RESEARCH ARTICLE

J. Serodio Æ S. Vieira Æ S. Cruz Æ F. Barroso

Short-term variability in the photosynthetic activityof microphytobenthos as detected by measuring rapidlight curves using variable fluorescence

Received: 9 June 2004 / Accepted: 18 October 2004 / Published online: 8 December 2004� Springer-Verlag 2004

Abstract Short-term variability in the photosyntheticactivity of microphytobenthos assemblages was studiedby measuring chlorophyll fluorescence rapid light curves(RLC), using pulse amplitude modulated (PAM) fluo-rometry. Measurements carried out on undisturbedsamples under dark–light cycles revealed large dieloscillations in both the initial slope of the RLC (a) andin the maximum relative electron transport rate (ETRm).Short-term variations in RLC parameters were alsoobserved, closely following changes in incident photonirradiance (E). Increases in irradiance were followed bydecreases in a and increases in ETRm, resulting in sig-nificant correlations between the light-saturationparameter Ek and E. These results were interpreted asresulting from the onset of reversible energy-dissipating,non-photochemical quenching mechanisms and ofcompensatory high light-induced activation of carbonmetabolism activity. Short-term RLC variability wasshown to result mainly from physiological causes and tobe detectable only by using short (10–20 s) light stepsduring RLC construction. Dark-adapted samples keptunder constant conditions exhibited apparently endog-enous rhythms in RLC parameters and in the maximumquantum yield, Fv/Fm, coincident with vertical migra-tory movements occurring during subjective photoperi-ods. These fluctuations appeared to result from theinteraction between migratory rhythms and the physio-logical responses, and from the endogenous activation ofprocesses affecting both the efficiency of energy transferfrom light-harvesting antennae to the photosystem II(PSII) reaction centres or from non-radiative pathways

(Fv/Fm, a) and the reactions downstream of PSII(ETRm).

Introduction

Microphytobenthos communities inhabiting estuarineintertidal sediments are exposed to a highly dynamicenvironment, characterised by considerable variabilityon a wide range of time scales. The estuarine intertidalenvironment is particularly variable and extremeregarding solar irradiance, due to the occurrence of longperiods of direct exposure to changeable degrees ofsunlight during low tide and of sudden dark–lighttransitions between extreme conditions during ebb orflood. Photosynthetic organisms respond to variabilityin light exposure through photoacclimation, the revers-ible phenotypic regulation of a variety of processes,including regulated changes in pigment content, com-ponents of the photosynthetic electron transport chainand carbon metabolism enzymes (MacIntyre et al. 2002).Photoacclimation may operate on a range of differenttime scales, from several days down to a few minutes, asin the case of the production of energy-dissipating pig-ments through the xanthophyll cycle (MacIntyre et al.2000; Muller et al. 2001).

As with other aquatic photosynthetic organisms, thephotoacclimation status of microphytobenthos commu-nities has been assessed through the construction ofphotosynthesis versus irradiance (P vs. I) curves, either onresuspended samples (Blanchard and Cariou-Le Gall1994; MacIntyre and Cullen 1995) or on undisturbedassemblages (Pinckney and Zingmark 1993; Serodio et al.2001). These studies became facilitated with the intro-duction of pulse amplitude modulated (PAM) fluorome-try (Schreiber et al. 1986) and the possibility of real-timeand non-destructive study of the photosystem II (PSII)activity in undisturbed biofilms (Kromkamp et al. 1998).Based on the relationship between the empirical fluores-cence index DF/Fm¢ (the effective quantum yield of stable

Communicated by M. Kuhl, Helsingør

J. Serodio (&) Æ S. Vieira Æ S. Cruz Æ F. BarrosoDepartamento de Biologia, Universidade de Aveiro,Campus de Santiago, 3810-193 Aveiro, PortugalE-mail: jserodio@bio.ua.ptTel.: +351-234-370787Fax: +351-234-426408

Marine Biology (2005) 146: 903–914DOI 10.1007/s00227-004-1504-6

charge separation at PSII, see notation inTable 1) and thequantum yield of photosynthesis (Genty et al. 1989),PAM fluorometry allows for the construction of lightcurves relating the rate of photosynthetic electron trans-port (ETR) and incident photon irradiance (E). By over-coming a number of methodological problems associatedto the measurement of photosynthetic rates on undis-turbed samples (Serodio 2003), fluorescence light curveswere readily adopted as proxy for traditional P vs. Ecurves, as a way of characterising the photoacclimationstatus of microphytobenthos biofilms (Kromkamp et al.1998; Barranguet and Kromkamp 2000; Perkins et al.2002; Underwood 2002).

