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L

Journal of Experimental Marine Biology and Ecology 249 (2000) 77–88

www.elsevier.nl / locate / jembe

Pigment signatures associated with an anoxic coastal zone:

Bahia Concepcion, Gulf of California

´ ´ ´

J. Bustillos-Guzman , D. Lopez-Cortes, F. Hernandez, I. Murillo

Northwest Center for Biological Research, POB 128, La Paz, B.C.S. Mexico Received 14 April 1999; received in revised form 29 February 2000; accepted 2 March 2000

Abstract

Bahia Concepcion is a coastal lagoon that has bottom anoxic conditions and high pigment concentrations during the summer. The phytoplankton responsible for this pigment increase is enigmatic, therefore we sampled the lagoon to analyze the pigment with a C8-HPLC system to look for signatures of phytoplankton groups. Analysis reveals a low pigment concentration in the mixed layer with a higher concentration of zeaxanthin and increasing values of chlorophyll a, peridinin, and fucoxanthin below, which peaked at the depth where oxygen dramatically decreases and H S increases. Below this depth, a high pigmentation was recorded and the most important2 signatures were six chlorophyll-like pigments that eluted between the fucoxanthin and the chlorophyll a, and one carotenoid that eluted just after the chlorophyll a. Spectral characteristics of these last pigments are very similar to pigments present in the Chlorobiales group. These results suggest that cyanobacteria, diatoms, and dinoflagellates are responsible for the chlorophyll a increases, though in highly pigmented samples, anoxygenic phototrophic bacteria are probably the main contributors to the increase in pigments.  2000 Elsevier Science B.V. All rights reserved.

Keywords: Bacteriochlorophylls; Coastal lagoons; Gulf of California; HPLC; Pigments

1. Introduction

Bahia Concepcion is one of the largest bays in the Gulf of California with about 400

km of surface and a maximum depth of 30 m (Lechuga-Deveze, 1994). Human influence is small because it is in a zone of low precipitation (between 150 and 250 mm per year), with practically no important towns (only small tourist settlements), and little maritime navigation. These characteristics allow the study of events (i.e. red tides, *Corresponding author. Tel.: 133-112-536-33; fax: 133-112-536-25.

E-mail address: jose@cibnor.mx (J. Bustillos-Guzman)

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biogeochemical cycles, etc.) in an unperturbed environment. Phytoplankton in the area is dominated by diatoms, but red tide species are occasionally present (Martinez-Lopez and Garate-Lizarraga, 1994). For summer, Lechuga-Deveze (1994) reported bottom pigment

concentrations of chlorophyll a (Chl a) between 7.1 and 45.5 mg l and chlorophyll b 21

(Chl b) values between 10.4 and 99.1mg l in more than a half of the area bay. These values are high when compared with normal stations where Chl a and Chl b

concentration maximum values, in general, are lower than 2mg l . Although there are several reports on the phytoplankton composition and abundance (Kiefer and Laker, 1975; Gilmartin and Revelante, 1978; Martinez-Lopez and Garate-Lizarraga, 1994; Lopez-Cortez, 1999; Lechuga-Deveze and Morquecho-Escamilla, 1999; Lechuga-De-veze et al., 2000), these high pigment concentrations are enigmatic because no one has reported species or groups of species that can be related to such an increase of biomass. Recently, Lechuga-Deveze (1994) analyzed the spectra of acetone extracts and sug-gested a population of prochlorophytes may be responsible for the high biomass. His reason is that highly pigmented samples have red-shift spectra when compared with normally pigmented ones, similar to that existing between the Chl a and divinyl chlorophyll a (DvChl a). Because of the difficulties of identifying and quantifying this picoplanktonic group with traditional methods of microscopy, this hypothesis has not been tested.

We have used high-performance liquid chromatography (HPLC) to analyze the phytoplankton pigment and look for markers of phytoplankton groups in these samples. Specifically, we tested for the existence of DvChl a, a marker of prochlorophytes (Goericke and Repeta, 1993), or other phytoplankton group fingerprints that may be responsible for this increase of biomass.

