Within the CODENET project work will be carried out on fifteen research tasks. Taken together these are intended both to maximise the potential of our collaboration and to give a provide a complimentary set of studies addressing our joint objectives. The notes here summarise the work to be undertaken and indicate the main participating teams.

      RT1. Culture isolation and maintenance (U. Caen, ETHZ)

      For all taxa new cultures will be isolated with a final target of 5-10 strains each, enough to assess first-order genotypic variation within the population. For Gephyrocapsa, a more extensive collection will be developed, to allow a case study in species level variation. Isolations will be based on sampling work in the Mediterranean, and N. Atlantic, via participation in local cruises from CSIC, and MNHN-UL. The ETHZ team will provide additional water samples from Bermuda, Hawaii, Puerto Rico, Canary Islands, and a N-S transect through the Atlantic (AMT-cruise 1997) and carry out some isolations from these samples. The principal culture collection, including duplicates of all strains, will be maintained at U. Caen, with free access to all network participants.

      RT2. Life-Cycle studies (U. Caen)

      We will use growth under varying conditions to induce life-cycle changes, flow cytometry to measure DNA content in different life-cycle stages and TEM fine structural studies to investigate the cytology of mitosis. N.B. For Coccolithus pelagicus, we can induce life-cycle changes (Rowson et al. 1986) and this experience should be directly applicable to Calcidiscus, and possibly Umbilicosphaera . Similarly experience with Emiliania (Green et al. 1996) should be directly applicable to Gephyrocapsa. Syracosphaera and Helicosphaera represent novel challenges.

      RT3. Molecular genetics (AWI)

      • A: Plastid genome studies for higher level phylogenetic analysis. Work comprises: measurement of plastid genome size; creation of a plastid genomic library to isolate and characterise specific gene order and arrangements for comparison with other algal genomes; localisation of specific genes within the nucleus or plastid with oligonucleotide probes to infer evolutionary pathways in plastid endosymbiosis.
      • B: Studies using slow evolving genes: sequencing of the 18S rRNA and * Tuf A genes to calculate evolutionary rates and to construct a molecular clock for the coccolithophorids. Synthesis of results from this data set and existing data from lsu RUBISCO to estimate time of origin.
      • C: Studies using fast evolving genes: sequencing of *non-coding regions (Intron regions in the calmodulin gene and spacer regions between the trnT and trnF gene, between the petB and D genes, and within the rRNA operon) to reconstruct the phylogenetic relationships among closely related keystone taxa. (AWI); construct a molecular clock to estimate time of divergence times of species.
      • D: Study of population level relationships: development of microsatellite probes to evaluate gene flow and dispersal and divergences in populations within the Gephyrocapsa/Emiliania complex. N.B. Microsatellites are being developed for Emiliania huxleyi (NERC funded to AWI and Univ Bristol, UK) that can be used in this project.

      RT4. Lipid biomarker composition and palaeothermal calibration (NIOZ, CSIC)

      Investigation of variation of lipids produced by coccolithophorids - including particularly biomarkers producing palaeothermal signals. Lipids will be analysed: (A) From the different taxa; (B) between strains of individual taxa; (C) from cultures of single taxa grown under varying conditions: (D) from suites of oceanographic and geological samples with well established coccolithophorid floras - see below RT11-14. Lipid analyses using gas chromatography will be carried out at NIOZ and CSIC (who already have a well established collaboration). The NIOZ analyses will also include study of stable carbon isotope fractionation into the specific lipids and into coccoliths.

      RT5. Photosynthetic pigment studies. (CSIC, NIOZ)

      HPLC analysis of chlorophylls and carotinoids from different taxa, and from cultures of single taxa grown under varying light conditions.

      RT6. Coccolith ultrastructure analysis (NHM)

      Culture material containing early growth stages will be used to precisely describe coccolith ontogeny and ultrastructure (cf. Young 1993), with additional quantitative study of crystallographic orientations. This work will be carried out in collaboration with Prof. S. Mann of Bath University.

      RT7. TEM fine structural studies (U. Caen)

      Material from culture samples will be to investigated by transmission electron microscopy in order to determine cytological characters, particularly flagellar and golgi body ultrastructure, and scale morphology.

