Colomban de Vargas–- ATIP-2004 Proposal 1

ATIP-2004 PROPOSAL:

ASSESSMENT OF SPECIES-LEVEL DIVERSITY AND EVOLUTION IN THE MARINE PHYTOPLANKTON USING MOLECULAR, MORPHOLOGICAL, AND FOSSIL DATA.

Introduction: pace of morphological versus genetic diversification in open ocean protists. The photic, epipelagic zone of the global Ocean is one of the largest and ecologically most influential compartments of Earth ecosystem. It is dominated by unicellular organisms. Among these, the auto- or mixo-trophic protists, also called phytoplankton, play fundamental roles in directing flows of biogeochemically and climatically important elements and gasses, determining their fate in terms of ocean-atmosphere exchange, conversion into organic matter, and deep-water sequestration (Falkowski 2004). The three most successful groups of phytoplankton in the Cenozoic Oceans (coccolithophores, diatoms, and dinoflagellates) have evolved convergent phenotypic traits, including the presence of hard skeletons enclosing the cell (calcareous coccoliths, siliceous frustules, and cellulose cysts, respectively). These micro-skeletons have accumulated in kilometers-thick layers of deep-sea sediments since the Jurassic (Haq and Boersma 1998) and thus built the most complete and continuous fossil record of any type of organism, a fundamental archive for reconstructing microbial evolution and studying the dynamics of Earth system. Despite the key ecological and geological importance of pelagic phytoplankton, their taxonomy has been almost exclusively based on morphological description of their micro-skeletons. However, the still very limited studies in molecular phylogenetics of pelagic protists at the ‘morphological species’ level over sufficiently large geographic scales (foraminifera - de Vargas et al. 1999, 2001, 2002; Darling et al. 1999, 2000, 2003; Bauch et al. 2003; haptophytes and diatoms - Medlin et al. 2000; Sàez et al. 2003; dinoflagellates -John et al. 2003; Scholin et al. 1995) have systematically shown that the morphological entities currently described as single species are in fact monophyletic assemblages of sibling species which diverged several million years ago according to molecular clock calculations. Preliminary results suggest that sibling species within a 'morphospecies' are in fact most often pseudo-cryptic species, i.e. that they do show stable morphological differences, but that these difference are subtle and have been overlooked or interpreted as ecophenotypic variations. In coccolithophores the genetic data to support this pattern are currently limited to a few species and very limited geographic coverage (Saez et al. 2003), but other data from integrated morphometric, biogeographic and culture studies suggest that the pattern is widespread (Geisen et al. 2004). These exciting new findings have wide-ranging implications for our concepts of phytoplankton taxonomy, ecology, and evolution. The pioneering molecular studies cited above strongly support the hypothesis that the actual number of phytoplanktonic species is much higher than the ~10,000 usually cited (Sournia, 1995). This means that, contrary to the traditional view, ubiquitous globally distributed phytoplankton species with high ecological plasticity may be rare or even absent in the pelagic realm. Instead, suites of sibling species may be common, each potentially adapted to highly defined ecological niches with restricted horizontal and/or vertical ranges. In addition, it seems that the patterns of gradual morphological differentiation (gradualism) frequently observed in the pelagic microfossil record (Benton and Pearson 2001; Jackson and Cheetham 1999) may be a misinterpretation resulting from adoption of over-broad species concepts in classical taxonomy. In fact, the morphology of planktonic protistan skeletons seems to remain extremely stable within a species-lineage after it has reached equilibrium. The deep-sea fossil record confirms that virtually identical morphotypes can last for tens of millions of years, certainly surviving the origination and potentially also the extinction of biological species, a process that has been recently reviewed and assigned to a concept of planktonic 'super-species' (de Vargas et al.

Colomban de Vargas–- ATIP-2004 Proposal 2

2004, and Fig. 1). As a corollary, tiny morphological differences may represent several million years of genetic isolation between phytoplankton species adapted to different ecological niches (de Vargas et al. 2002). Project goals The overarching goal of this project is to accurately assess the spatio-temporal dynamics of morphological versus genetic species-level differentiations during the last million years of phytoplankton evolution, i.e. the period of time that has given rise to the modern diversity. Broad objectives are: (1) to decipher the genetic, morphological, and ecological variations that separate species in the modern phytoplankton; and (2) to use both molecular clock and biostratigraphic data to determine the temporal scale(s) at which speciation occurs in the phytoplankton (model A versus B in Fig. 1). Figure 1: Morpho-genetic evolution of five theoretical planktonic super-species leading to 16 living biological species (modified from de Vargas et al. in press). The classical, morphological species of phytoplankton (1 to 5) are in fact monophyletic clusters of ssibling biologicaspecies (thin branches) that may be adapto different ecologicaPreliminary molecular phylogenetresults on coccolithop(Saez et al. 2003) suggest that the sibling species within a “morphospecies” typically originated in the late Miocene, between 14.8 and 5.3 Ma, as illustrated in modeA. However, the dynamics of speciation and the degree of disconnection between morphologand genetic evolutions may be much more intense and complex, as in model B. Star symbols correspond to homologous speciation nodes between evolutionary patterns A and B.