2. Material and methods

In August 1997, we sampled at several stations throughout the bay (see Lechuga-Deveze, 1994) to detect the ‘anomalous’ highly pigmented area. Once the area was located, one station was selected (Fig. 1) as representative of the ‘anomalous’ area and a detailed profile (at 0, 2, 4, 6, 8, 10, 12, 13, 15, 17, 19, 21, 22, 25, 26 and 27-m depth) was obtained for pigment, temperature, nitrates, oxygen and H S analysis. Additionally,2

irradiance (400–700 nm) striking an underwater quantum sensor (LI-COR) was measured using an integrating radiometer photometer LI-COR LI-188B. For pigments, seawater (1 l) was collected with Van Dorn bottles and transported on ice and in the dark to the laboratory where the water was filtered through GF / F glass fiber filters and immediately frozen at 2208C. For pigment extraction, filters were put in 2 ml of acetone, grinding in glass tubes and then centrifuged. The extract (200 ml) was mixed with 100 ml of 0.5 N ammonium acetate and injected through a 100-ml loop into an HPLC system (Hewlett Packard series 1100) with a diode-array detector. HPLC conditions were as in Vidussi et al. (1996) and permit the separation of DvChl a. Briefly, a hypersil MOS 10-cm long by 4.6-mm (I.D.) C8 column was used with the following solvent A (MeOH: 0.5 N aqueous ammonium acetate, 70:30% v / v) and solvent B (MeOH) gradient (min; percentage of solvent A, percentage of solvent B): (0; 75, 25),

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J. Bustillos-Guzman et al. / J. Exp. Mar. Biol. Ecol. 249 (2000) 77 –88 79

Fig. 1. Vertical variation of physical and chemical variables in Bahia Concepcion, Gulf of California: (A) temperature; (B) irradiance (I ) as a percentage of incident irradiance (I ), and dissolved oxygen (O ); (C)0 2

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(1; 50, 50), (15; 0, 100), (18.5; 0, 100) and (19; 75, 25). Pigments were detected with the diode-array absorbance signal set at 440 nm. Identification was made by comparing retention time and spectral characteristics with those of commercial pigment standards

(International Agency for C determinations, Denmark) and a rich prochlorophyte sample collected at the deep chlorophyll maximum from the Mexican Pacific Ocean (19852.2 N, 113811.99W) during the USA METOX01 cruise for the DvChl a. Quantification was done with the pigment response factor (HPLC peak area / pigment mass) obtained with the commercial pigment standards according to Mantoura and Repeta (1997). Nitrate and dissolved oxygen were measured according to Strickland and Parson (1972), sulfide by the iodometric method (Clesceri et al., 1989), and temperature and salinity with a Kahlsico salinometer model 140.

3. Results

3.1. Physical and chemical conditions

Using the physical and chemical variables (Fig. 1), three zones can be distinguished: (i) a surface mixed layer (upper 12 m) with the highest temperature (32.58C) and

dissolved oxygen (.6 ml l ), and relatively low nitrate concentration (lower than 0.4 21

mg at l ); (ii) an intermediate layer with temperature between 32.5 and 298C, 21

decreasing dissolved oxygen (from 6.6 to 1.0 ml l ) and stable nitrate concentration 21

(0.4–0.42mg at l ); and (iii) a bottom layer with temperature decreasing to 228C, very 21

low or undetectable dissolved oxygen (,1.0 ml l ), and increasing nitrate and H S2

concentration. In this last layer, irradiance was between 4 and 10% of the incident irradiance at the surface [Fig. 1(B)].

3.2. Pigments

Peridinin and fucoxanthin, pigment signatures of dinoflagellates (Jeffrey, 1974) and of diatoms (Liaaen-Jensen, 1985; Wright and Jeffrey, 1987) had a low concentration in the mixed layer, a slight increase between 12 and 19 m, and a sharp increase at 21 m [Fig. 2(C)]. A second increase of peridinin occurs at the bottom. Zeaxanthin concentration, an indicator of cyanobacteria and prochlorophyte abundance (Guillard et al., 1985; Goericke and Repeta, 1993) is relatively low and homogeneous in the upper 20-m layer

[Fig. 2(B); values ,0.2 mg l ) and decreases in the bottom layer. The general phytoplankton biomass signature, the Chl a, closely matches the variation of the peridinin and fucoxanthin [Fig. 2(A)], suggesting that diatoms and dinoflagellates are the main contributor to the phytoplankton biomass. To corroborate this, the contribution of the different phytoplankton groups to the Chl a was calculated by using a multiple linear regression analysis between diagnostic pigment (peridinin, fucoxanthin and zeaxanthin) and Chl a profile data (Barlow et al., 1993; Bustillos-Guzman et al., 1995). We found dinoflagellates, diatoms, and cyanobacteria contributed 73, 17 and 6% to Chl a.