      RT8. Phylogenetic synthesis (NHM, AWI)

      Cladistic methods will be used to combine the diverse data of phylogenetic significance obtained from studies of coccolith ultrastructure, biochemistry, cell fine structure, etc., to produce a synthetic interpretation of the relationships of the keystone taxa, and to extend the results to other taxa for which partial data sets are available. This will then be compared with results from molecular genetics (RT3) and geological studies (RT15), using both separate and combined analyses (cf. Smith 1994).

      RT9. Physiological characterisation (NHM, ETHZ)

      A range of culture experiments will be carried out in order to characterise the ecophysiology of the different taxa, and to investigate the variance in response between strains of single taxa. This work is fundamental to understanding the ecological diversity in the group and its relationship to genotypic diversity. Specific growth rates, biomass and coccolith production rates in response to varying light, temperature, and nutrient levels will be investigated. The principal methodology will be laboratory batch culturing. This method is straightforward and work in the EHUX project comparing batch culture, mesocosm, and field populations showed that it is an appropriate methodology for studying ecological response of coccolithophorids.

      RT10. Morphological work on cultured samples. (NHM, ETHZ, MNHN-UL)

      Morphometrics will be used to analyse variation in coccolith morphology in samples grown in culture. Approximately 50 samples will be analysed per species with the basic data set including: (A) Single samples grown under uniform conditions from all isolated strains. (B) A large suite of samples from a single strain grown under varying conditions. Sub-task distribution: MNHN-UL C. pelagicus ; NHM - U. sibogae, H. carteri and S. pulchra. ETHZ C. leptoporus and Gephyrocapsa.

      RT11. Study of plankton assemblages - ecological characterisation (ETHZ, FdA-VUA, CSIC, MNHN-UL)

      Phytoplankton filter sample collections are held at ETHZ, FdA-VUA, CSIC and MNHN-UL, including at ETHZ repeat sampled stations from Bermuda, Hawaii, Puerto Rico and the Canary Islands. Selected sample sets will be morphometrically analysed. Cruise opportunities will be available from ETHZ, CSIC, and MNHN-UL which will be used for special study of coccolithophorid development relative to hydrographic conditions and, in particular, other phytoplankton. Selected samples will be used for comparative lipid analyses (RT4D).

      RT12. Sediment trap studies - flux rates and seasonal succession. (ETHZ, FdA-VUA)

      These studies will be used to determine ecology of the species relative to the seasonal succession and to calculate species-specific carbonate fluxes. FdA-VUA has a large collection of sediment trap samples, including subsamples from the Honjo and Thunell collections specifically provided for studies of coccolithophorids. Additional trap samples from Bermuda and Hawaii will be provided by ETHZ. Morphometric studies using these will be carried out by ETHZ and FdA-VUA. Selected samples will be used for comparative lipid analyses (RT4D).

      RT13. Holocene sediment samples - global biogeography. (ETHZ, FdA-VUA MNHN-UL).

      An extensive collection Holocene samples has already been assembled at ETHZ (see FIGure). These will allow mapping of the global biogeography of the studied species relative to total nannofossil assemblage and in abundance per unit weight of sediment. Sub-task distribution: MNHN-UL C. pelagicus; FdA-VUA - U. sibogae, H. carteri and S. pulchra. (N.B. Calcidiscus and Gephyrocapsa studies have been carried out already at ETHZ - Bollman (in press), Knappertsbusch et al. (in press)). Selected samples will be used for comparative lipid analyses (RT4D).

      RT14. Geological sample studies - microevolution and ecological response. (NHM, ETHZ, FdA-VUA, MNHN-UL).

      Sequences of samples will be obtained from each of six DSDP/ODP sites (provisional selection Sites 552, 607, 704, 706-716, 803-807, 925-929 or 959-960) to provide coverage of the early Pliocene to Recent. Slides will be prepared using quantitative techniques to allow calculation of accumulation rates (specimens per unit area of sea bed per unit time) and microevolution of the taxa will be studied using morphometrics. Morphometric data and accumulation rates will be combined to calculate species-specific carbonate accumulation rates. Sub-task distribution: ETHZ - Calcidiscus and Gephyrocapsa; MNHN-UL C. pelagicus; NHM, FdA-VUA - H. carteri, U. sibogae, and S. pulchra. Selected samples will be used for comparative lipid analyses (RT4D).

      RT15. Macroevolutionary studies - divergence times of the lineages. (NHM, ETHZ).

      The evolutionary origins of the studied taxa will be reappraised by close study of critical intervals. This will be primarily based on extensive collections of geological samples already held by the participants.

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