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eses The proposed research seeks to test three related he mo

and ecological versus morphological diversity: sibling pelagic

rise to the modern

orphospecies is limited by the

hypotheses concerning t de and tempo of the evolution of species diversity in oceanic phytoplankton: A. Genotypic phytoplankton species are separated by subtle, or non-existent morphological differences, although they may occupy much more restricted and specific hydrographic spatio-temporal niches than previously assumed based on classical morphological taxonomy. B. Origin and distribution of diversity: the speciation events that gave phytoplankton species occurred relatively recently compared to the geological first appearance of the morphological “species”, and mostly through adaptation to a new or existing ecological niche; convergent speciation events leading to the colonization of defined water-masses and their associated food web occurred independently in different lineages. C. Control of diversity: the number of sibling species within a mrelatively low number of open ocean biotopes, in addition to the intense and global water circulation characterizing the pelagic realm.

Colomban de Vargas–- ATIP-2004 Proposal 3

Choice of the taxonomic group An accurate identification of the genetic and morphological boundaries defining the level of species will be the first essential step before subsequent assessment of biogeographic distribution and origination time using both molecular and morphological –including fossil- data. In order to maximize the likelihood of detecting this fundamental species level of morpho-genetic differentiation, we will target a phytoplankton group presenting highly organized and complex morphologies, as well as the most complete and continuous fossil record: the coccolithophores. The coccolithophores secrete remarkable biomineral structures, the coccoliths, forming composite skeletons around the living cells. The coccoliths are constructed of calcite crystal units, whose nucleation, orientation, and growth are precisely controlled (Young et al. 1999), and whose arrangements in different numbers and varieties of cycles display an amazing diversity (Aubry 1998). Extensive SEM-based study of coccolith morphology and biogeography across the global ocean has been undertaken (e.g. Kleijne 1993, Winter and Siesser 1994, Cros and Fortuno 2003, Young et al. 2003) with the result that morphological species concepts should not be too far from being an accurate representation of the actual biodiversity. The extreme richness of morphological variability found in coccoliths should allow us to ultimately detect subtle and stable morphological and/or chemical characters associated with the level of species.

The coccoliths are also one of the primary components of the calcareous ooze that has covered a significant part of the ocean floor for the last 225 My (Bown et al. in press). This archive will permit us to track the newly detected species-level morphological characters down into the sediment record in order to recognize their first appearance in geological time, and thus obtain a stratophenetic estimation of the species’ age that can be directly compared to the molecular clock inference. Background: The coccolithophores are the most abundant organisms of the nannoplankton (2-20 µm size) in the oligotrophic ocean (Andersen et al. 1996). Some species produce seasonal blooms observed from satellites (Iglesias-Rodriguez et al. 2002) and they direct flows of climatically major elements and gasses (i.e. carbon, dimethyl sulfide). Their excellent fossil record is widely used for stratigraphic (e.g. Bown 1998) and diverse paleoceanographic studies (e.g. Bijma et al. 2001; Bollman et al. 2002; Beaufort et al. 1997, 2001). Despite the tremendous importance of coccolithophores in Earth system biogeochemistry, their basic biology and biodiversity are still poorly understood (de Vargas and Probert, in press). The coccolithophores belong to the phylum Haptophyta, whose uncertain origin roots more than 800 Mya according to molecular clock estimations (Medlin et al. 1997, Ben Ali et al. 2001). The Haptophyta contain 2 classes and 5 orders (Edvardsen et al. 2000, Parke and Green 1976; see also Saez et al. 2004) of biflagellate cells with a characteristic third flagellum-like appendage, the haptonema, whose function is related to attachment and prey capture (Inouye and Kawachi 1994). The extant coccolithophores are divided into 4 orders (Young et al. 2003), the Coccosphaerales, Isochrysidales, Zygodiscales, and Syrachosphaerales, within the class Prymnesiophyceae – the group containing all haptophytes that secrete organic plate scales covering the cells. According to a preliminary 18S rDNA phylogenetic tree, the coccolithophores derived from the Prymnesiales sometime in the late Paleozoic, although the first coccoliths known from the fossil record date from the late Triassic, ~225 Ma. Subsequently, morphological diversity increased progressively to a peak of ~150 morphospecies at the end of the Cretaceous (Fig.2). The group suffered a 90% extinction at the K/T boundary but rapidly recovered in the Palaeogene. In contrast to the Mesozoic, the morphological diversity in the Cenozoic seems to have been principally driven by climate fluctuations - higher diversities being associated with warm climates - (Aubry 1998, Bown et al. 2004). The number of morphological species living in the modern ocean is ~280 (12 families and more than 50 genera, see Young et al. 2003). This number is ~5 times higher

Colomban de Vargas–- ATIP-2004 Proposal 4

than the quantity of fossil species living at anytime during the Neogene, which reflects the fact that most species do not produce coccoliths robust enough to be preserved at the Ocean bottom.

Figure 2: The morphological view of coccolithophore evolution based on deep-sea sediment data. The curve represents mean species richness at 3 million-year intervals. Modified from Bown et al. 2004. Thus, the fossil diversity represents only a fraction of the actual biodiversity. In addition a number of recent discoveries challenge the conventional morphological taxonomy and systematics in coccolithophores: (1) About a third of the modern coccolithophore diversity is classified in a single family, the Calyptrosphaeraceae, which contains all of the coccolithophores covered with 'holococcoliths'. The holococcolith structure consists of minute euhedral crystallites whose biomineralisation is thought to occur externally. Although they are structurally radically different from the intra-cellularly calcified 'heterococcoliths' (Fig.3), recent studies suggest that all holococolithophores are in fact the morphological expression of a coccolithophore cell during the haploid phase of its haplo-diploid life cycle (Geisen et al. 2002, Billard 2002, Houdan et al. 2004). (2) The few recent ‘blind’ molecular studies based on environmental 18S rDNA clones (Edvardsen et al. 2000, Fujiwara et al. 2001, Moon Van der Stay et al. 2000, Diez et al. 2001) have all revealed the presence of new, sometimes very divergent phylotypes within thethose new taxa actually belong to the picoplanktonic fraction (<3 µm) in which no coccospheres are known, suggesting that there is a considerable unknown diversity of ‘naked’ coccolithophores yet to be described, as has been demonstrated in the foraminifera (Pawlowski et al. 1999, 2003). This unknown diversity may contain both new species and haploid stages associated with calcified diploid stages of known species.

