKH$UDELDQ Sea and the Mediterranean Sea, where only few nifH clones of UCYN-A2 were reported (ManAharonovich et al., 2007). Previous reports showed that B. bigelowii usually occurred in low VDOLQLW\QXWULHQWULFKFRDVWDOZDWHUV.RQQRet al., 2007; Hagino et al.,DOWKRXJKLWZDVDOVR UHSRUWHGLQWKH6DUJDVVR6HD*DDUGHU+XOEXUW2XUVWXG\UHLQIRUFHVWKHSUHVHQFHRI B. bigelowiiLQV\PELRVLVZLWK8&<1$LQRSHQRFHDQVWDWLRQVFRQVLVWHQWZLWKWKH¿QGLQJRInifH copies of the UCYN-A2 phylotype in the open sea (Thompson et al., 2014). In the ocean, cyanobacterial diazotrophs generally show a consistent pattern in depth with higher DEXQGDQFHVLQWKHXSSHUHXSKRWLF]RQH*RHEHOet al., 2010; Moisander et al., 2010). Recurring VXPPHUEORRPVRIWKHVHGLD]RWURSKVDUHUHSRUWHGLQWKH1RUWK3DFL¿FVXEWURSLFDOJ\UH'RUHet Distribution of a prymnesiophyte–UCYN-A symbiosis 118 al., 2008) usually related to shallow mixing conditions that ensure the solar energy needed for QLWURJHQ¿[DWLRQ$UHFHQWVWXG\VKRZHGWKDWWKH8&<1$V\PELRVLVDSSHDUHGODUJHO\UHVWULFWHG WRWKHXSSHUZDWHUFROXPQZKHUH13UDWLRVZHUHEHORZ.UXSNHet al., 2014a). Our highly UHVROYHGYHUWLFDOSUR¿OHVVKRZWKDWLWKHSU\PQHVLRSK\WH±8&<1$V\PELRVLVRFFXSLHVQXWULHQW depleted surface waters, and ii) its abundance decreases in the shallow DCM. This pattern suggests DGHSHQGHQFHRQOLJKWLQWHQVLW\DQGRUDQRXWFRPSHWLWLRQRIWKHV\PELRWLFSRSXODWLRQE\RWKHU QRQGLD]RWURSKLFVSHFLHVWKDWEHQH¿WIURPWKHLQFUHDVHGQXWULHQWDYDLODELOLW\DWWKHQXWULFOLQH7KLV vertical pattern is comparable to that observed for UCYN-A1 nifH gene abundances in station ALOHA (Church et al.,QWKHVHYHUWLFDOSUR¿OHVEHWZHHQ4-106 nifH copies per L (roughly equivalent to 10-1 000 cells ml-1ZHUHTXDQWL¿HGLQWKHZHOOOLWQXWULHQWGHSOHWHGXSSHU waters, and abundances decreased in the DCM to 102-10 nifH copies per L (~1 cell ml-1). Environmental controls of cyanobacterial diazotrophs in the ocean In our study, no environmental factor explained unambiguously the prymnesiophyte–UCYN-A1 distribution in surface waters. Presence or absence of the symbiosis occurred under similar environmental conditions (Figure S4). Previous reports point to temperature as a driver of the distribution of cyanobacterial diazotrophs (Church et al., 2008; Moisander et al., 2010). Particularly for UCYN-A, nifH gene abundances have been reported to be higher in temperature ranges from 19 to 24ºC (Langlois et al., 2008; Church et al., 2008). Our study, with detections at a thermal range RI&UHYHDOHGDZHDNQHJDWLYHUHODWLRQVKLSEHWZHHQV\PELRVLVDEXQGDQFHDQGWHPSHUDWXUH agreeing with previous reports. Limiting nutrients such as iron or phosphate might also control the distribution of cyanobacterial diazotrophs (Moore et al., 2009; Sohm et