However, both photosynthesis and fluorescence lightcurves based on steady-state PSII activity (measuredafter a steady-state is reached under each light level), byrequiring from several minutes to a few hours to com-plete, may be inadequate to characterise the photoacc-limation status of microphytobenthos communities or todetect changes in this status as a response to short-termenvironmental variability. On the one hand, by allowingthe sample to acclimate to each light level during theconstruction of the curve, steady-state curves fail todescribe the light-acclimation status determined byshort-term light histories. On the other hand, resultsfrom the application of fluorescence imaging techniquesto intact microphytobenthos biofilms have shown thatsteady-state light curves can be confounded by changesin the composition of the microalgal biofilm resultingfrom natural migratory rhythms or by migratorymovements induced during the construction of the curve(Perkins et al. 2002). Moreover, changes in the verticaldistribution of microalgae due to vertical migration wereshown to potentially affect the light curve independentlyof alterations in the their true photosynthetic light re-sponse (Serodio 2004).

A significantly less intrusive alternative to steady-state light curves are the so-called fluorescence rapidlight curves (RLC; Schreiber et al. 1997; White andCritchley 1999), which, by typically applying light stepsof 10–20 s, can be completed within 1.5–2 min, beingexpected to minimise the confounding effects of verticalmigrations. However, despite these potential advantages

and its generalised use on other aquatic photoauto-trophs like seagrasses (Ralph et al. 1998, 2002b; Silvaand Santos 2003), corals (Ralph et al. 1999, 2002a), icemicroalgae (Kuhl et al. 2001; Glud et al. 2002) andmacroalgae (Longstaff et al. 2002; Gevaert et al. 2003),RLCs have not yet been applied to migratory micro-phytobenthic biofilms.

In the present study, RLCs were used to characteriseshort-term changes in the photosynthetic performance ofundisturbed microphytobenthos assemblages. The pho-tosynthetic response to short-term variability of irradi-ance was first studied by measuring RLCs at regularintervals on undisturbed samples along a 24 h dark–lightcycle and on samples exposed to the natural hourly vari-ability in irradiance. The short-term variability observedinRLCwas confirmed to resultmainly fromphysiologicaleffects, by testing the occurrence of diel variations in RLCparameters on microalgal suspensions and by comparingundisturbed and vertically homogenised sediment sam-ples. The influence of illumination time on the detection ofshort-term changes in RLC parameters was tested bycomparing light curves constructed by applying light stepsof different durations. The endogenous nature of RLCvariability and its relationship with endogenously con-trolled migratory rhythms were also investigated by car-rying out measurements on undisturbed samples keptunder constant conditions.

Materials and methods

Sampling and cultures

Undisturbed microphytobenthos samples were collectedon an intertidal mudflat located near Vista Alegre, Canalde Ilhavo, on the Ria de Aveiro, a mesotidal estuarylocated on the central west coast of Portugal. Thesampling site consists of fine muddy sediments (97%particles <63 lm), where microphytobenthos assem-blages are dominated throughout the year by diatoms ofthe genera Navicula, Nitzschia, Gyrosigma and Pleu-rosigma. Samples were collected using plastic corers(1.9 cm diameter) and were kept in an artificial tidal

Table 1 Parameter notationused in the text Notation Definition

a, b Initial slope and photoinhibition parameter of the ETR vs.E curve (lmol–1 m2s)

aP, Pm Initial slope and maximum photosynthetic rate of a photosynthesis vs.irradiance (P vs.E) curve

DF/Fm¢ Effective quantum yield of PSII (dimensionless)E Spectrally averaged photon irradiance of PAR (400–700 nm) (lmol m–2 s–1)Ek Light-saturation parameter of the ETR vs. E curve (lmol m–2 s–1)ETR Relative electron transport rate (=E·DF/Fm¢) (dimensionless)ETRm Maximum relative electron transport rate in a RLC (dimensionless)Fs, Fm¢ Steady-state and maximum fluorescence emitted by a light-adapted sample

(arbitrary units)DF Variable fluorescence (=Fm¢, Fs) (dimensionless)Fo, Fm Minimum and maximum fluorescence emitted by a dark-adapted sample

(arbitrary units)

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system until the measurements were carried out, on thenext day. The benthic diatom Cylindrotheca closterium(Ehrenberg) Lewin et Reinman, isolated from intertidalsediments of the Ria de Aveiro, was grown in unialgal,semi-continuous batch cultures at 15�C and 50 lmol m–2

s–1 in a 12 h light:12 h dark cycle, in natural seawaterenriched with f/2 nutrients (Guillard and Ryther 1962).

Fluorescence light curves

Variable fluorescence was measured on natural micro-phytobenthos samples using a PAM fluorometer com-prising a computer-operated PAM-control unit (Walz,Effeltrich, Germany) and an WATER-EDF-universalemitter–detector unit (Gademann Instruments, Wurz-burg, Germany). This instrument uses a modulated bluelight (LED-lamp peaking at 450 nm, half-band width of20 nm) as a source for measuring, actinic and saturatinglight, emitted at a frequency of 18 Hz when measuringFo or 20 kHz when measuring other fluorescenceparameters (further details in Serodio 2004). Measure-ments on C. closterium cultures were carried out using aPAM-210 fluorometer (Walz), which uses modulated redLEDs as a source for measuring (650 nm, 32 or 8 kHz)and for actinic and saturating light (665 nm). Sampleswere concentrated by centrifugation (3000 g, 5 min) andresuspended on fresh growth medium before each mea-surement.

