Extreme diversity in noncalcifying haptophytes explains a major pigment paradox in open oceans Hui Liua,b, Ian Proberta, Julia Uitzc, Herve´ Claustred, Ste´ phane Aris-Brosoue, Miguel Fradab, Fabrice Nota, and Colomban de Vargasa,b,1 aCentre National de la Recherche Scientifique, Unite´Mixte de Recherche 7144 and Universite´Pierre et Marie Curie Paris 06, Equipe Evolution du Plancton et Pale´o-Oce´ans, Station Biologique de Roscoff, 29682, France; bInstitute of Marine and Coastal Sciences, Rutgers University, New Brunswick, NJ 08901; cMarine Physical Laboratory, Scripps Institution of Oceanography, University of California at San Diego, La Jolla, CA 92093-0238; dCentre National de la Recherche Scientifique, Unite´Mixte de Recherche 7093 and Universite´Pierre et Marie Curie Paris 06, Laboratoire d’Oce´anographie de Villefranche/Mer, 06234, France; and eDepartment of Biology and Department of Mathematics and Statistics, University of Ottawa, Ottawa, ON, Canada K1N 6N5 Communicated by W. A. Berggren, Woods Hole Oceanographic Institution, Woods Hole, MA, June 2, 2009 (received for review December 18, 2008) The current paradigm holds that cyanobacteria, which evolved Several lines of evidence in fact argue for eukaryotic suprem- oxygenic photosynthesis more than 2 billion years ago, are still the acy over marine oxygenic photosynthesis. Flow cytometric cell major light harvesters driving primary productivity in open oceans. counts (7) show that picophototrophic protists (0.2–3 ␮m cell Here we show that tiny unicellular eukaryotes belonging to the size) are indeed 1–2 orders of magnitude less abundant than photosynthetic lineage of the Haptophyta are dramatically diverse cyanobacteria. However, biophysical and group-specific 14C- and ecologically dominant in the planktonic photic realm. The use uptake measurements suggest that tiny eukaryotes can, through of Haptophyta-specific primers and PCR conditions adapted for equivalent or higher growth rates of relatively larger cells, GC-rich genomes circumvented biases inherent in classical genetic dominate carbon biomass and net production in both coastal (8) approaches to exploring environmental eukaryotic biodiversity and oceanic (7) settings. High performance liquid chromatog- and led to the discovery of hundreds of unique haptophyte taxa in raphy (HPLC) analyses of group-specific accessory pigments 5 clone libraries from subpolar and subtropical oceanic waters. have further stressed the ecologic prevalence of phototrophic Phylogenetic analyses suggest that this diversity emerged in Pa- Ј protist taxa. In particular, 19 -hexanoyloxyfucoxanthin (19-Hex) ECOLOGY leozoic oceans, thrived and diversified in the permanently oxy- was originally estimated to account for 20–50% of total chlo- genated Mesozoic Panthalassa, and currently comprises thousands rophyll a (Chla) biomass in tropical Atlantic and Pacific sites (9) of ribotypic species, belonging primarily to low-abundance and and has since been consistently reported in open ocean photic- ancient lineages of the ‘‘rare biosphere.’’ This extreme biodiversity zone waters, e.g., (10, 11), suggesting a ubiquitous occurrence of coincides with the pervasive presence in the photic zone of the haptophytes in upper layers of the water column. Surveys of -world ocean of 19؅-hexanoyloxyfucoxanthin (19-Hex), an acces- genetic diversity based on environmental ribosomal DNA librar sory photosynthetic pigment found exclusively in chloroplasts of ies over the last decade have unveiled an unexpected diversity of haptophyte origin. Our new estimates of depth-integrated relative tiny eukaryotes in all oceans (12). Paradoxically, most picoeu- abundance of 19-Hex indicate that haptophytes dominate the karyotic sequence diversity from photic layers represented novel chlorophyll a-normalized phytoplankton standing stock in modern heterotrophic (13) and parasitic (14) protists within phyla tradi- oceans. Their ecologic and evolutionary success, arguably based on tionally thought to be dominated by photoautotrophs. This bias mixotrophy, may have significantly impacted the oceanic carbon indicated that marine protist diversity might be significantly pump. These results add to the growing evidence that the evolu- skewed toward heterotrophic taxa (15), as appears to be the case tion of complex microbial eukaryotic cells is a critical force in the for prokaryotes. However, the paucity of haptophyte nuclear functioning of the biosphere. rDNA sequences in these surveys contrasts strikingly with the abundance of 19-Hex in marine waters. Haptophyta ͉ photosynthesis ͉ protistan biodiversity ͉ Here we use a combination of previously undescribed genetic, eukaryotic biodiversity pigment, and microscopy data to unveil a dramatic and ancient diversity of unique