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J. Bustillos-Guzman et al. / J. Exp. Mar. Biol. Ecol. 249 (2000) 77 –88 81

Fig. 2. Vertical variation of pigment concentration in Bahia Concepcion, Gulf of California: (A) chlorophyll a; (B) zeaxanthin; (C) fucoxanthin and peridinin.

3.3. Unidentified pigments

At least six chlorophyll-like pigments (Chl-like) and one carotenoid were recorded. The Chl-like pigments eluted between the diadinoxanthin and Chl b and the carotenoid eluted after the Chl a (Fig. 3). The first three Chl-like pigments, eluting after the diadinoxanthin, have the same absorption spectrum form: a soret (blue) and a red (Qy) maximum at 476 nm and 658 nm with a soret:red ratio (r) of 3.68 [Fig. 4(A)]. The other Chl-like pigments have a soret at 444 nm, a Qy at 660 nm, and an r of 4.94 [Fig. 4(B)].

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Fig. 3. Typical chromatogram (440 nm) from highly pigmented (A) and normal samples (B). Peak identification: 1, chlorophyll c1 and c2; 2, peridinin; 3, fucoxanthin-like pigment; 4, diadinoxanthin; 5–11, unknown chlorophyll-like pigments; 12, chlorophyll b; 13, chlorophyll a; 14, unknown carotene; 15, unknown pigment; 16,b-carotene; 17, fucoxanthin; 18, zeaxanthin; 19 and 20, unknown pigments. X-axis units are min.

Y-axis units are milliabsorbance units.

The carotenoid has maximum absorbance at 450 nm and 476 nm with a 450 / 476 ratio of 1.15 [Fig. 4(C)]. Because we do not have the response factor for these unknown pigments, the response factor of Chl b and pheophytin b were used to quantify the first and second group of Chl-like pigments, and that ofb-carotene for the carotene because their spectra match closely. Pheophytin b was prepared according to Jeffrey (1997) to obtain its response factor. The vertical distribution shows Chl-like pigments start increasing below 19-m depth, peaking at 25 m, and then decreasing [Fig. 5(A) and (B)]. The carotenoid pigment also increases together with the Chl-like pigments but peaked at 23 m [Fig. 5(C)]. All Chl-like pigments show the same quantitative variation with depth.

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J. Bustillos-Guzman et al. / J. Exp. Mar. Biol. Ecol. 249 (2000) 77 –88 83

Fig. 4. Vertical variation of chlorophyll-like pigments in Bahia Concepcion, Gulf of California: (A) pigments 5, 6 and 7; (B) pigments 9, 10 and 11; (C) carotenoid 14.

4. Discussion

One of the surprises of this research was that the bulk of pigment that contributes to the high pigmentation of the samples were six Chl-like pigments and that these pigments differ from Chl a and DvChl a in retention time (they eluted before them) and spectral characteristics. Chl a and DvChl a have an on-line soret maximum at 432 nm and 442 nm, respectively [Fig. 5(A), DvChl a spectrum not shown]. Our first conclusion is that the high concentration of red-shift pigment is not associated with prochlorophyte or Chl

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Fig. 5. Absorption spectra of chlorophyll a and the unknown chlorophyll-like pigments taken by the diode-array spectrophotometer as they elute from the column: (A) chlorophyll a; (B) pigments 5, 6 and 7; (C) pigments 9, 10 and 11; (D) carotenoid 14. X-axis units are nm. Y-axis units are milliabsorbance units.