haptophytes. Most of

y analyzed at a fine scale in the

Figure 3: Disconnection between genetic and morphological differentiation in coccolithophores. The linearized phylogenetic tree, based on the Tuf-A genemorphospecies are actually separated by millions of years of evolution according to the molecular clock calibrated on fossil data (Saez et al. 2003). SEM images on the right illustrate the extreme morphological conservation of coccoliths existing between sibling species that display high genetic differences in both nuclear and chloroplastic genes. (3) The five morphospecies that have been geneticall

shows that different varieties within 5

european project CODENET (Saez et al. 2003) were all revealed to be in fact monophyletic groups of sibling species, separated by extremely subtle morphological differences despite

Colomban de Vargas–- ATIP-2004 Proposal 5

millions of years of isolation according to molecular clock calculations (Fig 3). The CODENET data also provides preliminary indications that the sibling species within a morphological ‘species’ may inhabit different biogeographical ranges or seasonal niches, as has been shown for planktonic foraminifera (de Vargas et al. 1999, 2001, 2002; Darling et al. 1999, 2000, 2003). This is reflected in morphometric studies based on worldwide surface sediment data (Calcidiscus leptoporus, Knappertsbusch et al. 1997, 2000; Gephyrocapsa spp., Bollmann 1997, 1998), which show the presence of morphological variants occupying different ecological subdivisions of the total biogeographic range of the morphospecies. However, the genetic data from CODENET were strongly limited by a major technical barrier,

r phylogeny of the coccolithophores based

t-evolving DNA markers (D1-D2 regions of the LSU rDNA, ITS rDNA and

crete morphological and/or chemical characters of the calcareous liths that

d C. In our goal 1, we

i.e. the need to culture these organisms in order to obtain sufficient material for genetic analyses. This has prevented analyses of a high number and diversity of samples because the culture process is slow and labour-intensive. Moreover, species living in oligotrophic waters, as with most pelagic protists, are particularly fragile and have not yet been successfully isolated into laboratory culture. As stated previously, the vast majority of coccolithophore diversity is found in the oligotrophic, stratified Ocean. Here we propose new comparative morphological and genetic methods that will allow a rapid survey of global coccolithophore diversity. Whilst this project is primarily focused on investigating species-level differentiation within a few important morphospecies, it will also produce abundant data of direct relevance for the analysis of coccolithophore macro-evolution, global diversity (in particular the amount of ‘naked’ coccolithophores), and life cycles.

Proposed Research, preliminary and expected results: Practical Goals Our specific goals are to: 1- Reconstruct a comprehensive morpho-moleculaon 28S rDNA gene sequences and SEM analyses, compare it with the strato-phenetic knowledge of the group, and calibrate the molecular tree with geological dates wherever possible. 2-Use faschloroplastic Tuf-A gene sequences) to recognize and reconstruct the origins and phylogenetic relationships between the sibling species within 4 classical, model morphospecies, and analyze their worldwide biodiversity, biogeography, and depth distribution. 3-Identify dispermit accurate discrimination between the sibling species within each of the 4 model morpho-species; track those characters in the sediment record and compare the morphological versus molecular-clock estimations of species’ origins.

Fulfilment of those goals will permit testing of our hypotheses A, B, anwill include in the analysis a maximum number of extant species with known stratigraphic range. The total diversity of living morphospecies is not high and we expect to sample all important species and significant clades without difficulty. We have already obtained the largest molecular data set on coccolithophores, which consists of 75 LSU rDNA sequences (~2000pb length) from most of the species cultivated at the Algobank of Caen, the largest repository for living coccolithophores (de Vargas et al., in prep). However, a third of these species are strictly coastal with no or very poor fossil records (Pleurochrysidaceae, Hymenomonadaceae). New and abundant genetic data from the oligotrophic ocean produced during this project will allow to enlarge, consolidate and stabilize the global coccolithophore phylogenetic tree, a sine qua non condition for reliable and multi-nodes calibrations with the fossil record, and further accurate estimations of DNA substitution rates. The choice of the best calibration points in this new strato-molecular framework will allow us to estimate, using molecular clocks, the age of the sibling species.

Colomban de Vargas–- ATIP-2004 Proposal 6

The worldwide molecular dataset acquired in goal 2 will permit to estimate the biogeographic and/or hydrographic partitioning of the genetic diversity in coccolithophores, and generate hypotheses concerning the ecological history of the species complex: which hydrographic conditions were propitious for speciation of the ancestral sibling species, and which ecological niches where subsequently colonized and when?