photosynthetic picoplanktonic protists within xygenic photosynthesis, the most complex and energetically the Haptophyta. This diversity could account for the mysteri- Opowerful molecular process in biology, originated in cya- ously high concentration of 19-Hex in the photic layer of the nobacteria more than 2 billion years ago in Archean oceans (1). world oceans, our calculations indicating that haptophytes con- Ϸ Marine photosynthesis still contributes Ϸ50% of total primary tribute 2-fold more than either cyanobacteria or diatoms to production on Earth (2). This revolutionary process was inte- global oceanic Chla standing stock. The phylogenetic position of grated, at least once, into an ancestral phagotrophic eukaryotic these tiny haptophytes implies that they are photophagotrophic, lineage through the evolution of chloroplasts, which themselves coinciding with the recent discovery of dominant bacterivory by were redistributed to a large variety of aquatic eukaryote small eukaryotic phytoplankton in the oceans (16). Mixotrophy lineages via permanent secondary and tertiary endosymbioses may provide a competitive advantage over both purely phototro- (3). Despite this evolutionary trend from photosynthetic pro- phic microalgae (including cyanobacteria) and aplastidial pro- karyotes to eukaryotes, particularly visible in today’s coastal tists, and the extreme genetic diversity of tiny haptophytes oceans where microalgae such as diatoms and dinoflagellates are matches the cellular and behavioral complexity inherent in this omnipresent, cyanobacteria have been repeatedly claimed as the mixed mode of nutrition. champions of photosynthesis in open ocean waters (4). This hypothesis followed the introduction of flow cytometry and Author contributions: C.d.V. designed research; H.L., I.P., J.U., H.C., M.F., and F.N. per- molecular genetic approaches to biological oceanography in the formed research; H.L., J.U., H.C., S.A.-B., F.N., and C.d.V. analyzed data; and I.P. and C.d.V. 1980s, which revealed astonishing concentrations of minute wrote the paper. cyanobacterial cells of the genera Procholorococcus and Syn- The authors declare no conflict of interest. echococcus in marine waters (5). The physiology, ecology, and 1To whom correspondence should be addressed. E-mail: [email protected]. functional and environmental genomics of these prokaryotes are This article contains supporting information online at www.pnas.org/cgi/content/full/ subjects of ongoing intensive study (6). 0905841106/DCSupplemental. www.pnas.org͞cgi͞doi͞10.1073͞pnas.0905841106 PNAS ͉ August 4, 2009 ͉ vol. 106 ͉ no. 31 ͉ 12803–12808 Downloaded by guest on September 27, 2021 fragments from bulk DNA extracted from the 0.2- to 3-␮m fraction of seawater collected at 4 offshore stations in the Arctic and Indian oceans (Fig. S1, Table S1). Standard eukaryotic rDNA analyses of mv5_19 mv5_21 these samples yielded Ϸ0.4–0.7% haptophyte sequences (10, 11). In contrast, our data reveal hundreds of previously undescribed rDNA sequences from tiny haptophytes. Rarefaction curves for individual clone libraries (Fig. 1) indicate that current sequencing mv18_59 effort is far from exhaustive, notably in subtropical waters where genetic diversity is particularly dramatic. Estimates of the number of unique ribotypes using the Chao1 estimator were 1098–1147 and Number of different OTUs 325–509, respectively, for the Indian and Arctic ocean samples (with rather large confidence intervals, see Table S2). The frequency distribution of unique ribotypes (Fig. 1) indicates higher species 020406080 z11_11 z61_43 richness in subtropical waters, with a substantial number of 0 50 100 150 orphan and deep-branching genotypes (see below) in both warm and cold waters. This parallels recent observations for Number of sequences sampled marine prokaryotes of a ‘‘seed bank’’ of ancient and rare taxa, Fig. 1. Rarefaction analysis for each environmental clone library based on termed the ‘‘rare biosphere’’ (17). unique LSU rDNAs sequences (OTUs); 72, 85, 65, 56, and 37 OTUs, respectively, were obtained from the Indian ocean (Mv 19, 21, 18) and subarctic (z 11, 43) Taxonomy and Evolutionary History of the Previously Undescribed clone libraries (Fig. S1). The pie charts show, for each library, the amount of Diversity. The 674 novel environmental LSU rDNA sequences identical sequences in each retrieved OTU. were aligned with 64 orthologous gene sequences from clonal culture strains representing a cross-section of known haptophyte biodiversity. Phylogenetic analyses indicate that all environmen- Results and Discussion tal sequences belong to the Haptophyta (Fig. 2), a eukaryotic A Massive Unique Diversity of Oceanic Picohaptophytes. We first phytoplankton division classically considered as nanoplankton