al., 2011b). Indeed, KLJKHUDEXQGDQFHVRIGLD]RWURSKVKDYHEHHQOLQNHGWRRFHDQLFDUHDVZKHUHWKHVHQXWULHQWVDUH VXSSOLHGIURPGXVWGHSRVLWLRQRIDGMDFHQWGHVHUWDUHDV5LGDPHDQG*XLHX7\UUHOOet al., &DSRQHet al., 2005; Mahowald et al., 2009). The patches of symbiosis observed in this study in the eastern North Atlantic, the western coast of Australia or even near Hawaii, could be explained by deposition events, since high dissolved Fe concentrations have been reported in these areas (Brown et al., 2005; Langlois et al., 2012). Patchiness seems to be a common feature of diazotrophic populations (Church et al.*RHEHOet al., 2010) and UCYN-A is regarded as very dynamic over small spatio-temporal scales (Robidart et al., 2014). The regional dominance of different diazotrophic groups can be related to distinct temperature and nutrient requirements (Church et al., 2008; Langlois et al., 2008). For instance, in the tropical North Atlantic, Trichodesmium was dominant in the warmer western side whereas UCYN-A was PRUHDEXQGDQWLQWKHFRROHUHDVWHUQVLGHQHDU&DSH9HUGH,VODQGV*RHEHOet al., 2010). During the MALASPINA expedition, a similar picture for the distribution of UCYN-A and Trichodesmium within this basin was observed, with these two diazotrophic groups dominating in different regions (see Fernández-Castro et al. in press for a Trichodesmium distribution map). In addition, N2 Chapter 3 119 ¿[DWLRQUDWHVPHDVXUHGGXULQJWKH0$/$63,1$H[SHGLWLRQGLGQRWFRUUHODWHZLWKTrichodesmium abundances (Fernández-Castro et al., in press), but we noticed that regions of enhanced N2¿[DWLRQ rates as in the western South Atlantic or the eastern Indian ocean were coupled to the presence of the prymnesiophyte–UCYN-A association. A recent latitudinal study along the eastern Australian coast also described a community shift from Trichodesmium in the north to unicellular diazotrophs in the south where the highest N2¿[DWLRQUDWHVZHUHUHSRUWHG5DHVet al., 2014). Different ecological strategies between the two related lineages Our results obtained by FISH counts and metabarcoding sequences in a large set of marine samples demonstrated that the two types of prymnesiophyte–UCYN-A symbiosis were widespread in the photic layer of the world ocean. We observed that UCYN-A2 host was homogeneously distributed at both surface and DCM but in low abundance, whereas UCYN-A1 host was only detected in some hotspots of very high abundance at surface, suggesting different ecological strategies. We hypothesize that UCYN-A1 host could follow the r strategy, with fast growth under favourable FRQGLWLRQVVXFKDVXQSUHGLFWDEOHHYHQWVRIGXVWGHSRVLWLRQRUWKHRQVHWRIWKHUPDOVWUDWL¿FDWLRQ XVLQJVXUIDFHOLJKWIRUWKHHQHUJ\GHPDQGLQJSURFHVVRIQLWURJHQ¿[DWLRQ2QWKHRWKHUKDQGWKH 8&<1$KRVWFRXOGIROORZWKHNVWUDWHJ\DOVRNQRZQDVVWUHVVWROHUDQWVSHFLHVE\5H\QROGV 1997), persisting at low abundances and being more competitive in stable and low resource FRQGLWLRQV,QWKLVVHQVHWKHVPDOO8&<1$KRVWVHHPVH[FOXGHGLQWKH'&0OLNHO\EHFDXVH RWKHUVSHFLHVDUHPRUHHI¿FLHQWLQZDWHUVZLWKKLJKHUQXWULHQWVDQGGLPPHUOLJKWZKLOHWKLVGHSWK limitation could be less severe in the UCYN-A2 host, which seems to be less affected by changes in the light regime (Thompson et al., 2014). A better understanding of the role of UCYN-A GLD]RWURSK\DORQJWKHYHUWLFDOSUR¿OHPD\FHUWDLQO\EHQH¿WIURPWUDQVFULSWLRQDOSUR¿OHVRIWKHVH populations, including the mechanisms of the host to regulate UCYN-A activity. Overall, further studies on the biogeochemical role of the UCYN-A diazotrophy may need to include contrasted ecological differences among the different lineages. Acknowledgements )LQDQFLDO VXSSRUW KDV EHHQ SURYLGHG E\ WKH 6SDQLVK 0LQLVWU\ RI (FRQRP\ DQG &RPSHWLWLYLW\ WKURXJK SURMHFW &RQVROLGHU,QJHQLR 0DODVSLQD  &6' WR &0' )/$0( &*/WR50DQG3$1*(120,&6&*/%26WR6$*$0&DEHOOR ZDVUHFLSLHQWRID6SDQLVK)3,JUDQW%(6:HDFNQRZOHGJHDOOWKHWHFKQLFLDQV UHVHDUFKHUV FUHZ DQG FKLHI VFLHQWLVWV RI WKH GLIIHUHQW FUXLVHV IRU FROODERUDWLRQ :H WKDQN 0 *DOLQGRDQG3GHOD)XHQWHIRUQXWULHQWDQDO\VLV5/RJDUHVIRUVHTXHQFHDGYLFH56LPyDQG -0*DVROIRUXVHIXOFRPPHQWVRQWKHPDQXVFULSWDQG%)HUQiQGH]&DVWURDQG%0RXULxRIRU sharing unpublished data. Distribution of a prymnesiophyte–UCYN-A symbiosis 120 Supplementary material Figure S1 Map of stations in the MALASPINA (a) and the Tara-Oceans (b) circumnavigation H[SHGLWLRQV7KHVWDWLRQVLQWKH,1'(0$5(6FUXLVHDUHVKRZQLQWKH5HJLRQ5LQSDQHO(a). ÝS ÝS EQ ÝN ÝN ÝWÝWÝ ÝE Ocean Data View 77 76 74 71 69 68 66 64 61 58 55 52 49 47 45 44 41 38 35 32 30 28 26 24 22 19 17 15 12 9 8 63 146 144 141 139 137 134 131 130 127 126 124 120 116 113 110 107 104 101 100 98 95 92 89 86 83 81 A B CD R1 R1 ÝS ÝS EQ ÝN ÝN ÝWÝWÝ ÝE Ocean Data View 123 125 124 122 98 111 100 102 109 36 38 41 42 45 52 64 65 66 67 68 70 72 76 78 82 84 85 R2 34 32 30 25 23 22 18 16 9 11 7 420 R2 a) b) Chapter 3 121 Figure S2 (SLÀXSUHVFHQFH PLFURVFRS\ LPDJH RI SU\PQHVLRSK\WHOLNH FHOO QRW ODEHOHG E\ WKH UPRYM69 probe harbouring a labeled UCYN-A cell, co-occuring with a labeled host cell (from MALASPINA station 68). Upper panel correspond to DAPI signal (blue-labeled nucleous) and lower panel corresponds to the combined signal of the UPRYM69 probe (green-labeled host under EOXHOLJKWH[FLWDWLRQDQG8&<1B$SUREHUHGODEHOHGV\PELRQWXQGHUJUHHQOLJKWH[FLWDLRQ Figure S3 (SLÀXSUHVFHQFHPLFURVFRS\LPDJHRID%ELJHORZLLFHOOZKHUHWKH8&<1$V\PELRQW seems to be detached