Measurements on undisturbed sediment samples werecarried out using a 6-mm-diameter Fluid Light Guidefibre optics bundle or a 1.5-mm-diameter plastic fibre.The relative position of the fibre optics and the sedimentsurface was controlled using a micromanipulator(MM33, Martzhauser, Germany), and the measure-ments were made at a constant distance of 1 mm. The 6-mm fibre optics bundle was positioned perpendicularlyto the sediment surface and was used to deliver the ac-tinic light provided by the fluorometer. When using the1.5-mm fibre, actinic cold white light was provided by ahalogen lamp (Volpi Intralux 5000-1, Volpi, Switzer-land) and delivered to the sample by a fibre opticsbundle connected to a 5-cm-diameter light ring (Stan-dard Ringlight, Volpi), providing a homogeneous shade-free light field. Sediment samples were sectioned withminimum disturbance into plastic rings 5 mm deep(same diameter of sampling corers); maintained in aPetri dish containing water collected at the sampling site;and were placed below the centre of the light ring, at afixed vertical distance from it (ca. 2 cm). Photon irra-diance incident on the sample surface was measuredcontinuously using a PAR micro-sensor (Spherical Mi-croQuantum Sensor US-SQS/W, Walz) positioned nearthe inner border of the light ring and connected to thefluorometer control unit. The sensor was calibrated tomeasure the irradiance level at the sample surface. Thefibre was positioned at a 45� angle, which had beenshown in previous tests not to significantly shade the siteof measurement.

RLCs were constructed by exposing the samples to 8–11 increasing actinic light levels, delivered by the PAMfluorometers: modulated blue light, in the case of theWATER-EDF-universal and the PAM-control units, ormodulated red light, when using the PAM-210 (cul-tures). Maximum actinic light levels attained were 920(6-mm fibre), 1707 (1.5-mm fibre), or 1250 lmol m�2 s�1

(PAM-210). Unless stated otherwise, the exposure peri-od under each light level was 10 s. RLCs were con-structed by calculating, for each level of actinic light, therelative electron transport rate from the delivered actinicirradiance and the effective quantum yield of PSII byETR=E·DF/Fm¢. The light response of microalgae wascharacterised by fitting the model of Platt et al. (1980) toETR versus E curves and by estimating the parameters a(initial slope of the light curve), ETRm (maximum ETR)and ß (photoinhibition parameter). Although a decreaseof ETR under high irradiances was observed in only asmall minority of the RLCs, justifying the use of asimpler model without the photoinhibition parameter ß,it was shown that the removal of ß from the modelresults in the overestimation of ETRm (MacIntyre et al.2002). The light-saturation parameter Ek was calculatedby Ek=ETRm/a. The model was fitted iteratively usingMS Excel Solver. Curve fit was very good (r>0.90) in allcases.

Short-term variability in undisturbedmicrophytobenthos

Short-term variability in RLCs was characterised bycarrying out hourly measurements on undisturbed mi-crophytobenthos samples along a 24 h dark–light cycle.The start and the end of the photoperiod were set tomatch sunrise (during low tide) and the time of flood,respectively. Photon irradiance was kept at a constantlevel of 150 lmol m�2 s�1 during the photoperiod. Tostudy the photosynthetic response to short-term vari-ability in irradiance, RLCs were measured at regularintervals on samples kept in the laboratory, but exposedto irradiance levels matching the in situ levels. Mea-surements were carried out on 2 days in opposite situ-ations along one spring–neap tidal cycle, 20 (springtides) and 27 (neap tides) March 2003. During daytimelow tide periods, the photon irradiance incident on thesample was set to match the level measured outside atthat moment (PAR sensor LI-193SA and LI-250 lightmeter, Li-Cor, Lincoln, Nebraska, USA) and was keptconstant for 15–20 min. During this period, effectivequantum yield, DF/Fm¢, was measured every 4–5 min,using the 1.5-mm fibre. The actinic light was then swit-ched off (other ambient actinic light levels were negligi-ble), and a RLC was recorded 10–15 s thereafter. Thesample was maintained in the dark for 5 min, afterwhich one saturating pulse was applied and the maxi-mum efficiency of PSII, Fv/Fm, was determined. Thesample was again exposed to actinic light matching theirradiance level measured outside.

