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-AlióRamon Massana

Institut de Ciències del Mar, CSIC

Passeig Joan de Borbó, s/n, 08039 Barcelona, Catalunya, Spain.

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

- 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

ABC

November 1997

May 1998

September 1999

Table 3. During the MTP-III-MATER/HESP/97, MTP-II-MATER/HESP/04-98 andMTP-I-MATER/HESP/99 cruises, on board the R/V Hesperides, stations (A, B, C, in acoastal-offshore gradient) located across the Western Alborán Sea Gyre (SW

Mediterranean) were sampled in November 1997, May 1998 and September 1999(fraction between 0,2-5 µm in size).

System Station Coordinates Depth Date

(Meters)

MEI B0a 36º 14’N/ 4º 15’W 5 9/11/97MEII A1 36º23’4N/4º14’20W 5,65 5/2/98MEII A2b 36º 23’ N/ 4º 15’ W 5, 25 5/5/98MEII A3 36º22’35N/4º14’40W 5.50 5/8/98MEII A4 36º23’9N/4º14’38W 5,45 5/11/98MEII B1 36º14’26N/4º15’12W 5,40 5/3/98MEII B2a 36º 14’ N/ 4º 15’ W 5, 25 5/6/98

MEII B3 36º13’55N/4º15’4W 5,25 5/9/98MEII B4 36º14´25N/4º15’ 28W 5,25 5/12/98MEII C1 35º59’50N/4º15’49W 5,50 5/4/98MEII C2c 36º 0’ N/ 4º 15’ W 5, 35 5/7/98MEII C3 36º0’38N/4º16’57W 5,55 5/10/98MEII C4 36º0’11N/4º15’58W 5,50 5/13/98MEIII A1 36º 22’N/4º14’82W 5,36 9/17/99MEIII A2 36º23’N/4º15’01W 5,35 9/21/99MEIII A3 36º23’43N/4º15’10W 5,40 9/24/99MEIII B1 36º14’38N/4º15’21W 5,36 9/16/99MEIII B2 36º14’27N/4º15’32W 5,30 9/20/99

MEIII B3 36º13’25N/4º15’17W 5,10 9/23/99MEIII C1 36º0’55N/4º16’44W 5,40 9/15/99MEIII C2 35º59’50N/4º16’4W 5,50 9/19/99MEIII C3 35º59’03N/4º15’07W 5,35 9/22/99_____________________________________________________________________

a coastal nearby upwelling areab frontal upwelling areac gyre area (offshore)

Molecular methodsFlow citometry MicroscopyPigment analysisHPLC

Fingerprinting

DGGET-RFLP

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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

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

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

1998Cruise

<5 µmStation AStation BStation C<5 µm<5 µm>5 µm>5 µm>5 µm

MATER 98MATER97 MATER 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

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)

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

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.

Mantoniella

Ostreococcus

Unidentified prymnesiophyte

Novel marine alveolate

MATER 98MATER97 MATER 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

CHEMTAX (CHEMical TAXonomy) approachStationDepth(m)fractionCyano Green (Prasino)Hapto Crypt DiatomsPelagoDinofl

ME97B0 5 < 5 µm 9 43 34 9 2 4 0 100ME98A1 5 < 5 µm 11 34 27 11 16 2 0 100ME98A2 5 < 5 µm 3 27 43 9 12 5 0 100ME98A3 5 < 5 µm 6 19 55 12 6 3 0 100ME98A4 5 < 5 µm 17 18 42 8 15 0 0 100ME98B1 5 < 5 µm 0 28 63 0 0 0 9 100ME98B2 5 < 5 µm 12 18 56 8 0 5 0 100ME98B3 5 < 5 µm 56 24 7 10 0 3 0 100ME98B4 5 < 5 µm 38 14 34 9 0 4 0 100ME98C1 5 < 5 µm 16 41 20 10 10 4 0 100ME98C2 5 < 5 µm 49 22 18 9 0 1 0 100ME99A1 5 < 5 µm 99 0 0 0 0 1 0 100ME99A2 5 < 5 µm 81 17 1 0 0 1 0 100ME99A3 5 < 5 µm 52 35 8 3 0 2 0 100ME99B1 5 < 5 µm 67 31 2 0 0 0 0 100ME99B2 5 < 5 µm 51 27 16 1 0 4 0 100ME99B3 5 < 5 µm 11 55 14 4 15 1 0 100ME99C1 5 < 5 µm 26 14 31 0 29 0 0 100ME99C2 5 < 5 µm 63 22 11 0 0 4 0 100ME99C3 5 < 5 µm 69 20 9 0 0 3 0 100

97-B0 98-B3 98-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--------------------

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 andprymnesiophytes as the main components of the picoplankton during our cruises in the Alborán Sea.

Conclusions