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J. Bustillos-Guzman et al. / J. Exp. Mar. Biol. Ecol. 249 (2000) 77 –88 85 a-containing algae. It is also evident from the spectral characteristics of the unknown pigments that their presence in samples will interfere with the calculation of Chl a and Chl b. This will result in an overestimation of chlorophylls when using spectro-photometric measurements at some specific wavelengths and explains the high con-centration of Chl a and Chl b reported by Lechuga-Deveze (1994). We can now examine in detail these unknowns and other pigments and the physical and chemical conditions. The hydrographic structure corresponds well with the one previously described when high pigmented samples were recorded by Lechuga-Deveze (1994). According to Lechuga-Deveze et al. (2000), these conditions are caused because a change of wind direction and the warming of air temperature during the end of winter and beginning of spring induce the formation of a sharp thermocline that insulates the surface layer from bottom waters. Phytoplankton and zooplankton biomass develop profusely during the transition from mixed to stratified water column and supply organic matter that, when it sinks, exhausts the dissolved oxygen during the summer. The recurrence of anoxic water near the bottom and highly pigmented samples suggest that these conditions are a summer feature of this bay.

Distribution of pigments in samples seems to be associated with the physical and chemical structure of the water column. Thus, pigment signatures of cyanobacteria (zeaxanthin) were in the oxygenic, nitrate-poor, warmer surface water whereas di-noflagellate and diatom pigment (peridinin and fucoxanthin) maxima were at the base of the oxycline. The increase of cyanobacteria in surface waters has been related to their capacity to photosynthesize at high light intensities (Kana and Glibert, 1987), or to absorb photons in the green domain (Ong et al., 1986). Diatom and dinoflagellate pigments allow absorption in the spectral blue band (Bricaud et al., 1988; Antoine, 1995) and high nutrient levels are a prerequisite for proliferation (Cortes-Altamirano and

Nunez-Pasten, 1986; Goldman, 1993; Bustillos-Guzman et al., 1995). Even though we did not record the highest nitrate concentration with the highest fucoxanthin and peridinin concentration, a mechanism of upwelling flux of nutrients at the base of the oxycline may be acting as has been suggested for other zones (Jorgensen, 1989; Goldman, 1993).

At the bottom, Chl-like and the carotenoid were dominant and were in a zone where oxygen concentration was very low or undetectable and H S appeared. The origin of2

these pigments is unknown but a comparison of the red (Qy) and soret band absorbance maxima of bacteriochlorophylls with the three pigments eluting after the diadinoxanthin show Qy and a soret band quite similar to bacteriochlorophyll e (BChl e) and its homologous forms (Gloe et al., 1975; Frigaard et al., 1996). According to Truper (1970), Pfennig and Truper (1981) and Pfennig (1989), a group of phototrophic bacteria exists that photosynthesize under the above conditions. They also note that these anoxyphotobacteria comprise the order Rhodospirillales or ‘purple bacteria’ that have bacteriochlorophyll a or b, and the order Chlorobiales with bacteriochlorophyll c, d or e as the main pigments, depending on the species. The presence of bacteria from the Chlorobiacea group associated with the high biomass is therefore highly possible. This is also suggested by the presence of the carotenoid because its spectrum is similar to isorenieretane, an antenna carotenoid characteristic of this order (Liaaen-Jensen and Andrewes, 1972). Indeed, anoxygenic photobacteria are abundant and conspicuous in salt marshes, closed bays, and in estuaries where plant material is decaying and light

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intensities are too low to support growth of other phototrophic organisms (Pfennig and Truper, 1981; Schlegel and Jannasch, 1981; Schaub and van Gemerden, 1996). These conditions correspond well with that described here and others for the bay (Lechuga-Deveze, 1994; Lechuga-Deveze et al., 2000).

A second group of bacteriochlorophylls (BChls) or secondary homologues are also present in the Chlorobiaceae group and elute after the main set of BChls (Borrego and Garcia-Gil, 1994; Borrego et al., 1999). These could be the pigments eluting before the Chl a [peaks 8–11; Fig. 3(A)], but spectral characteristics are different to the similar BChl e set [peaks 6–7; Fig. 3(A)]. According to Otte et al. (1993) and Borrego et al. (1999), BChl e and their homologues have similar spectra. The absorption spectra of peaks 8–11, however, resemble the spectrum of bacteriopheophytin e (BPhtin e) and homologues (Gloe et al., 1975; Simpson and Smith, 1988; Borrego and Garcia-Gil, 1994). These pigments differ from BChl e in that they are Mg-free derivatives and their absorption maxima are at shorter wavelengths (Gloe et al., 1975). In our highly pigmented extracts, maximum absorption of Bphtin e is 32 nm lower than its parents. Bacteriopheophytins and homologues form an important part of the bacteriochlorophyll– protein complexes (Oelze, 1985). However, the high content of Bphtin e in our samples has to be considered with caution because Bphtin e can be an artifact of extraction as shown by Gloe et al. (1975) who observed that acetone extracts from Chlorobium cell material can contain up to 50% of this pigment and the response factor used to calculate the Bphtin e concentration is not correct.