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Physiological variability of RLCs

Although indices calculated from ratios of fluorescenceparameters are expected to reflect solely the physiolog-ical status of the sample, it has been shown thatparameters of RLCs measured on sediment samples canbe significantly affected by the variations in microalgalbiomass associated to vertical migratory movements(Serodio 2004). To distinguish physiological effects fromthe potential artefacts associated to migratory rhythmsas the cause for the short-term variability in RLCs, twotypes of experiments were carried out. First, in order totest the occurrence of diel RLC variations in the absenceof changes of biomass, hourly measurements were madeon a microalgal suspension during a complete 24 hdark–light cycle, using log-phase cultures of the benthicdiatom C. closterium. Second, migratory effects onRLCs were evaluated by comparing the hourly variationof RLC on undisturbed (migratory effects present) andon vertically homogenised (slurries, migratory effectsabsent) microphytobenthos samples along a dark–lighttransition coincident with sunrise during low tide. Slur-ries were prepared by resuspending repeatedly the top2 mm of sediment on site water using a syringe. Theslurry was let to settle on a sedimentation chamber for2 min, and fluorescence was measured from below, byplacing the 6-mm fibre next to the chamber’s glass base(Serodio et al. 2001). The formation of non-homoge-neous vertical profiles of biomass due to migratorymovements was avoided by resuspending the slurriesbefore each measurement. After the measurement ofeach RLC, the sample was darkened for 2 min and theFo level was measured to trace changes in microalgalbiomass in the photic zone of the sediment (Serodioet al. 1997). In both experiments, a constant photonirradiance of 50 lmol m�2 s�1 was applied during thephotoperiod.

Exposure time

The influence of illumination time on the parameters aand ETRm was studied by comparing light curves con-structed by applying light steps of 10, 20 and 40 s. Lightcurves were measured hourly along a dark–light cycle onundisturbed samples, using the 6-mm fibre. The startand the end of the photoperiod were set to match thesunrise during low tide and flood tide, respectively.Photon irradiance was kept constant at 150 lmol m�2

s�1. To prevent cumulative actinic effects, light curves ofdifferent exposure times were measured in separatesamples.

Endogenous variations in undisturbedmicrophytobenthos

The endogenous nature of the short-term variability ofRLC parameters suggested by previous results was testedbymeasuringRLCs at constant time intervals (20 min) on

undisturbed samples during 1–3 days under constantconditions in the laboratory (darkness, 20�C, air expo-sure).Fo andFv/Fmwere determined from the first pulse ofeach light curve, applied before the actinic light wasturned on. The samples were kept moist by being partlyimmersed (not covering the surface) in site water. Mea-surements were made using the 1.5-mm fibre.

Results

Short-term variability in RLC parameters

A clear diel pattern was observed in the RLCs measuredon undisturbed microphytobenthos samples throughouta 24 h dark–light cycle (Fig. 1). Diel oscillations wereobserved both for a and ETRm, the largest changestaking place around the dark–light transition: less thanan hour after the start of the light period, a and ETRm

had increased by 72.6% and 118.2% (Fig. 1, points b,c). Considering the extreme values observed during the24-h cycle, a and ETRm varied by 133.6% and 352.8%,respectively. After the initial increase, a remained vir-tually constant during the entire photoperiod, starting todecrease only after the start of the dark period. Incontrast, ETRm displayed a more variable pattern dur-ing the photoperiod, with maximum values occurring atleast 2 h after its start, and starting to decrease beforethe actual end of the light period. As a consequence, Ek

followed closely the variation of ETRm throughout the24-h cycle (P<0.001). Following the start of the darkperiod, both parameters decreased to values similar tothose measured before the photoperiod, although thisdecrease was more gradual than the increase observedafter the dark–light transition.

The measurement of RLCs on samples subjected tothe natural short-term variability in irradiance revealedlarge variations in the photosynthetic light responsefollowing changes in light conditions. Despite the dif-ferent timing of tidal flooding and light exposure, thesame general pattern was observed on both days, with adecreasing markedly with irradiance above 300–500 lmol m�2 s�1 (Figs. 2, 3A). On the other hand,ETRm increased steeply with E until ca. 300 lmol m�2

s�1, after which it continued to increase in a much lesspronounced way (Fig. 3B). Nevertheless, in both casesthe variation with irradiance could be described by asimple linear regression model. The inverse variation ofa and ETRm led to a highly significant correlation be-tween Ek and E (Fig. 3C).

Fv/Fm was also affected by irradiance, althoughoverall variations were relatively small. On 20 March, atthe time irradiance had reached its maximum value, Fv/Fm had decreased by only 9.1%. After that, the cumu-lative effects of the prolonged exposure to high light ledto a stronger decline, although only by 17.6%. On 27March, Fv/Fm remained reasonably constant throughoutthe high tide period, with the exception of a slight in-crease, during the first 2 h in the morning, and of some

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decline, largely reversible, when irradiance reached val-ues >500 lmol m�2 s�1 (Fig. 2B).

Physiological variability of RLCs

Suspensions of the benthic diatom Cylindrotheca clos-terium also showed a diel pattern in the RLC param-eters a and ETRm when subjected to a dark–lightcycle. Despite the lower irradiance level to which thesample was exposed, the range of variation of bothparameters was comparable to those observed inundisturbed sediment samples (Fig. 1): 146.7% for aand 289.0% for ETRm. Both RLC parameters pre-sented the same overall behaviour, with a varying lessthroughout the light period (Fig. 4A) and with ETRm

taking a longer time to reach maximum values andstarting to decrease sooner and more markedly beforethe transition to darkness (Fig. 4B). Due to the par-allel oscillation of a and ETRm, Ek varied by only28.7% between light and dark periods.