from the host. The image corresponds to the combined signal of the DAPI stain (blue-labeled nucleus), the UBRADO69 probe (grenn-labeled host under blue light excitation) DQG8&<1$SUREHUHGODEHOHGV\PELRQWXQGHUJUHHQOLJKWH[FLWDWLRQ UCYN-A labeled host non-labeled host UCYN-A 5 µm 5 µm Chapter 4 Chapter 4 131 Chapter 4 *OREDO GLVWULEXWLRQ DQG LQWUDVSHFL¿F YDULDELOLW\ RI PDULQH SHODJRSK\WHV Ana M. Cabello, Ramiro Logares, Sarah Romac and Ramon Massana. 6XPPDU\ Pelagophytes are important components of marine photosynthetic picoeukaryote assemblages and are widely distributed across the oceans. Molecular surveys based on ribosomal genes suggest WKDWSHODJRSK\WHVKDYHDORZHYROXWLRQDU\GLYHUVL¿FDWLRQZLWKPRVWHQYLURQPHQWDOVHTXHQFHV being nearly identical to cultured species, in particular Pelagomonas calceolata. To assess the inWUDVSHFL¿FYDULDELOLW\ZLWKLQWKLVVSHFLHVDQGYDOLGDWHWKHSUHYDOHQFHRIRWKHUJHQHUDZHDQDO\]HG SHODJRSK\WHS\URWDJVFRQWDLQLQJWKH¿QDOSDUWRIWKH6U'1$aESDQGWKHFRPSOHWH,76 UHJLRQaESLQDZLGHDUUD\RIPLFURELDODVVHPEODJHVIURPWKHHSLSHODJLFRFHDQLQFOXGLQJ open ocean sites in major basins and European coastal sites. Most pelagophyte species were found in our dataset and we detected a novel and still-uncultured species. The majority of pyrotags aDI¿OLDWHGWRPelagomonas calceolata and Aureococcus spp. P. calceolata was dominant LQPRVWVDPSOLQJVLWHVZKLFKFRQ¿UPVWKHEURDGGLVWULEXWLRQRIWKLVVSHFLHVLQPDULQHV\VWHPV whereas Aureococcus VSSWHQGHGWREHUHVWULFWHGWRFRDVWDOVLWHV$QDO\VLVRI,76VHTXHQFHVD SK\ORJHQHWLFPDUNHUPRUHUHVROXWLYHWKDQWKH6U'1$LQGLFDWHGORZLQWUDVSHFL¿FYDULDELOLW\ within P. calceolata and Aureococcus spp. and suggested the existence of cosmopolitan species without geographical differentiation. Overall, our study highlighted the low genetic diversity of pelagophytes in the global surface ocean. Chapter 4 133 Introduction Marine photosynthetic protists include a huge taxonomic diversity, with representatives in all but RQHWKHRSLVWKRNRQWDGLYLVLRQVRIWKHHXNDU\RWLFWUHHRIOLIH&DURQet al.1RWet al. They are important members of the eukaryotic plankton, and form the majority of cells within WKHSLFRDQGQDQRVL]HIUDFWLRQVP1RWet al.0DVVDQD7RJHWKHU with cyanobacteria, photosynthetic protists are responsible for half of the primary production on (DUWK)LHOGDQGDUHPDLQSOD\HUVRIWKHFDUERQELRORJLFDOSXPS$VUHYHDOHGE\PROHFXODU VXUYH\VLQLWLDOO\EDVHGRQ6DQJHUVHTXHQFLQJ'tH]et al.0RRQYDQGHU6WDD\et al. 0F'RQDOGet al./HSqUHet al.6KLet al.DQGUHFHQWO\RQKLJKWKURXJKSXW VHTXHQFLQJCheung et alGH9DUJDVet al.0DVVDQDet al.WKLVDVVHPEODJHLV GRPLQDWHGE\GLQRÀDJHOODWHVGLDWRPVKDSWRSK\WHVPDPLHOORSK\WHVSUDVLQRSK\WHVFKU\VRSK\WHV DQGSHODJRSK\WHV$PRQJWKHPSHODJRSK\WHVDUHRQHRIWKHPDMRUFRQWULEXWRUVWRWRWDOFHOOFRXQWV LQWKHVPDOOHVWVL]HIUDFWLRQP1RWet al.