In summary, the use of HPLC to analyze pigments in this research has helped us to advance the identification of the cause of the high pigmentation of the bottom of more than half the bay. Our analysis suggests that an important population of autotrophic anoxygenic bacteria is the main cause of the general pigment increase whereas Chl a increase is mainly linked to dinoflagellates and diatoms in Bahia Concepcion during summer. This bacterial population may play an important role in the nutrient reminerali-zation and anoxygenic photosynthesis in this and other coastal lagoons with similar physical and chemical characteristics.

Acknowledgements

This study was part of the IAYCG-11 project from CIBNOR and partially supported ´

by CONACYT (Mexico). We wish to thank Robert Blankenship, Daniel Brune and Michael Lince from Arizona State University and C. Borrego (Institute of Aquatic Ecology, Spain) and N.U. Triqaard for helpful discussions, the two referees for their critical review of the manuscript, and Ellis Glazier who edited this English-language paper. [RW]

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Fig. 4. Vertical variation of chlorophyll-like pigments in Bahia Concepcion, Gulf of California: (A) pigments 5, 6 and 7; (B) pigments 9, 10 and 11; (C) carotenoid 14.

4. Discussion

One of the surprises of this research was that the bulk of pigment that contributes to

the high pigmentation of the samples were six Chl-like pigments and that these pigments

differ from Chl a and DvChl a in retention time (they eluted before them) and spectral

characteristics. Chl a and DvChl a have an on-line soret maximum at 432 nm and 442

nm, respectively [Fig. 5(A), DvChl a spectrum not shown]. Our first conclusion is that

the high concentration of red-shift pigment is not associated with prochlorophyte or Chl

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a-containing algae. It is also evident from the spectral characteristics of the unknown

pigments that their presence in samples will interfere with the calculation of Chl a and

Chl b. This will result in an overestimation of chlorophylls when using

spectro-photometric measurements at some specific wavelengths and explains the high

con-centration of Chl a and Chl b reported by Lechuga-Deveze (1994). We can now examine

in detail these unknowns and other pigments and the physical and chemical conditions.

The hydrographic structure corresponds well with the one previously described when

high pigmented samples were recorded by Lechuga-Deveze (1994). According to

Lechuga-Deveze et al. (2000), these conditions are caused because a change of wind

direction and the warming of air temperature during the end of winter and beginning of

spring induce the formation of a sharp thermocline that insulates the surface layer from

bottom waters. Phytoplankton and zooplankton biomass develop profusely during the

transition from mixed to stratified water column and supply organic matter that, when it

sinks, exhausts the dissolved oxygen during the summer. The recurrence of anoxic water

near the bottom and highly pigmented samples suggest that these conditions are a

summer feature of this bay.

Distribution of pigments in samples seems to be associated with the physical and

chemical structure of the water column. Thus, pigment signatures of cyanobacteria

(zeaxanthin) were in the oxygenic, nitrate-poor, warmer surface water whereas

di-noflagellate and diatom pigment (peridinin and fucoxanthin) maxima were at the base of

the oxycline. The increase of cyanobacteria in surface waters has been related to their

capacity to photosynthesize at high light intensities (Kana and Glibert, 1987), or to

absorb photons in the green domain (Ong et al., 1986). Diatom and dinoflagellate

pigments allow absorption in the spectral blue band (Bricaud et al., 1988; Antoine,

1995) and high nutrient levels are a prerequisite for proliferation (Cortes-Altamirano and

˜

Nunez-Pasten, 1986; Goldman, 1993; Bustillos-Guzman et al., 1995). Even though we

did not record the highest nitrate concentration with the highest fucoxanthin and

peridinin concentration, a mechanism of upwelling flux of nutrients at the base of the

oxycline may be acting as has been suggested for other zones (Jorgensen, 1989;

Goldman, 1993).

