Latest development in HPLC analysis of
pigments
and
first overview of fractionated samples from
Roscoff
Mikel Latasa and Kees van Lenning
Institut de Ciencies del Mar, Barcelona.
PICODIV I Workshop, Roscoff, 27-06-00
How can pigments be used in the framework of PICODIV?
1.- To classify isolated strains of phytoplankton because of their
chemotaxonomical capabilities.
2.- To estimate natural biodiversity. Pigments should help us to
estimate the contribution of the different algal classes to total
phytoplankton stock.
CHEMOTAXONOMICAL MARKERS
Algal classes
Major Pigments
Prochlorophyceae
Divinyl chlorophyll a, monovinyl chlorophyll b, divinyl
chlorophyll b, zeaxanthin, MgDVP, alpha-carotene
SynechococcusMonovinyl chlorophyll a, zeaxanthin, beta-carotene.
type
Phycobiliproteins.
Chlorophyceae
Monovinyl chlorophyll a, monovinyl chlorophyll b, lutein
(zeaxanthin), neoxanthin, violaxanthin, antheraxanthin,
alpha- and beta-carotene.
Prasinophyceae
Monovinyl chlorophyll a, monovinyl chlorophyll b,
prasinoxanthin, zeaxanthin, neoxanthin, violaxanthin,
MgDVP, alpha- and beta-carotene
Dinophyceae
Monovinyl chlorophyll a, chlorophyll c2, peridinin,
diadinoxanthin, diatoxanthin, beta-carotene
Prymnesiophyceae Monovinyl chlorophyll a, chlorophyll c2, chlorophyll c3,
19'hex-fucoxanthin (19'but-fucoxanthin, fucoxanthin),
diadinoxanthin, diatoxanthin, alpha and beta-carotene
Pelagophyceae
Monovinyl chlorophyll a, chlorophyll c2, chlorophyll c3,
19'but-fucoxanthin (19'hex-fucoxanthin, fucoxanthin),
diadinoxanthin, beta-carotene
Bacillarophyceae
Monovinyl chlorophyll a, chlorophyll c2, chlorophyll c1,
fucoxanthin, diadinoxanthin, diatoxanthin, betacarotene
Chrysophyceae
Monovinyl chlorophyll a, chlorophyll c2, chlorophyll c1,
chlorophyll c3, fucoxanthin, violaxanthin, betacarotene
Cryptophyceae
Monovinyl chlorophyll a, chlorophyll c2, alloxanthin,
diadinoxanthin, alpha and beta-carotene.
Phycobiliproteins.
Other classes:
Other pigments:
Bolidophyceae, Eustigmatophyceae,
Micromonadophyceae, Euglenophyceae,
Raphidophyceae...
Vaucheriaxanthin, loroxanthin, heteroxanthin,
siphonaxanthin, siphonein, uriolide, gyroxanthin...
+ "new discoveries"
The new techniques are offering new results: new pigments
New methods
Mono- and divinyl chlorophyll c3
4-keto-19'hex-fucoxanthin
Monogalactosyl diglycerol chl c2
Other non-polar chl c
Application in CODENET
"Old" methods
Coeluted as chlorophyll c3
Coeluted with 19'hex-fuco
Non-polar chlorophyll c
Unidentified
400
A
500
10
456 nonpolar Chl c
Wavelength (nm)
600
700
400
Absorbance
Absorbance
Absorbance
0
2
*
* * *
*
20
A
446 472
Wavelength (nm)
500
600
30
19'-HFx
700
nonpolar Chl c
Carotenes
Chl a allomer
Chl a
19'-Hexanoyloxyfucoxanthin
Diadinoxanthin
4-Keto-19'-hexanoyloxyfucoxanthin
Fucoxanthin
Chl c
DV-Chl c3
Absorbance 440 nm (mAU)
Chlide a
*
40
400
A
500
50
448 472
4-keto-19'-HFx
586 632
Wavelength (nm)
600
700
The most common exceptions:
1.- Some strains of Phaeocystis (Prymnesiophyceae) present
pigment characteristics of the diatoms (Vaulot et al. 1994)
2.- The dinoflagellates and their endosymbionts.
a) Gymnodinium galatheanum, Gyrodinium aureolum, and
Gymnodinium breve have plastids possessing 19'hexfucoxanthin instead of peridinin (Liaaen-Jensen 1985, Tengs et
al. 2000)
b) Peridinium balticum has fucoxanthin and an unknown
pigment (Whiters et al. 1977)
c) Lepidodinium viride has chlorophyll b (Watanabe et al. 1990)
The less common exceptions:
3.- Prasinophyceae with pigment composition of Chlorophyceae
4.- Prasinophyceae and Chlorophyceae with "abnormal"
pigmentation (siphonaxanthin, loroxanthin...)
Questions:
* are these exceptions some wrong classifications by pigments
or clues to revisit former classifications?
* how many exceptions among picophytoplankton species?
Conclusion: We need to isolate and screen picophytoplankton
species for pigments.
MONITORING BIODIVERSITY
From measuring pigment concentrations to estimate phytoplankton
diversity.
Individual pigment markers:
sufficient and necessary
Divinyl chlorophyll a (Prochlorophyceae)
Alloxanthin (Cryptophyceae)
Peridinin (Dinophyceae)
sufficient but not necessary
Prasinoxanthin (Prasinophyceae)
necessary but not sufficient
Fucoxanthin (Bacillariophyceae)
19 But-fucoxanthin (Pelagophyceae)
[19 Hex-fucoxanthin (Prymnesiophyceae)]
Zeaxanthin (Cyanophyceae)
Lutein (Chlorophyceae)
To estimate biodiversity we must know the contribution of each
group to the phytoplankton standing stock.