-DUGLOOLHUet al.*UREet al. DQGDUHJOREDOO\GLVWULEXWHGLQWKHVHDERWKDWFRDVWDODQGRSHQRFHDQVLWHV.LUNKDPet al. This group has been suggested to be a major player in the process of nitrate assimilation (Dupont et al., 2015). Due to their relevance in the world’s ocean, pelagophytes deserve further studies on their diversity and ecology. Pelagophytes are marine VWUDPHQRSLOHDOJDHLQWHJUDWHGLQWKH&ODVV3HODJRSK\FHDH$QGHUVHQet al.EDVHGRQJHQHWLFXOWUDVWUXFWXUDODQGSLJPHQWGDWD'H<RHet al.6DXQGHUVet al., 7KHFODVVLQFOXGHVWZRRUGHUV0RVWPHPEHUVZLWKSODQNWRQLFFRFFRLGIRUPVDUHSODFHG in the order Pelagomonadales, whereas members presenting colonial forms growing on shallow ZDWHUVVXEVWUDWHVDUHDOOSODFHGLQWKHRUGHU6DUFLQRFKU\VLGDOHV$GOet al.:\QQHet al., 3ODQNWRQLFSHODJRSK\WHVUDQJHLQFHOOVL]HVEHWZHHQDQGP9DXORWet al. DQGDUHUHSUHVHQWHGE\JHQHUDZLWKRQHVSHFLHVHDFKPelagomonas calceolata, Pelagococcus subviridis, Aureococcus anophagefferens, Aureoumbra lagunensis and Ankylochrysis lutea. P. calceolata seems to be the most abundant and widespread pelagophyte. 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U78033_Sarcinochrysis marina 0.05 Figure 1. 0D[LPXPOLNHOLKRRGSK\ORJHQHWLFWUHHRIWKHSHODJRSK\WH9W\SHVLGHQWL¿HGLQ RXUGDWDVHWFRGHGDV278QXPEHUDQGWKHQXPEHURIS\URWDJVWKH\LQFOXGHDQG*HQ%DQNFORVHVWUHODWLYHVLQEROG'LIIHUHQW9W\SHVZLWKLQDJHQXVDUHLQGLFDWHGDVWWRUW%RRVWUDS YDOXHVIURPUHSOLFDWHVDERYHDUHVKRZQ Results General description of the dataset :HDQDO\]HGSHODJRSK\WHVSHFL¿FS\URWDJVRISDUWLDO6U'1$DQGFRPSOHWH,76IURP(XURSHDQ FRDVWDOVLWHVDQGRSHQRFHDQVLWHVIURPWKH$WODQWLFVLWHVWKH,QGLDQVLWHVDQGWKH3DFL¿F VLWHV$WHDFKVWDWLRQ VXUIDFH DQGRU'&0 GHSWKVZHUH VDPSOHGIRU WKHSLFRSODQNWRQDQG DGGLWLRQDOO\IRUWKHQDQRSODQNWRQLQFRDVWDOVLWHV,QRUGHUWRDVVLJQWD[RQRPLFDOO\WKHVHTXHQFHV obtained and compare the different samples based on pelagophyte diversity, we retained only 278VLQFOXGLQJFRPSOHWH9DQG,76UHJLRQV278VDQGIXUWKHUFOXVWHUHGWKHPEDVHGRQ WKH9UHJLRQVLQFHDODUJHYDULDELOLW\ZDVH[SODLQHGE\WKH,76UHJLRQ7KH¿QDOQRUPDOL]HG WDEOHRI278VUHSUHVHQWHGVDPSOHVIRUWKHSLFRSODQNWRQDQGIRUWKHQDQRSODQNWRQ Low global diversity of marine pelagophytes 144 :HSHUIRUPHGWKHVDPHDQDO\VLVIRUWKH,76YDULDQWVLQAureococcus. $WRWDORIDQG 278VZHUHGH¿QHGZLWKLQW\SHVDQGUHVSHFWLYHO\DQGZHVHOHFWHGWKHPRVWDEXQGDQW278V !RIWRWDOS\URWDJVRIHDFKW\SHWREXLOGDSK\ORJHQHWLFWUHH)LJD6HTXHQFHGLYHUJHQFH EHWZHHQDOORIWKHPZDVRQDYHUDJH7KHWUHHFOXVWHUHGGLIIHUHQWLDOO\WKH,76YDULDQWVRI each Aureococcus9W\SHDQGGLVSOD\HGWZRGLVWLQFWFODGHVZLWKLQWKHW\SHYDULDQWFODGHV $DQG%6HTXHQFHGLYHUJHQFHZDVZLWKLQW\SHDQGZLWKLQW\SHZLWKLQ FODGH$RQDYHUDJH7RH[SORUHLIWKHYDULDQWVRIWKHVHFODGHVKDYHDQHFRORJLFDOPHDQLQJZH