MINUTES OF THE PICODIV MEETING HELD AT
SBR ROSCOFF JULY 2002
Note: All presentations are available as ppt or pdf files from the PICODIV web site
Attending
 SBR: Vaulot, Not, Romari, Simonnet, Biegala, Groisillier, Doussal,
 UW: Scanlan, Fuller, Hastings
 AWI: Medlin, Valentin, Mehl
 ICM: Pedros-Allio, Massana, Latasa, Guillou
 UO: Throndsen, Eikrem
I
WORKPACKAGE I (CULTURES)
Three short presentations were given:
- Valentin: current status of isolation of cultures from Helgoland
- Guillou: current status of isolation of cultures from Blanes
- Vaulot and Le Gall: current status of the Roscoff Culture Collection
The emphasis of year 3 is to publish the description of novel species obtained in culture. The following
list of the papers is planned for each taxonomic group (in bold/red are the high priority papers). For each
new taxon the following information will be used: ultrastracture (from UO), photosynthetic pigments (from
ICM), for cyanobacteria 16S sequence (UW) and for eukaryotes 18S nuclear sequence (from AWI, SBR,
ICM) and 16S plastid sequence (from UW). Additional gene sequences (rbcL) will be eventually acquired
as needed.
 Cyanophyceae
- Fuller et al Ultrastructural diversity amongst the marine Synechococcus genus. This paper
will combine ultrastructural from Jahn and Wenche and phylogenetic information from Dave
and Nick. It will rely on the phylogenetic analysis published in the probe paper planned in Wpk
3.
 Prasinophyceae
- Guillou et al. Genetic diversity within Prasinophyceae based on 18S sequences.
- Guillou et al. Mamiellales combining TEM, pigment data and phylogenetic information. The
description of a new Mamiella species (RCC 391) will be included in this paper.
- Eikrem et al. will describe a new order within the Prasinophyceae (CCMP 1205)
- Latasa et al. will discuss pigment composition within the Prasinophyceae
 Telonema
- Kamran et al. Telonema as the common ancestor of alveolates and stramenopiles to be
submitted to Nature.
- Eikrem et al. Ultrastructural analysis of Telonema subtile
 Dictyochophyceae
- Eikrem et al. Description of a new species (Florenciellia legallia = RCC 446). It could be
interesting to complement this paper with some information on the genetically closely
related strain RCC 505 from Blanes (full 18S, pigments).
- Later, it could be interesting to do an analysis of Rhizochromulina related strains including the
flagellate RCC 332.
 Prymnesiophyceae


II
Chrysochromulina (lower priority): Two potentially novel species are present in the RCC (RCC
390 and 400). More data need to be acquired (full sequences, pigments, details of scales).
Chrorarachniophyceae
- RCC 365 is a small flagellate that could be interesting to study. However until now it has not
been possible to fix (full sequence will be obtained).
- RCC 337 is a larger strain that is probably related to Chlorarachnion (full sequence will be
obtained)
Other taxonomic groups (lower priorities)
- Dinophyceae: Exuviella like (RCC 303). This strain could correspond to Prororentrum nux
recently described by Zingone in Phycologia
- Eustigmatophyceae: Nannochloropsis (RCC 357 + Blanes strains). A new species has been
described that could correspond to one of this strain.
- Trebouxiophyceae: Nannochloris like. Many strains.
- Chlorophyceae: Chlamydomonas like (two strains RCC 300 and 537)
WORKPACKAGE II (CLONE LIBRARIES)
Three short presentations were given:
- Valentin: Analysis of sequences from a novel group closely related to the Rhodophyta
- Guillou: Analysis of natural populations (fractionated and following enrichment) by DGGE
- Groisillier: Strategy planned for the analysis of the novel alveolate groups.
Dave pointed some of the issue that still remain
 There are still some problems with the cyanobacteria/plastid clone libraries since the existing
primers do not appear to be really specific. This could be solved by using a 23S rRNA primer
 It could be interesting to construct other plastid/cyano libraries
- Low oxygen zone off Chile
- Arabian Gulf
 Some eukaryotic clone libraries planned from cruises of opportunity (MINOS, PROSOPE, Red Sea)
remain to be constructed after samples have been prescreened by DGGE.