The comparison of undisturbed samples and slurriesfurther confirmed that short-term variations in RLCsmay be caused by changes at the physiological levelindependently of changes in the vertical distribution of

microalgae. In spite of the elimination of the effects ofvertical migration, confirmed by the constancy of theFo value throughout the experiment (Fig. 5C), slurries

Fig. 1A–D Typical fluorescence rapid light curves (RLC) measuredon undisturbed microphytobenthos samples (A, B), and dieloscillation in the RLC parameters a (C) and ETRm (D) over a24-h period. Constant photon irradiance of 150 lmol m–2 s–1

during the light period. Curves in panels A and B resulted fromfitting the model of Platt et al. (1980) to mean values of threereplicated measurements. Panels C and D show mean values ofRLC parameters (three replicates) and error bars representing onestandard error. Black horizontal bars represent darkness

Fig. 2A, B Hourly short-term variation of photon irradiance (E),Fv/Fm, and the RLC parameters a and ETRm on undisturbedmicrophytobenthos samples under in situ light conditions on2 days during one spring–neap tidal cycle, 20 (A, spring tide) and27 (B, neap tide) March 2003

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exhibited marked hourly changes in RLC parametersthat were comparable to those observed for theundisturbed samples (Fig. 5A, B) and also to thepatterns displayed in Fig. 1. The parameters estimatedfor the two types of samples were significantly corre-lated when considering the entire data set (both a andETRm: r=0.990, P<0.001 and r=0.928, P=0.741,respectively).

Exposure time

When measured on dark-adapted samples, a and ETRm

were substantially dependent on the duration of the lightexposure period used in the construction of the RLCs(Fig. 6). Exposure times of 40 s were sufficient to in-crease a and ETRm to values close to those measuredafter light adaptation (92.2% and 81.8%, respectively,by averaging the data of both dark periods). In contrast,when using exposures of 10 or 20 s, a and ETRm re-mained significantly below the values obtained byapplying 40 s [at maximum, averaging over each darkperiod, 57.6% (10 s) and 69.3% (20 s) and 48.5% (10 s)and 68.3% (20 s), respectively]. The effects of exposuretime were much reduced in the case of light-adaptedsamples, particularly in the case of a, which only mar-ginally differed from the values estimated from steady-state light curves (99.1%, 10 s and 96.5%, 20 s). Dif-ferences between ETRm estimated from RLCs and fromsteady-state light curves were also significantly reduced,although to a lesser extent (86.1%, 10 s and 83.6%,20 s). Ek was much less affected by exposure time,reaching 87.1% (10 s) and 87.3% (20 s) of steady-statevalues during the light period, differing by 91.0% (10 s)and 113.8% (20 s) from the values estimated using 40 sduring the dark periods. These results also showed thatthe difference between RLC parameters measured by

Fig. 3A–C Linear relationships between a (A), ETRm (B) and Ek

(C) and E, measured on undisturbed microphytobenthos samplesunder in situ light conditions on 20 (squares) and 27 (circles) March2003

Fig. 4A, B Diel oscillation of the RLC parameters a (A) and ETRm

(B) in a suspension of the benthic diatom Cylindrotheca closterium.Constant photon irradiance of 50 lmol m–2 s–1 during the lightperiod. Means of three replicates. Error bars represent onestandard error (three replicates). Black horizontal bars representdarkness

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using short illumination times (10 or 20 s) on dark-adapted samples and after full light-activation variedwith time, decreasing with the approach of the photo-period and decreasing after the start of the dark period(Fig. 5). For example, values reached by using 20 s oflight exposure are, 1 h later, reached by applying only10 s.

Endogenous variations in undisturbedmicrophytobenthos

Figure 7 illustrates the short-term variability in RLCparameters and in Fo and Fv/Fm measured on undis-

turbed microphytobenthos assemblages maintained un-der constant conditions in the dark. All parametersexhibited strong persistent oscillations, closely syn-chronised with the start and the end of the periods ofdiurnal low tide observed at the sampling site (subjectivephotoperiod, delimited by sunrise during low tide andthe afternoon flood), which were gradually attenuatedthroughout the 3 days of the experiment. Fo displayedlarge (ca. fivefold), asymmetrical peaks, resulting from avery rapid increase (ca. 400% in 60 min) and a slowerdecrease to pre-dawn values, starting almost immedi-ately after reaching maximum values (Fig. 7A). Opticalmicroscope observations of the surface of replicatedsamples revealed that peaks in Fo coincided with thesurfacing of large numbers of the motile diatom Pleu-rosigma angulatum (Quekett) W. Smith. In contrast toFo, Fv/Fm, a and ETRm displayed almost symmetricaloscillations, with the maximum values being reachednear the middle of the subjective light period (Fig. 7C,D). These parameters declined during the second half ofthe photoperiod, when Fo was already below half itspeak maximum. Fv/Fm and a displayed a very similarpattern, covarying throughout the whole duration of the