UHSUHVHQWHGWKHLUFRQWULEXWLRQLQWKHGLIIHUHQWVDPSOHV)LJE&ODGH%PDLQO\UHSUHVHQWHG E\RQH278ZDVGRPLQDQWRUHYHQWKHXQLTXHUHSUHVHQWDWLYHLQPRVWVDPSOHV2QWKH RWKHUKDQGFODGH$DSSHDUHGZHOOUHSUHVHQWHGLQDIHZFRDVWDOVLWHV*LMyQDQG%ODQHVDQGZHUH virtually absent in the others. They also appeared in the two open ocean sites where Aureococcus W\SHZDVGHWHFWHG5HJDUGLQJ,76YDULDQWVRIAureococcusW\SHVDQGQRGLIIHUHQWLDOVLJQDO ZDVGHWHFWHG7\SHZDVYLUWXDOO\UHSUHVHQWHGE\RQO\RQH,76YDULDQWZKLOHW\SHZDVRQO\ GHWHFWHGLQWKHSLFRDQGQDQRVL]HIUDFWLRQRIDVLQJOHVLWHLQ5RVFRII Aureococcus1_ID6363_8978 Aureococcus1_ID1962_277 Aureococcus1_ID5503_230 Others OSLO09 OSLO10 GIJON BLANES NAPLES10 VARNA 0 20 40 60 80 100 pyrotags contribution (%) OTU2760_780 OTU5820_177 OTU7936_333 OTU1564_932 Aureococcus3 Aureococcus2 Clade 1A Clade 1B 69 60 66 100 52 A B NNN S D A1 I3 I4N N N N N N N S D S D S S S S D S D D S D Figure 7. 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al. Shi et al.,QWKHVHVWXGLHVPelagomonas calceolataVHHPHGWREHWKHXQLTXHDEXQGDQW PHPEHURIWKLVJURXS2XUUHVXOWVFRQ¿UPWKDWLQVRPHFLUFXPVWDQFHVLQGHHGP. calceolata was the XQLTXHSHODJRSK\WHVSHFLHVVXFKLQODUJHRFHDQLFDUHDVRIWKH3DFL¿F2FHDQ+RZHYHUE\XVLQJ SHODJRSK\WHVSHFL¿FSULPHUVZHDOVRKLJKOLJKWHGRWKHUJHQHUDWKDWPLJKWEHHTXDOO\LPSRUWDQWRU even dominate the assemblages like AureococcusVSSRUWKH1RYHOJURXS ,QWKHSUHYLRXVVWXGLHVRQSLFRHXNDU\RWLFFRPPXQLW\FRPSRVLWLRQLQ(XURSHFRDVWDOZDWHUVWKH few pelagophyte clones detected were identical to P. calceolata ERWK LQ WKH (QJOLVK &KDQQHO 5RPDULDQG9DXORWDQGWKH0HGLWHUUDQHDQ6HD'tH]et al.0DVVDQDet al. Marie et al.0F'RQDOGet al.%\FRQWUDVWRXUGDWDVKRZWKDWLWLVLQWKHVHFRDVWDO waters where most pelagophyte genera are represented, in particular the unprecedent distribution and abundance of 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HUURUV$ SODXVLEOH H[SODQDWLRQ IRU WKH DEVHQFH RI Aureococcus spp. within the 7DUDGDWDVHWLVWKDWLWVPRVWDEXQGDQWW\SHSUHVHQWVRQHPLVPDWFKZLWKWKHHXNDU\RWLFSULPHU Low global diversity of marine pelagophytes 146 5$PDUDO=HWWOHUet al.XVHGLQ7DUDWKHIRUZDUGHXNDU\RWLFSULPHU)PDWFKHG DOOSHODJRSK\WHV7KLVELDVFRXOGDOVRH[SODLQWKHSUHYLRXVUHVXOWVXVLQJFORQLQJDQGVHTXHQFLQJ LQFRDVWDOZDWHUV0DVVDQDet alVLQFHWKHVDPHPLVPDWFKH[LVWHGLQWKHSULPHUXVHG,Q DUHFHQWVWXG\RQ(XURSHDQFRDVWDOZDWHUVXVLQJS\URVHTXHQFLQJDQGHXNDU\RWLFSULPHUVIURP WKH9UHJLRQRIWKH6U'1$0DVVDQDet alWKHWZRGRPLQDQW278VZHUHLGHQWLFDO to P. calceolata and Aureococcus anophagefferensW\SHEHLQJWKHODWWHUVSHFLHVGRPLQDQWRU XQLTXHLQDIHZRIWKHVDPSOHVGDWDQRWVKRZQ Thus, our work offers a new picture of pelagophyte assemblages in coastal and open ocean waters 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