1. A spatio-temporal comparison of picoeukaryotes in the Alborán Sea (SW Mediterranean) by denaturing gradient gel electrophoresis and HPLC pigment analysis Beatriz Díez Mikel Latasa Carlos Pedrós-Alió Ramon Massana Institut de Ciències del Mar, CSIC Passeig Joan de Borbó, s/n, 08039 Barcelona, Catalunya, Spain.

2. Location of stations along the studied Alborán Sea area (SW Mediterranean Sea)

3. - Study of temporal (short and long-term) variability of picoeukaryotes in Alborán Sea - Study of spatial variability of picoeukaryotes in Alborán Sea - Identification of the dominant groups Objetives

4. ABCABC November 1997 May 1998 September 1999

6. The DGGE protocol follows the protocol of Díez et al. (2001) 0.75 mm-thick vertical gel containing 6% (w/v) polyacrylamide (37:5:1) acrylamide:bisacrylamide) Linear gradient of denaturing agents, increasing from 45% at the top to 65% at the bottom of the gel (100% denaturing agents is defined as 7M urea and 40% deoinized formamide) 600-800 ng PCR product Electrophoresis performed at 100v/cm 16h Stain: 1 x TAE buffer + Syber Gold for 45 min and visualized with a FluorS MultiImager and the MultiAnalyst Imaging software (Biorad Laboratories) EUK516 R EUK1F

7. The HPLC method follows the protocol of Zapata et al. (2000) adapted to our system

8. In general, % of Chl a <5µm was smaller at station A than at B and C

9. 1998 Cruise

10. MATER 98MATER97MATER 99 Spatial and temporal DGGE transect (5-0.2µm surface) 18 12 16 15 15 20 20 15 17 19 17 11 17 14 11 13 16 14 18 10 18 16 OTUs number B0 A1 A2 A3 A4 B1 B2 B3 B4 C1 C2 C3 C4 A1 A2 A3 B1 B2 B3 C1 C2 C3

11. ME98B3 ---------------------- +------ ME98B4 ----------------- | | +----- | MER98B1 ----------- | | +------ | ME98B2 ----------- | +---- ME98A2 ----------------- | | +----- | | ME98A4 --------------- | | | | +-- | | | ME98A3 --------------- | | | +------ | ME98A1 ---------------- | | +------ | ME98C3 ---------------- | +------------------ ME98C2 ------------- | | +----- | | ME98C1 ------------- | | | +-------------- | ME98C4 ------------------ | +--------------- ME99C3 --------------- | +-------- | ME99C2 --------------- | | +------- | ME99A3 ------------- | | | +------ | | | ME99A2 ------------- | | | | +---- | | ME99C1 --------------- | | | +---- | | ME99A1 --------------- | | +-------------------- ME99B2 ------------- | +----------- | ME99B1 ------------- | | +------ ME99B3 --------------------- | +--- ME97B0 --------------------- DGGE Binary Matrix (<5µm) 1998: Stations A and B appear closer to each other than to station C 1999: A and C appear closer to each other than to B 1997 station B appears closer to 1999 station B (both cruises in autumn)

12. ME98B1 -------- +-- ME98C1 ------ | | +-| | ME98A4 -----||| | +-|| | ME98A1 ----- || | +- | ME98C3 ------- | +-------------------- ME98A2 ----- | | | | | ME98A3 ----+ | | +-- | | ME98B2 ----- | | | +- | | ME98C2 ----- || | | +--| | | ME98B4 ----- | | | +-- | ME98B3 -------- | +---------------------- ME97B1 ----- | +------- | ME99B3 ----- | | +------------------ ME99A3 ----- | +- | ME99C2 -----| | +--- | ME99C3 -----| | | +- | | ME99B2 ----- | | +--- ME99A1 ----- | | | ME99A2 ----+ | +-- | ME99B1 ----- | | +-- ME99C1 ------- HPLC Binary Matrix (<5µm) 1997 station B grouped together with 1999 stations (both in autumn) It was very difficult to find clear relations among samples within of the two principal clusters

13. C398 ---------------- +----- B097 ---------------- | +-------------- C198 ----------- | | +---- | | A298 ----------- | | | +------ | B298 --------- | | +-- | | A398 --------- | | | +---- | C298 ----------- | +---------------------- B198 -------------------- | | +- | | B498 ------------ || | | +----- || | | A198 ------------ | || | | +---| | | B398 ----------------- | | | +-------------- | A498 -------------- | | +------- | B199 -------------- | +-------- B399 ---------------- | +--------- | C199 -------------- | | | +-- | | A399 -------------- | | +-------------------------------- B299 ----------- | +--- | A199 ----------- | | +--- | C299 ----------- | | | +--- | | A299 ----------- | | +-------- C399 ----------------- HPLC Binary Matrix (WW) Also two principal clusters appear separating 1998 and 1999 samples.

14. Mantoniella Ostreococcus Unidentified prymnesiophyte Novel marine alveolate MATER 98MATER97MATER 99 B0 A1 A2 A3 A4 B1 B2 B3 B4 C1 C2 C3 C4 A1 A2 A3 B1 B2 B3 C1 C2 C3 Novel stramenopiles

15. CHEMTAX (CHEMical TAXonomy) approach

16. 97-B098-B398-B4 5 5 25 50 100 250 500 5 25 50 100 250 500 VERTICAL PROFILES Two principal clusters divide samples from surface to 100m and deeper samples (250 to 500m) B0-5 ------------------------------------- +----------- B4-5 --------------------------------- | | +---- | B3-5 ----------------------------- | | +-- | | B3-25 ----------------------------- | | | +-- | B4-25 ------------------------------- | +---------- B4-50 ------------------------------ | +------ | B3-50 ------------------------- | | | +----- | | B3-100 ------------------------- | | +------------ B4-100 ------------------------------------ B4-250 ---------------------------- +--- B3-250 ---------------------------- | +--------------------------- B3-500 -------------------- | +----------- B4-500 --------------------

17. Picoeukaryotes are ubiquitous in this area Distribution of picoeukaryotes was dependent on the physical conditions. Both DGGE and HLPC data showed that differences in picoplankton composition were larger between years than among stations. Both techniques agreed in presenting chlorophyll b-containing algae and prymnesiophytes as the main components of the picoplankton during our cruises in the Alborán Sea. Conclusions