 Should we probe clone libraries from underrepresented groups (e.g. Haptophytes)? This is not a
priority at this point.
The emphasis of year 3 will be on the analysis of clone libraries from coastal sites and the preparation of
papers (some are already in advanced stage). Moreover, we will now obtain full sequences for some of the
most interesting groups. We decided that the study of each targeted group will be coordinated by one
person who will then request full length sequences from the other partners as needed.
 Telonema --> Kamran (Oslo)
 Red group --> Klaus (AWI)
 Prasinophyceae --> Laure (Barcelona)
 Stramenopiles --> Ramon (Barcelona)
 Alveolates --> Agnès (Roscoff)
 Daniel also mentionned that he communicated OLIPAC and Roscoff sequences from two
taxonomic groups (one uncultivated, apparently related to Acanthareans, and Cercozoa) to
David Moreira for phylogenetic analysis.
III
WORKPACKAGE III (PROBES)
One short presentation was given:
- Doussal: Validation of probes for a novel group closely related to the Rhodophyta
The status of the different probes is as follows
 Validated and used on field samples
- Synechococcus (UW): paper in preparation
- Stramenopiles (ICM): paper submitted
- Prasinophyceae (SBR): paper in preparation
- Cryptophyceae Crypt13 (SBR): validated (description will be included in time series paper)
 In progress
- Hetero01 (AWI): signal and specificity problems
- Env Rhodophyta-like (SBR): almost validated
- Prymnesiophytes (AWI): in progress, FISH-TSA remains to be done
- Cryptophytes (AWI): no testing planned in immediate future
 To be designed
- Alveolates (SBR): will be designed late 2002
- Eustigmatophytes , Dictyochophytes (SBR): already designed, not tested
- Telonema, Chlorarachniophytes, Chrysophyceae: nothing planned yet
IV
WORKPACKAGE IV (PROBE MEASUREMENT)
The progress and plans for the different technologies were reviewed.
 DNA chips (AWI): Linda please fill in with your current plans
 FISH-TSA with flow cytometry (SBR). Although a protocol for cell concentration and FISH
hybridization compatible with flow cytometry has been worked out, some discrepancies
between microscopy and cytometry counts have appeared for non-Chlorophyta taxonomical
groups. Attempt to solve this problem is currently under way. Also questions emerge
concerning how applicable this approach will be. The most interesting prospect appear to
couple FISH-TSA with sorting to enrich samples in specific taxonomic groups and perform
molecular analyses such as clone libraries on the sorted material.
 Quantitative PCR (SBR). Progress has been done in the first semester of 2002 (DEA Bastien
Simonnet). However still a lot of issues remain to be solved.
- Although we used mainly the SYBR approach, the use of Taqman probes should be tested
further.
- Primer should optimized with two major criteria
• amplification efficiency: it should be as close as possible to 100%
• specificity: non-target groups should not be amplified
- Although initially we had planned to make primers for all eukaryotic taxonomic groups, we
have now narrowed our targets:
• General eukaryotic primers. It is critical to get good data for the total eukaryotic
population and several combination of primers should probably used together
• Prasinophyceae. The probes
- Normalization of data: we plan to use cloned DNA to determine gene copy number
- Finally we will perform some tests on:
• Mixed populations
• Natural samples
V
WORKPACKAGE V (TIME SERIES)
Two short presentations were given:
- Latasa: Improvements of the CHEMTAX running conditions
- Eikrem: TEM of natural samples: results and problems
The tables below summarize the measurements that will be done on the samples from the different
seasonal series, the exchange of samples between partners and the minimum set of probes to be applied
(click here to download the corresponding Excel file).
It was emphasized that we should proceed as quickly as possible with the exchange of samples, in
particular for DNA samples. We agreed on a deadline on Sept 15, 2002 to proceed with sample exchange.