Finally, our large-scale morpho-molecular comparative analysis on extant coccolithophore will be the key to track the sibling species in continuous and complete deep-sea fossil records, and check if the sibling species detected through molecular phylogenetics have an even more complex evolutionary history than the one revealed using molecular data (Fig. 1B). In other words, we will test for instance if the living morphospecies enclose a few biological species that appeared some time in the Miocene (Fig1A), or if the latter have short stratigraphic ranges resulting from more recent speciation events in the Pliocene or Pleistocene, from ancestral species that underwent extinction, possibly as a result of the latest climate fluctuations. Target morphological species: The model morphological species have been selected for (a) having a very wide biogeographic distribution and high frequency of occurrence; (b) representing a diverse and complete set of ecologies among the coccolithophores including eutrophic, oligotrophic and deep-photic species; and (c) having a good fossil record widely used in paleo-ecologic and/or stratigraphic analyses. Gephyrocapsa species complex This has been the numerically dominant group of coccolithophores since its origination ~4 Ma. Different morphological variants of Gephyrocapsa largely dominated the nannoplankton assemblages during most of the Pleistocene (Fig. 4 and Fig. 5), until they gave rise to Emiliania ~250 000 ya, the latter therefore being considered as part of this species complex. Gephyrocapsa spp show a preference for mesotrophic environments but occur in virtually all samples, and sometimes create massive blooms that play major ecological roles in dymethyl sulfide production and carbon export (Bijma et al. 2001). Figure 4: Acme interval in DSDP hole 610B. The graphs refer to percentage relative abundance of nannoplankton species. Although these data represent a single core, the pattern is very widespread. (modified after Hine and Weaver 1998). On conventional taxonomy there are 3 common Gephyrocapsa species (oceanica, muellerae, and ericsonii) whilst Emiliania contains the single species E. huxleyi. However morphological data supported by laboratory culture work, biogeographic analysis, and immunological work indicate that all four species consist of at least 2-3 ecologically discrete morphotypes (Young and Westbroek 1991; Bollmann 1997; Geisen et al. 2004). For instance, the 6 discrete Gephyrocapsa morphotypes described by Bollmann (1997) based on global surface sediment data clearly occupy different latitudinal provinces. The high frequency of this taxon, its simple isolation into culture, and the availability of the complete DNA sequence for the E. huxleyi nuclear genome (250 Mbp) (Betsy Read, de Vargas et al, in progress), make it an ideal target. Calcidiscus leptoporus C. leptoporus is a relatively large, moderately common, meso-oligotrophic coccolithophore with an exceptionally good fossil record, extending back to the early Miocene (ca 23 Ma, Fig. 5). The species displays morphological variants that have been the subject of taxonomic debates for more than 80 years (Quinn et

Colomban de Vargas–- ATIP-2004 Proposal 7

al. 2004). C. leptoporus has proven an ideal test taxon for the study of microevolution and diversity within the coccolithophores (e.g. Knappertsbusch et al. 1997, Renaud et al. 2002, Saez et al. 2003, Quinn et al. 2004). Morphological work based on global sediment samples has shown that the morphospecies includes at least 3 discrete morphotypes with separate but overlapping biogeographic ranges (Knappertsbusch et al. 1997). First genetic analysis of 2 of those morphotypes indicated that they diverged ~11 Ma (Fig.3 and Saez et al. 2003). In this project we propose to investigate the biogeographic distribution and ecological significance of the established morphotypes through sampling along transects in different oceans. This will test the alternative hypotheses that the morphotypes represent 3 well-separated and globally distributed species or are components of a much more complex mosaic of related taxa. C. leptoporus is also one of the morphospecies on which we have

positively tested the single-cell PCR amplification protocol (see below). Figure 5: Both morpho-species Gephyrocapsa and Calcidiscus have reliable stratigraphic records with known ancestors, which allows accurate calibration of the molecular tree, and further assessment of the evolutionary history of the sibling, pseudocryptic species within each clade. Such excellent fossil frameworks exist for most coccolithophore families and will be used to calibrate our global coccolithophore tree.

Florisphaera profunda F. profunda is the dominant deep-photic coccolithophore (e.g. Okada and Honjo 1973, Kinkel et al. 2000). The liths it produces are enormously abundant in the fossil record and first recorded at least 7 Ma (Okada and Matsuoka 1996). The ancestor of F. profunda may be Minylitha convallis, a short-lived (9 to 7.2 Ma) morphospecies of the late Miocene (Aubry, pers comm); however the relationship of this group to other coccolithophores is totally unknown. Florisphaera liths are morphologically simple and easy to recognize, and are a key indicators of surface oligotrophy and nutricline depth in palaeoceanographic studies (e.g. Molfino and McIntyre 1990, Ahagon et al. 1993, Kinkel et al. 2000, Beaufort et al. 1997, 2001). Despite being globally distributed, Florisphaera has been conventionally regarded as monospecific. Quinn et al. (in press), however, have shown that the species varies greatly in size, shape, and ornament of the liths. At least 5 discrete morphotypes can be recognized, whose presence and abundance in the water column seem to be determined by variations in nutrient concentrations. As with

Colomban de Vargas–- ATIP-2004 Proposal 8

Umbellosphaera (below), this taxon has not been maintained in culture and essentially nothing is known of its biology despite its great ecological and geological interest. Our worldwide morphogenetic study of F. profunda will (1) determine the phylogenetic origin of the ‘species’, (2) estimate the age and ecological niches of the morphological variants, (3) test for genetic isolation between the basins in a deep photic species. These results are expected to greatly enhance the power of Florisphaera as a paleo-ecological marker. Umbellosphaera tenuis Umbellosphaera is the dominant coccolithophore in oligotrophic waters (e.g. Okada and Honjo 1973, Kinkel et al. 2000) and indeed one of the most widespread and common phytoplankton taxon in the oligotrophic open ocean. The genus has a reliable fossil record through the Pliocene but only became abundant in the Quaternary, possibly as result of ecologically replacing Discoaster spp. Umbellosphaera is conventionally regarded as consisting of only two species but Kleijne (1993) and Young et al. (in press) have argued that at least 6 morphotypes can be consistently distinguished. This genus has not been maintained in culture, there is no molecular data on it and the distribution of the morphotypes is very poorly known. It is particularly attractive for our study since the oligotrophic ecology means that it occurs in a set of semi-isolated populations across the sub-tropical oceans of the world.