At the bottom, Chl-like and the carotenoid were dominant and were in a zone where

oxygen concentration was very low or undetectable and H S appeared. The origin of

these pigments is unknown but a comparison of the red (Qy) and soret band absorbance

maxima of bacteriochlorophylls with the three pigments eluting after the diadinoxanthin

show Qy and a soret band quite similar to bacteriochlorophyll e (BChl e) and its

homologous forms (Gloe et al., 1975; Frigaard et al., 1996). According to Truper

(1970), Pfennig and Truper (1981) and Pfennig (1989), a group of phototrophic bacteria

exists that photosynthesize under the above conditions. They also note that these

anoxyphotobacteria comprise the order Rhodospirillales or ‘purple bacteria’ that have

bacteriochlorophyll a or b, and the order Chlorobiales with bacteriochlorophyll c, d or e

as the main pigments, depending on the species. The presence of bacteria from the

Chlorobiacea group associated with the high biomass is therefore highly possible. This is

also suggested by the presence of the carotenoid because its spectrum is similar to

isorenieretane, an antenna carotenoid characteristic of this order (Liaaen-Jensen and

Andrewes, 1972). Indeed, anoxygenic photobacteria are abundant and conspicuous in

salt marshes, closed bays, and in estuaries where plant material is decaying and light

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intensities are too low to support growth of other phototrophic organisms (Pfennig and

Truper, 1981; Schlegel and Jannasch, 1981; Schaub and van Gemerden, 1996). These

conditions correspond well with that described here and others for the bay

(Lechuga-Deveze, 1994; Lechuga-Deveze et al., 2000).

A second group of bacteriochlorophylls (BChls) or secondary homologues are also

present in the Chlorobiaceae group and elute after the main set of BChls (Borrego and

Garcia-Gil, 1994; Borrego et al., 1999). These could be the pigments eluting before the

Chl a [peaks 8–11; Fig. 3(A)], but spectral characteristics are different to the similar

BChl e set [peaks 6–7; Fig. 3(A)]. According to Otte et al. (1993) and Borrego et al.

(1999), BChl e and their homologues have similar spectra. The absorption spectra of

peaks 8–11, however, resemble the spectrum of bacteriopheophytin e (BPhtin e) and

homologues (Gloe et al., 1975; Simpson and Smith, 1988; Borrego and Garcia-Gil,

1994). These pigments differ from BChl e in that they are Mg-free derivatives and their

absorption maxima are at shorter wavelengths (Gloe et al., 1975). In our highly

pigmented extracts, maximum absorption of Bphtin e is 32 nm lower than its parents.

Bacteriopheophytins and homologues form an important part of the bacteriochlorophyll–

protein complexes (Oelze, 1985). However, the high content of Bphtin e in our samples

has to be considered with caution because Bphtin e can be an artifact of extraction as

shown by Gloe et al. (1975) who observed that acetone extracts from Chlorobium cell

material can contain up to 50% of this pigment and the response factor used to calculate

the Bphtin e concentration is not correct.

In summary, the use of HPLC to analyze pigments in this research has helped us to

advance the identification of the cause of the high pigmentation of the bottom of more

than half the bay. Our analysis suggests that an important population of autotrophic

anoxygenic bacteria is the main cause of the general pigment increase whereas Chl a

increase is mainly linked to dinoflagellates and diatoms in Bahia Concepcion during

summer. This bacterial population may play an important role in the nutrient

reminerali-zation and anoxygenic photosynthesis in this and other coastal lagoons with similar

physical and chemical characteristics.

Acknowledgements

This study was part of the IAYCG-11 project from CIBNOR and partially supported

´

by CONACYT (Mexico). We wish to thank Robert Blankenship, Daniel Brune and

Michael Lince from Arizona State University and C. Borrego (Institute of Aquatic

Ecology, Spain) and N.U. Triqaard for helpful discussions, the two referees for their

critical review of the manuscript, and Ellis Glazier who edited this English-language

paper. [RW]

References

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Barlow, R.G., Mantoura, R.F., Gough, M.A., Fileman, T.W., 1993. Pigment signatures of the phytoplankton composition in the northeastern Atlantic during the 1990 spring bloom. Deep Sea Res. 40, 459–477. Borrego, C.M., Garcia-Gil, L.J., 1994. Separation of bacteriochlorophyll homologues from green