Fig. 5A–C Comparison of the evolution of the RLC parameters a(A) and ETRm (B) and variation of minimum fluorescence, Fo (C)during a dark–light transition (coincident with sunrise during lowtide) on undisturbed and on vertically homogenised samples(slurries, black dots). Constant photon irradiance of 50 lmol m–2

s–1 during the light period. Error bars represent one standard error(three replicates). Black horizontal bars represent darkness

Fig. 6A, B Effects of the illumination time used in the constructionof the light curve (light steps of 10, 20, or 40 s) on the parameters a(A) and ETRm (B), measured on undisturbed microphytobenthossamples. Constant photon irradiance of 150 lmol m–2 s–1 duringthe light period. Dark–light transition coincident with sunriseduring low tide. Error bars represent one standard error (threereplicates). Black horizontal bars represent darkness

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experiment (r=0.906, P<0.001). Significant correla-tions were also found between a and ETRm when con-sidering the subjective photoperiods (in all cases,P<0.001). Coincident with the increase in Fo at the startof the subjective photoperiods, Fv/Fm and a decreasedfor ca. 1 h and only then began to display the cyclicpattern paralleled by ETRm. During subjective high tideor night, considerable high-frequency variability wasobserved in ETRm (Fig. 7D). This noisy variability wasconfirmed to result from the low number of data pointsin the light-saturated part of the curve and consequent

increase in the error involved in the determination ofETRm.

Discussion

RLCs and short-term photoacclimation

The study of the photoacclimation of benthic micro-algal communities has been based on the constructionof steady-state light-response curves, either by mea-suring photosynthetic rates (Lamontagne et al. 1986,1989; Blanchard and Montagna 1992; Blanchard andCariou-Le Gall 1994; MacIntyre and Cullen 1995;Hartig et al. 1998) or variable fluorescence (Hartiget al. 1998; Kromkamp et al. 1998; Barranguet andKromkamp 2000; Underwood 2002), usually only afew times along each diel cycle. While informative on

Fig. 7A–D Free-running variation of minimum fluorescence, Fo

(A); maximum quantum yield, Fv/Fm (B); and the RLC parametersa (C) and ETRm (D), measured in an undisturbed microphytoben-thos sample during 3 days (sampling interval: 20 min). Verticaldotted lines define the subjective light period, delimited by sunrise/sunset or ebb/flood at the sampling site. Black and white horizontalbars represent subjective night and submersion during high tide,respectively

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the potential response of a sample if let to adapt todifferent light levels, steady-state light curves requireillumination times long enough to alter its light-accli-mation status to particular ambient conditions andrecent light history. Chlorophyll fluorescence RLCsattempt to detect adjustments in the functioning of thephotosynthetic apparatus following short-term varia-tions in ambient conditions (White and Critchley1999), and are generally assumed to characterise thephotoacclimation status attained immediately beforethe start of the curve (Schreiber et al. 1997; Ralphet al. 1999; Glud et al. 2002). The rapid assessment ofthe photophysiological status provided by RLCs isparticularly welcomed in the case of microphytoben-thos, as short-term responses to changes in light levelduring the construction of steady-state light curvesalso include vertical migratory movements that signif-icantly affect the determination of fluorescenceparameters (Perkins et al. 2002; Serodio 2004). Thelow intrusiveness of RLCs was confirmed in this study,by showing that by using illumination times of 10–20 sit is possible to detect variations in light curveparameters associated to short-term photoacclimationthat cannot be detected using longer exposure times.The observation of diel rhythms in RLC parameterson suspensions of a benthic diatom and on sedimentsamples, independent of changes in the vertical distri-bution of the microalgae, further indicate that RLCscan be used to detect short-term changes in physio-logical status of microphytobenthos biofilms.

The application of RLCs on undisturbed micro-phytobenthos assemblages, by permitting a low intru-siveness and a high measuring frequency, revealedimportant short-term changes in the photoacclimationstatus. Although similar short-term variability in RLCparameters have been observed on other aquatic pho-tosynthetic organisms, such as seagrasses (Ralph et al.1998, 2002b; Silva and Santos 2003), corals (Ralph et al.1999) and macroalgae (Longstaff et al. 2002; Gevaertet al. 2003), their occurrence in microphytobenthosassemblages has never been reported. The control ofmicrophytobenthos photosynthesis has been consideredto rely to a large extent on the capacity to, by under-going vertical migratory movements within the photiczone of the sediment, regulate light exposure andabsorption (‘‘behavioural photoacclimation’’, Under-wood 2002). The results of the present study show thatshort-term photoacclimation of microphytobenthoscommunities is also controlled by the rapid operation ofphysiological mechanisms that optimise the photo-chemical processing of absorbed light energy. The vari-ation of a and ETRm, closely following changes inirradiance levels prevailing during the recent light his-tory, indicates a large plasticity in the photosyntheticlight response through the rapid regulation of Ek. Thisconcurs with the concept of photoacclimation throughshifts in Ek to match prevailing irradiance on time scalesof hours to days (‘‘Ek-dependent variation’’; Behrenfeldet al. 2004).