Collection of sample materiel and field work Living plankton: The open Ocean is characterized by intense mixing rate on a global scale, with no obvious barriers to gene flow between water-masses. In these conditions, the acquisition of a worldwide morphological and molecular dataset covering the latitudinal and depth ranges of the target morphospecies is a sine qua non condition to distinguish the morphological variation due to ecophenotypy from that due to reproductive isolation and evolution. Our targeted biological sampling strategy has three components (Fig. 6): (1) 2-week missions to collect in waters around tropical islands located in the 3 main oceanic basins. At least 3 daily cruises will be undertaken during each of these missions, in order to thoroughly sample the local coccolithophore diversity, and compare it from basin to basin on a worldwide scale. We have been offered laboratory space from 3 marine stations in Puerto-Rico, Hawaii, and Reunion Island. These marine laboratories all present an important common advantage: they border narrow shelf areas leading to very steep shelf breaks. Deep open-ocean waters with permanent vertical stratification and typical open-ocean oligotrophic phytoplankton assemblages can thus be reached only a few miles offshore with a small boat at low cost. Real-time CTD and chlorophyll data will be acquired with an autonomous profiler. Samples will then be collected with plankton nets (mesh-size: 5 and 10µm, ring-diameter: 50 cm) and 10l Niskin bottles at 4 different depths, below the Deep Chlorophyll Maximum (DCM), at the DCM, in the Middle Photic Zone, and in the Upper Photic Zone. The ‘Niskin water’ will be filtered on 0.8µm polycarbonate membrane filters for subsequent morpho-genetic analyses of the total coccolithophore assemblage, while the ‘net material’ will principally serve for single-cell morpho-genetic analyses (see below). (2) Three basin-scale transects in the Indian, Atlantic, and Pacific oceans (Fig. 6). All 3 cruise tracks cross large oligotrophic central water-masses, in addition to passing through a broad range of waters with different vertical stability and productivity levels. Relatively frequent sampling stations every 200-300 km will allow us to estimate the biogeographic distribution patterns for each coccolithophore sibling species. At each station, water samples from 6 different depths will be filtered on different kind of membranes for total-assemblage morpho-genetic analyses (see below). CTD casts will provide standard oceanographic data (salinity, temperature, chlorophyll) at each station, and allow us to characterize the sampled water-masses. Note that the material from the Indian Ocean transect was already collected in May-June 2003, and the material from the Pacific (BIOSOPE) and Atlantic (AMT-17) will be collected respectively in October 2004 and September 2005.

Colomban de Vargas–- ATIP-2004 Proposal 9

Figure 6: Location of proposed sampling sites for data collection of living and fossil coccolithophores. In addition to the materiel collected during the 4 ‘island missions’ (stars), nannoplankton samples will be collected on filters at 70 pelagic stations and 6 depths per station, during basin-scale cruises in the Indian, Pacific, and Atlantic oceans. The little rhombohedra in the Indian and Atlantic oceans depict the stations from which we already got filtered samples for morphogenetic analyses; a total of 253 samples were collected during the last 2 years. (3) Time series sampling to test for seasonal changes in coccolithophore morpho-genetic diversity will be undertaken in Greece and Puerto-Rico with the help of our local collaborators. Dr. Maria Triantaphyllou (University of Athens) is leading a long-term survey of coccolithophore distribution and seasonality along 2 transects offshore the island of Andros, Aegean Sea (Triantaphyllou et al. 2002a, 2002b). Dr. Triantaphyllou systematically splits the filters into two pieces and preserved one half in GITC buffer (de Vargas et al. submitted) for comparative genetic analyses. Half of the worldwide coccolithophore morphological diversity is found offshore Andros, and numerous morphotypes display interesting adaptation patterns to particular depths or seasons. A comparison of this ‘greek dataset’ to the 3 ‘oceanic islands data sets’ will permit to test if the relative disconnection of the Mediterranean Sea from the global ocean system since the Messinian salinity crisis 5 Ma led to some extent of mediterranean endemism at the genetic level. Prof. Nilda Aponte –Director of Marine Sciences at the University of Puerto-Rico- has also expressed great interest in collaboration, and will help coordinating time-series nannoplankton sampling at different seasons, a few miles offshore the marine station of Magueyes Island. More than 100 coccolithophore morphospecies inhabit the permanently stratified Puerto Rican waters, and previous studies have shown distinct seasonal biodiversity fluctuations (Jordan and Winter 2000). Finally, we will attempt to PCR amplify DNA extracted from dried filters collected by Drs. Jacques Giraudeau (Université of Bordeaux, France) and Luc Beaufort (CEREGE, France). Typically, micropaleontologists studying living coccolithophore assemblages filter seawater through a membrane, which is then air-dried and stored in a Petri-Slide. This preservation method may be suitable for DNA analysis, as demonstrated by positive PCR results from single foraminifer after 2 years of dry preservation (Holzmann and Pawlowski, 1996). Dr. Giraudeau has a collection of 2200 dried filters from 12 huge cross-oceans transects (France-New York – Panama-New Caledonia) that took place at different seasons during the last 3 years. During each cruise, surface waters samples were collected at 200 sites. Dr.