Method
Population
Flow cytometry
Flow cytometry
Phytoplankton counts
Pigments HPLC
DGGE
SSCP
Dot blots
FISH
picoplankton
bacteria
FISH
FISH
FISH
FISH
QPCR
DNA chips
General probes
Prasinophytes
Red group
Stramenopiles
General set
General probes
cyanos
cyanos
Roscoff
2000-2001
2001-2002
Test period
24/07/00
05/03/01
to 16/02/01
to 1/10/02
SBR
SBR
SBR
SBR
SBR
SBR
Mikel
Mikel
Ramon
Klaus
Dave
Dave
Fabrice
Fabrice
Fabrice
SBR
Fabrice
Fabrice
Fabrice
Ramon
SBR
AWI
Helgoland
2001-2002
Blanes
2001-2002
SBR
SBR
Helgoland
Mikel
Ramon
Klaus
Dave
Dave
ICM
ICM
Renate
Mikel
Ramon
Klaus
Dave
Dave
Fabrice
Fabrice
Fabrice
Fabrice
Ramon
SBR
AWI
Ramon
SBR
AWI
Remarks
FISH on selected samples from the Blanes
site for Prochlorococcus since these FISH
probes are already developed. FISH Syn
probes for use with Roscoff and Helgoland
samples will likely be outside the PICODIV
timeframe
See Excel sheet in same file
Corresponds to samples already processed
Sending lab
Receiving lab ---> Roscoff
Roscoff
Warwick
Syn cultures
Sequences
Bremerhaven
FISH euks
DNA QPCR
Sample flow cyto
Cultures
Sequences
FISH euks
DNA QPCR
Cultures
Sequences
Barcelona
Warwick
FISH proks
DNA for probes
Bremerhaven
DNA SSCP
DNA Microarray
FISH proks
DNA for probes
FISH proks
DNA for probes
DNA SSCP
DNA Microarray
Barcelona
FISH strameno
DNA DGGE
Samples HPLC
Cultures HPLC
Oslo
Samples TEM
Culture TEM
FISH strameno
DNA DGGE
Samples HPLC
Cultures HPLC
Samples TEM
Culture TEM
Samples TEM
Culture TEM
Minimum set of wide probes
Euk
Chlorophyta
Mamiellales
Bolidophyceae
Pelagophyceae
Haptophyta
Cryptophyceae
Dinophyceae + Apicomplexa
VI
A
A
A
A
A
A
A
Euk1209R+CHLO01+NCHLO01
CHLO02
PRAS04
BOLI01
PELA01
PRYM02
CRYPT13
DINO_B or DINO01
x
x
x
x
x
x
x
x
Pub
Pub
SBR
Pub
Pub
Pub
SBR
AWI/SBR
x
x
x
x
x
x
x
ARCTIC CRUISE (WENCHE AND CARLOS)
Carlos gave an overview of the cruise (available on the web site). Preparation is going well and the data
acquired should complement very well those from the coastal sites since the cruise will sample a variety of
cold water ecosystems. Four people from PICODIV will be on board: Wenche (UO), Carlos, Ramon
(ICM) and Fabrice (SBR).
VII
PUBLICATIONS
A list of the PICODIV scientific papers in preparation or planned was discussed and is available in a
separate file. Carlos agreed to contact the editors of Limnology and Oceanography to try to have as a
special issue of the journal dedicated to the use of molecular approaches to study the diversity of plankton.
VIII
FINAL PICODIV MEETING
The final PICODIV meeting will take place in Norway. Jahn did a presentation of the facilities at
University of Oslo and at the Biological Station on the Oslo fjord. It was finally agreed that the final
PICODIV meeting will take place at the University of Oslo (better facilities and access to Internet) during
4 days between Wed 26 March and Sat 29 March 2003. This meeting will be mainly dedicated to the
preparation of the final report.
IX
PICOPLANKTON SYMPOSIUM
It was tentatively decided to organize a Picoplankton symposium after the end of the project. As Daniel
had a preliminary contact with Gerri Miceli who is in charge of the Gordon Research Conference, Daniel
and Bill Li (Bedford, Canada) will send in September a short proposal to the Gordon Research Conferences
for a meeting to be held in spring 2004 in Roscoff. This meeting entitled "Photosynthetic Picoplankton:
from ecology to genomics" could be the first of a series called "Microbiology of the Ocean". As a
function of the Gordon answer, other matching funds will sought for.