Short-term photoacclimation under high light

The decrease of photosynthetic rates under high light isusually attributed to the operation of non-photochemicalquenching processes that include rapidly reversible,photoprotective energy-dissipating mechanisms (‘‘en-ergy-dependent quenching’’, qE) and, to a variable butlesser extent, slowly reversible changes involving dam-ages to the photosynthetic apparatus (‘‘photoinhibitoryquenching’’, qI) (Quick and Orton 1984). The relativeconstancy of Fv/Fm throughout the photoperiod,including mid-day periods of high irradiance, is consis-tent with the hypothesis that the high light-inducedquenching of a is caused by the activation of photopro-tective mechanisms, possibly associated to the reversibleconversion of pigment diadinoxanthin into diatoxanthinin the photosystems’ antennae. Decreases in Fv/Fm havebeen associated to the operation of energy-dependent,non-photochemical quenching and to the lack of pho-toinhibitory, damaging effects on the photosyntheticapparatus (Falkowski et al. 1994; Schofield et al. 1998;Ralph et al. 2002b). These processes increase the mag-nitude of energy dissipation in the pigment bed,decreasing the transfer efficiency of captured excitationenergy to the reaction centres, and, therefore, the Fm

emission. Considering the likely underestimation of Fv/Fm due to the relatively short dark-adaptation applied(necessary to minimise the potential migratory responseand effects on the fluorescence parameters), the relativelyhigh and constant values measured throughout the dayfurther confirm the operation of a rapidly reversibleprocess. Although results showing a decrease of a with Ecould be found in the literature, they appear to havepassed unnoticed, as they were not mentioned or dis-cussed (Ralph et al. 1998; Longstaff et al. 2002; Gevaertet al. 2003; Silva and Santos 2003). The rapid relaxationunder low light of the down-regulation by energy-dependent quenching processes explains the fact thatquenching of a has not been observed on long, steady-state light curves measured under different ambient lightconditions (Blanchard and Montagna 1992; Blanchardand Cariou-Le Gall 1994; Serodio et al. 2001).

An expected consequence of the operation of PSIIdown-regulation would be the parallel decrease of a andETRm as excitation energy captured by the antennae isdiverted from photochemistry into non-radiative dissi-patory pathways and a higher irradiance level is neededto reach ETRm. Accordingly, decreases of ETRm underhigh light have been attributed to dynamic down-regu-lation of photosynthesis through reversible non-photo-chemical quenching (Ralph et al. 2002a, 2002b).However, the observed increases in ETRm stronglysuggest that some sort of compensatory mechanism isactivated under high light, which, together with the de-crease in a, causes the photosynthetic light response tosaturate at higher values (higher Ek). The most likelypossibility is the light-induced increase of the activity ofthe enzymes of carbon metabolism, which would resultin an effectively protective way of photochemically dis-

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sipating excitation energy otherwise excessive andpotentially damaging to PSII (Ralph et al. 1999; Gevaertet al. 2003). The simultaneous high light-induced de-crease in light-limited photosynthesis and the increase inlight-saturated photosynthesis was observed in steady-state light curves in phytoplankton (Behrenfeld et al.1998). These results were interpreted as denoting aphotoacclimation strategy toward the optimisation ofresource allocation among the different components ofthe photosynthetic apparatus, considering the large andrapid changes in light exposure that phytoplankton cellsare subjected to in their natural environment. Tests withcultures showed that only microalgal populations grownunder low light could develop the physiological capacityto perform compensatory increases in light-saturatedphotosynthesis under high-light conditions (Behrenfeldet al. 1998). Despite the possible discrepancies betweenphotosynthetic rates and fluorescence measurements(Hartig et al. 1998; Barranguet and Kromkamp 2000;Glud et al. 2002; Serodio 2003), the results obtainedsuggest that microphytobenthos cells may behavephysiologically as photoacclimated to relatively lowlight, enabling the plasticity necessary to cope with thesudden and large changes in the light exposure thatcharacterise the estuarine intertidal environment. In thecase of microphytobenthos, the development and per-sistence of low light-acclimation characteristics in anenvironment where exposure to direct sunlight occursdaily could be facilitated by the migration-mediatedregulation of light exposure.

Unlike a, ETRm measured on undisturbed micro-phytobenthos assemblages is potentially affected bychanges in microalgal distribution in the photic zone ofthe sediment, associated to depth-integration effects(Serodio 2004), that might also have contributed to theobserved results. In particular, a downward movement ofmicroalgae leading to the formation of a sub-surfacebiomass maximum, an expected migratory response toexcessive irradiances, would result in an increase of depth-integrated ETRm, independent of physiological changes(Serodio 2004). The changes inRLCparameters observedwithin 1 week may also have been caused by photoaccli-mation or changes in taxonomic composition, indicatingthe need formore detailed data on the temporal variabilityon hourly to seasonal time scales. In summary, theresponse of microphytobenthos to high irradiance can behypothesised to result from the simultaneous operationof three distinct processes: (1) down-regulation ofphotosynthesis through energy-dissipating mechanisms,detectable by a decrease in a; (2) compensatory increasein carbon metabolism activity, leading to an increasein ETRm; and (3) downwardmigration, increasingETRm,while not affecting a.