Colomban de Vargas–- ATIP-2004 Proposal 10

Beaufort has a personal collection of more than 200 filters distributed in the Atlantic, Pacific and Indian Oceans that will also be available for our study. Fossil plankton: The ODP sediment cores (Fig. 6) in which we plan to track the morphological first-appearance of the coccolithophore sibling species have been selected based on several criteria: (1) they have very good Neogene nannofossil records including our target morphospecies; (2) they have been the subject of previous high quality nannofossil investigations and thus have accurate age models; (3) they come from relatively shallow water, and are sedimentologically suitable (i.e. high sedimentation rate –between 2.2 and 4.9 cm/kyr-, minimal lateral transport, reworking, winnowing or bioturbation of sediment). However, the choice of cores will ultimately depend on our morpho-molecular results. The possible discovery of a provincialism between the sibling species higher than suspected may reorient our initial ODP sites selection. Target genes: Most recent molecular surveys of pelagic protistan biodiversity have used the 18S rDNA gene. This gene has revealed an astonishing number of totally new phylotypes, including novel classes or phyla according to genetic distances (e.g. Lopez-Garcia et al. 2001, Moon-van de Staay et al. 2001, Massana et al. 2002, Moreira and Lopez-Garcia 2002, Vaulot et al. 2002). Often, variations in the 18S gene are assumed to unravel species diversity, just as genetic distances in the 16S gene are routinely used by microbiologists to delineate ‘species’ in bacteria. However, the 18S rDNA is subject to considerable purifying selection and is not appropriate for species-level phylogenies. The few estimations of 18SDNA substitution rates in marine protists using fossil calibrations indicate mean values of ~0.035, 0.1, and 0.04 %/Ma, respectively for the diatoms, coccolithophores, and dinoflagellates (Medlin et al. 1997; Saez et al. 2003; John et al. 2003). This means, for a DNA fragment of 1000 bp, the occurrence of 1 to 3 DNA substitutions for 3 million years of evolution. Indeed, the 18S rDNA most often cannot discriminate any evolutionary events occurring in the last 2 My, and is a very inaccurate marker for the last 5 My. In our project, we will use the 5’ fragment (~1500 bp) of the 28S rDNA gene for the global coccolithophore phylogeny (goal 1). This DNA fragment contains the D1 and D2 domains that evolve between 5 and 10 times faster than 18S rDNA (unpublished data). The resolution power of the 28S rDNA should allow accurate detection of all Pliocene speciation events (5.32-1.81 Ma), and the majority of Pleistocene events. Additional nuclear and chloroplastic markers –protein gene- will be used in case of obvious disagreement between the rDNA and fossil morphological data. The genetic analyses at the sibling species level (goal 2) may require faster evolving molecular markers. Our parsimonious approach will consist of initially testing whether the 28S rDNA is adequate to resolve this taxonomic level (the youngest of the 4 target morphospecies appeared 4 Ma). Fast evolving nuclear ITS rDNA and chloroplastic Tuf-A sequences (Saez et al. 2003) will be used either to confirm the 28S rDNA data, or as a substitute if sibling speciation events turn out to have been associated with more recent climatic events (i.e. the last glaciations). Note that we may have to look for even faster-evolving genetic markers, in the nucleus, chloroplast, and mitochondria. Full genomes (mitochondria, chloroplast, and nucleus) sequences of the coccolithophore Emiliania huxleyi are completed and will provide a huge amount of information and potential genetic markers for our project. Methods The success of our research goals will depend on our ability to rapidly analyze a large number of samples from the global ocean. For a first-order, rapid survey of the morphological diversity in our samples, we will use the Automatic System of Coccolith Recognition SYRACO recently developed by Beaufort and Dollfus (Dollfus 1997, Beaufort and Dollfus in press), a technique based on artificial neural network (ANN) recognition that has greatly proved its efficiency (Beaufort et al. 1997, 2001).

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Molecular techniques for examining microbiological diversity and phylogeny are now extremely powerful, but the step consisting in identifying the organism hidden behind a DNA signature still represents a major difficulty. This is particularly true for studies of natural biodiversity, since we know that the vast majority of unicellular organisms do not grow after isolation into existing culture media. One of the major innovations of our project is the use of simple new protocols to systematically combine morphological and genetic analyses. Single-cell morpho-genetic analyses Freshly collected coccolithophore cells are isolated by hand –using ultra-fine Pasteur pipettes- under an inverted or dissecting microscope. The cells are directly transferred to the DNA extraction buffer GITC* in a 96-well filtration plate (Fig. 7). Vacuum filtration is applied, and the coccoliths are retained on the filters while the extracted DNA is individually recovered into a 96-wells underdrain plate (Fig. 7). The filters are rinsed with a few drops of pH 8.5 water. The coccoliths and DNA extracts form the basis for subsequent comparative genetic and morphological analyses in the laboratory. Single-cell analyses will be mainly applied during the island missions.