Until now, the best approaches can estimate the contribution of each
group to the chlorophyll standing stock ----> pigment diversity
Approaches:
1) Considering one pigment marker per group.
Multiple lineal regression (Gieskes and Kraay 1988).
2) Taking into account the contribution of pigments shared by
different groups.
a) Mathematical algorithms (Everitt et al 1990, Letelier et al
1993).
b) CHEMTAX (Mackey et al 1996)
Possible failures of this approach:
a) the ascription pigment to taxa is based on few isolates in
some of the groups.
b) uncertainties in the "seed" pigment ratios (CHEMTAX's
error limit is ±35%) because of:
- changes due to physiological state
- changes due to intraclass variation
CONCLUSIONS:
Pigment analysis is a very useful tool to classify isolated
phytoplankton strains.
New HPLC techniques are revealing new pigments which can
improve the classification of phytoplankton strains.
The agreement between the pigment composition of
picophytoplankton and their correspondent algal groups is largely
unknown.
The step from pigment concentrations to quantification of the
contribution of the different groups to total algal stock is not
immediate.
A definite push pursuing this goal has been the development of
CHEMTAX.
There is an urgent need for isolating and culturing field species of
(phyto)plankton and, above all, picoplankton.
0
10
//
2
Chl c
*
*
20
Zeaxanthin
Lutein
Alloxanthin
*
*
30
*
40
Carotenes
Chl a epimer
Chl a allomer
MV + DV-Chl b
*
Chl a
Fucoxanthin
Neoxanthin
Prasinoxanthin
Violaxanthin
19'-Hexanoyloxyfucoxanthin
Diadinoxanthin
19'-Butanoyloxyfucoxanthin
Peridinin
1
Chl c
DV-Chl c3
Chlide a MgDVP
Absorbance 440 nm (mAU)
FIRST RESULTS of pigment analysis from Roscoff
50
R00301 GF/F
R00329 < 3 um
R00301
R00329 > 3 um
R00301
Chl b
Zeax
Allox
Per19but
19hex
Chl b
Chl b
Per
Per
19but
19but
19hex
Fuco
Fuco
Zeax
Zeax
Allox
Allox 19hex
19hex
Fuco
Fuco
Fuco
Otros
Otros
Per
19but
Per
Allox
19but
Zeax
19hex
Chl b
Allox
Zeax
Chl b
R00313 GF/F
R00313 < 3 um
R00313 > 3 um
Per
Per
19but
19but
Fuco
Per
Fuco
Chl b
Fuco
Chl b
Zeax
Otros
19hex
19hex
19hex
Allox
Allox
19but
Allox
Zeax
Zeax
Chl b
R00329 GF/F
R00329 < 3 um
Per
Per
Chl b
R00329 > 3 um
19but
19but
Chl b
19hex
Zeax
Allox
Fuco
Zeax
Otros
Otros
Per
Fuco
19hex
Fuco
Fuco
Allox
19hex
19but
Per
Allox
19but
Zeax
19hex
Chl
Alloxb
Zeax
Chl b
R00412 GF/F
R00412 < 3 um
Per
Per
Chl b
R00412 > 3 um
Per
19but
19but
Chl b
Fuco
Chl b
Otros
Zeax
Fuco
Fuco
Allox
19hex
19hex
Allox
19hex
Zeax
19but
Zeax
Allox
R00427 GF/F
R00427 < 3 um
R00427 > 3 um
Per
Per
19but
19but
Per
19hex
Fuco
19but
Chl b
Fuco
Chl b
Zeax
Allox
Allox
Zeax
19hex
Allox
Zeax
19hex
Fuco
Otros
Chl b
Total chl a, from GF/F filters and from the sum of
the fractions
700
600
-19%
GF/F
Sum
42%
-27%
ng / L
500
400
300
34%
7%
200
100
0
R00301
R00313
R00329
R00412
R00427
Sample
Percentage of the total chl a
in the < 3 um fraction
100
80
%Chla < 3 um
%
60
40
20
0
R00301
R00313
R00329
Sample
R00412
R00427
Shannon's index
Pigment diversity in the different size fractions
3.0
GF/F
< 3 um
> 3 um
2.5
2.0
1.5
1.0
0.5
0.0
R00301
R00313
R00329
Sample
R00412
R00427
No Prochlorococcus pigment signatures in any of the samples.
The dominant groups in terms of pigments are always diatoms and
green algae. The green algae seem to be Prasinophytes and not
Chlorophytes.
Other main players are Cryptophytes, Haptophytes and
Dinoflagellates.
Cyanobacteria, Crysophytes and Pelagophytes are always minor
components (if at all).
Diatoms dominate completely (more than 75% in pigment biomass)
the > 3 um fraction.
The < 3 um fraction is dominated mostly by green algae and
diatoms.
CONCLUSIONS
The agreement, in terms of chlorophyll, between the sum of the < 3
um and > 3 um fractions and the total fraction (GF/F) should be
improved.
The picophytoplankton made between 40% and 80% of the total
chlorophyll a in this coastal site (median is 50%).
There were not important shifts in pigment composition in spite of a
3-fold change in chlorophyll concentration
Diatoms were an important component of the < 3 um fraction
The pigment diversity was much higher in the < 3 um fraction
These are preliminary data and all these conclusions are based on
raw pigments. The application of CHEMTAX will most likely modify
the estimated contribution of each group.