Figure 7: GITC*: a direct morpho-genetic analysis of single planktonic protist cells. This technique was developed for foraminifers by de Vargas et al. (2003, submitted), and recently successfully applied to coccolithophores and other protists. A. The Vacuum manifold that allows simultaneous filtering of a large number of single cell samples, and separation of the shell (on the filtration plate) from the DNA extraction (underdrain plate). B. The filters from A are punched and individually analyzed by SEM. illustration shows a SEM zooming on a single coccolith of Umbilicosphaera sibogae. C. PCR amplification products (rDNA) on agarose gel from single cell analyses. The GITC* buffer rapidly dissolves all organic components of the cell but does not affect the CaCO3 skeleton. The first example shows the result of 38 PCRs on 38 single specimens of the foraminifer G. truncatulinoides. Success rate is ~90%; SEM pictures show skeletons that have been recovered after DNA extraction. The second example shows our preliminary results on the coccolithophore species C. pelagicus and C. leptoporus, using a gradient from 1 to 500 cells for the PCR reactions. This protocol can be applied on a large number of cells at low cost, and allows a rapid morpho-genetic survey of protist diversity on large geographic scales (de Vargas et al. 2001, 2003, submitted).

Total-assemblage morpho-genetic analyses For each location, water samples (10l from Niskin Bottles; note that there are typically between 500 and 5000 coccolithophore cells per liter of oligotrophic water) are filtered through both polycarbonate and cellulose acetate filters. The cellulose filters are used to rapidly detect the presence/absence and abundance of our target taxa using SYRACO (see below). The polycarbonate filters are used for total

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DNA extraction, FISH (Fluorescent In Situ Hybridization), and SEM morphological and ultrastructural analyses. This approach will mainly be used for the basin-wide cruises, where a single student can easily collect the material. Total DNA will be extracted from the frozen filters using the DNeasy Plant MiniKit (Quiagen), and used for 2 kinds of analyses. Firstly, and principally, the DNA extractions from around the world where our target taxa have been localized and quantified in parallel using SYRACO will serve as template for PCR amplification using primers specific for the selected morpho-species. Thus, we will be able to rapidly detect the worldwide genetic diversity within each morpho-species, while in parallel examining their morphological and ultrastructural characteristics by performing SEM analyses. Secondly, if morphological analyses of the coccoliths reveal interesting features on a particular filter, we will use taxonomically more or less specific primers (targeting an order, family, or genus) to PCR amplify genes from the group of interest. On the other hand, if new 28S rDNA genes are sequenced and do not cluster with any morphological group, specific oligonucleotide probes will be designed and used in combined FISH-SEM analyses as described in Stoeck et al (2003), in order to associate a morphology to the DNA sequence. PCR, cloning, and sequencing Oligonucleotide primers with different specificities (haptophyte, coccolithophore, or different taxonomic levels within the coccolithophores) will be designed progressively throughout the project. We already have a set of 41 coccolithophore-specific primers within the 28S rDNA. The PCR products will be purified and directly sequenced whenever possible - for instance for the single-cell DNA extraction and species-specific PCR. If required, the PCR fragments will be ligated into the pGEM-T Vector System and cloned in XL-1 ultra-competent homemade cells before sequencing. Once genetic differences are accurately identified between sibling species, simple PCR-RFLP protocols will be used to rapidly assay at low cost a large quantity of samples from all over the world, as in de Vargas et al. 1999, 2001. Phylogenetic analyses and molecular clock calibration The 28S rDNA, ITS rDNA, and Tuf-A DNA sequences will be aligned using ClustalX and the alignments will be further improved de visu (Genetic Data Environment, Larsen et al. 1993). For each DNA alignment, hierarchical likelihood ratio tests (hLRT) will be performed to estimate the DNA substitution model that best fits the data set (ModelTest, Posada and Crandall 1998). Maximum likelihood analyses (using PAUP*, Swofford 2000) and Bayesian statistics (MrBayes, Huelsenbeck and Ronquist 2001) will be performed on unambiguously aligned sites to reconstruct phylogenetic trees. In parallel, relative rate tests (RRTree, Robinson-Rechavi and Huchon 2000) and likelihood ratio tests between trees reconstructed with and without constraint of a molecular clock (PAUP*, Lintree, Takezaki et al. 1995) will be used to analyze the relative rates of DNA substitution between the sequences, and eventually eliminate the sequences whose substitution rate significantly differs from the mean rate calculated using the total alignment, so that a global molecular clock can be approximated for the remaining sequences. The best DNA trees will be compared to phylogenetic schemes proposed from palaeontological studies of fossil coccolithophores and the program StratCon (Huelsenbeck 1994) will be used to test the consistency of phylogenetic trees with the stratigraphic record. All homologous phylogenetic nodes observed between the DNA and fossil trees will be used as absolute calibration points for molecular clock computation. Alternatively, programs allowing for different rates of DNA substitutions across lineages (i.e., taking into account a "relaxed" molecular clock) will be tested (QDate –Rambaut and Bromham 1998; multidivtime –Thorne et al. 1998) and the resulting tree calibrations will be compared to those obtained with the assumption of a global molecular clock. Morphological analyses: Analysis of morphological variation within the morphospecies will be undertaken by a combination of (1) light microscope based study of size/shape/weight variations of coccoliths using automated measurement of digital images captured with SYRACO. SYRACO is the first integrated system to automatically recognize coccoliths at low

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taxonomic level (morpho-genus to morphological sub-species levels depending on the taxa). The routine version of SYRACO is able to identify the 11 most abundant living taxa, at a rate of 98% and at a speed of more than 1000 individuals per minute. It saves the image of the identified specimens in taxon specific files which permit further rapid morphological analyze; (2) SEM examination of fine ultrastructural variations. Our attention here will focus on the detection of discrete and stable morphological characters of the coccolith (geometry, structure, cycles, ornaments) that characterize a sibling species. The morphological analyses will be conducted in close and continuous interconnection with the genetic analyses. Filters - from single-cell or total-assemblage samples - where genetic differences are detected will be examined with particular caution. Together with the molecular-clock and biogeographic analyses, the detection of the morphological characters discriminating between the sibling species will allow us to select the appropriate ODP cores and search for the appearance of the morpho-characters in the relevant sediment layers. This work is labor intensive but ideally suited to the use of undergraduate and DEA students; it will also be coordinated by at least 3 laboratories (de Vargas, Roscoff; Beaufort, CEREGE Aix; Probert, Algobank Caen).

Significance During the past decades, most research programs aiming at studying the pelagic biosphere have focused on global matter/energy fluxes and remote sensing, avoiding the problem of understanding the natural history and ecology of planktonic organisms at the species level. There are very few studies in evolutionary systematics of oceanic plankton compared to coastal marine, fresh water, and terrestrial organisms and most often microplankton is divided into rough and unrealistic functional groups. However the species –and their populations- are the fundamental biological units on which natural selection and genetic drift act, and the species ultimately control biological fluxes. Today, molecular and morphological technologies are capable of analyzing planktonic protists at the individual level, and we propose here, for the first time, to investigate species-level systematics and biogeography in an entire group of phytoplankton over global scale. We will take advantage of the remarkable quality of pelagic fossil record to transfer our morpho-genetic information to the study of speciation in Neogene deep-sea sediments. Thus, we will try to solve the paradox existing between the amazingly stable morphological evolution in the deep-sea fossil record –displaying similar micro-skeletons over ranges of million years-, contrasting with the hectic biological/organismic turnover occurring in the modern ocean, and the frequent climatic fluctuations that have strongly affected the pelagic ecosystems on relatively short time scales. Our study will unravel for the first time which time scales control speciation dynamics in a group of oceanic phytoplankton. It will provide essential ecological and genetic data to the vast community of scientists using microfossils as a routine tool to analyze oceanic and climatic changes, and thus greatly improve future exploitation of the deep-sea fossil record as an archive of the past. Finally, considering the projected severe climatic changes in the near future, we believe that one shouldn’t wait to start assessing how pelagic micro-diversity evolved over the last few million years, and therefore may react in the close future. Project Management and collaborations. This project will be principally realized at the Marine Station of Roscoff. I will take overall responsibility for coordinating scientific and administrative aspects of the proposed research, and will ensure continuing communication and meetings between all participants. In addition to the ATIP-postdoc, a PhD student will be fully involved in the project: Mr. Miguel Frada, from the University of Lisbon, just got a FCT (Fundação para a Ciência e a Tecnologia) fellowship for 4 years of work in my laboratory and thus will be an additional free of charge investigator. Multiple collaborations are planned in different areas of the project. I will lead the molecular part in Roscoff in close collaboration with Dr. Daniel Vaulot and his “Oceanic Plankton” team,

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international experts in the application of flow cytometry, FISH, and genomics to the study of pelagic microbes. My current collaboration with Dr. Betsy Read (Cal State University, San Marco, USA) for the annotation of the Emiliania huxleyi nuclear genome -starting in Winter 2004- will ensure a front position for the discovery and application of new molecular markers. The sampling missions and coccolithophores isolation/culturing will be realized with Dr. Ian Probert and Prof. Chantal Billard from the University of Caen. Dr. Probert is a leading expert in the culture, taxonomy and ecophysiology of extant coccolithophores. He was responsible for the development of the largest and most diverse culture collection of living coccolithophores existing today, maintained at the AlgoBank of Caen. The collection will be largely increased during the project to become a worldwide reference. The oceanographic cruises will involve collaboration with Dr. Herve Claustre (BIOSOPE, CNRS, Villefranche-sur-Mer) and Prof. Patrick Holligan (AMT, Southampton Oceanography Center). The morphological analyses will be achieved in collaboration with Dr. Luc Beaufort from the CNRS-CEREGE, Aix. Dr. Beaufort is an internationally renowned paleoceanographer; he designed the first integrated optical system to automatically recognize coccoliths (SYRACO). Beaufort will screen all the collected samples for coccoliths/coccospheres diversity and abundance of the target taxa. He will also actively participate in SEM analyses, together with Dr. Probert and myself. Concerning the micropaleontological analyses, I intend to continue my collaboration with Dr. Aubry at Rutgers (NJ, USA), and Dr. Young from the NHM of London. They are among the few most experienced and respected specialists of nannoplankton morphological systematics and fossil evolution. Finally, this project will indirectly involve the numerous international collaborations I have established during my PhD in Switzerland, and post-doc and professorship in the US (Harvard, WHOI, and IMCS at Rutgers). In particular, I will keep a formal partnership with the Institute of Marine and Coastal Sciences, Rutgers, where I am currently working, in order to pursue my involvement in the International Census of Marine Life (CoML, http://www.coml.org/coml.htm). Research on plankton biodiversity/biogeography is the weakest component of the CoML and our project will serve as a pilot study to estimate on a worldwide scale the amount of unknown diversity in a group of phytoplankton. All genetic, morphological, stratigraphic, and biogeographic data obtained during the project will be integrated and stored into a web-site fully interoperable with the Ocean Biogeographical Information System (OBIS) network of databases. Living, morphological and genetic material will be preserved in culture and tissue collections and made available to the wider scientific community.