Endogenous variations

One of the advantages of using fluorescence RLCs is thepossibility to probe the dark-adapted state of the pho-

tosynthetic apparatus (White and Critchley 1999). Themeasurement of RLCs in undisturbed microphytoben-thos assemblages revealed apparently endogenouslycontrolled changes in photophysiological parametersduring dark–light transition and under constant condi-tions. The increase in RLC parameters preceding thestart of the photoperiod represent a ‘‘pre-activation’’ ofthe photosynthetic apparatus and may be interpreted asan adaptation to the periodic variability of the intertidalenvironment, complementing the role of endogenouslycontrolled migrations in the anticipation of events likethe beginning of diurnal low tide. The results show thatthis activation, indicated by the light dose necessary toobtain full light-activation, increases as the start of thelight period approaches. Light-activation was in all casessurprisingly fast, being virtually completed by theexposure to light during the construction of RLCs withillumination times of 40 s (a total light exposure of ca.5 min). In contrast, pea plants required >15 min of200 lmol m�2 s�1 to reach a steady-state in RLCparameters (White and Critchley 1999).

Endogenous rhythms in the photosynthesis versuslight response (steady-state) of microalgae have beenwidely reported, both for the parameters describing light-limited (aP) as light-saturated parts (Pm) of the light curve(Prezelin 1992). Variations in light-limited photosynthe-sis are known to be associated to changes in light-har-vesting complexes (concentration of photosyntheticallyactive accessory pigments, PSII:PSI ratio) and/or theactivity of the photosynthetic light reactions and PSII(non-photochemical quenching and photoinhibition)(Behrenfeld et al. 2004). A striking feature of the resultspresented here (and of all similar experiments conducted)is the extreme resemblance of the patterns of a and Fv/Fm

(Fig. 7). Strong correlations between aP and Fv/Fm havebeen found in diatoms, and interpreted as indicating thecontrol by common physiological processes: the efficiencyof energy transfer from the light-harvesting antennae tothe reaction centres and the number of functional PSIIreaction centres (Green et al. 1991; Geider et al. 1993;Falkowski and Kolber 1995). Variations in Fv/Fm in thedark were also observed in cyanobacteria-dominatedphytoplankton, being attributed to state-transitions, theregulated decoupling between antennae and PSII (Beh-renfeld and Kolber 1999). However, state-transitionshave never been observed in diatoms (Owens 1986; Tingand Owens 1993; Lavaud et al. 2002), the dominantgroup in the studied sample, and the fluorometer we used,by utilising blue excitation light, is expected to be rela-tively insensitive to cyanobacteria.

Fluctuations in Fv/Fm and a may be considered toresult from endogenously controlled changes in thecomposition of light-harvesting complexes associatedwith the cell cycle of the dominant migratory species.Endogenous oscillations in the effective absorption crosssection of PSII were shown to be associated to the cellcycle of green algae (Strasser et al. 1999), and the cellcycle of benthic diatoms was shown to be synchronisedwith the migratory rhythm (Saburova and Polikarpov

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2003). In higher plants, oscillations in light-limitedphotosynthesis were shown to result from the expressionof genes for chlorophyll a/b–binding proteins, con-trolled by a biological clock (Martino-Catt and Ort1992). In benthic diatoms, diel variations were shown inthe levels of mRNA from genes encoding fucoxanthin,chlorophyll a/c–binding proteins of the light-harvestingcomplexes (Meyer et al. 2003). On the other hand, as Fv/Fm is affected by the cellular nutrient status, related tothe production of PSII reaction centres (Falkowski andKolber 1995), the marked oscillations during subjectivephotoperiods could have been enhanced by the surfacingof motile microalgae, presenting increasing Fv/Fm and avalues due to recent nutrient uptake during burial in thenutrient-rich sub-surface layers of sediment.

Although RLCs may vary due to physiological causesalone, the observed variations could also result fromeffects of the endogenous migratory rhythm on thephysiological status of the microalgae. In diatoms, Fv/Fm may decrease in the dark, due to non-photochemicalquenching resulting from the formation of the trans-thylakoidal proton gradient, attributed to chlororespi-ration (Ting and Owens 1993; Jakob et al. 1999) and tothe reverse operation of ATPase (Dijkman and Kroon2002). As chlororespiration is inhibited by anaerobiosis,the decrease in Fv/Fm and a coincident with the surfacingof large numbers of microalgae (large and sudden in-crease in Fo) could be explained by the operation of thisprocess, as the microalgae become exposed to oxygenwhen reaching the surface. The following increase in Fv/Fm and a, coinciding with the start of the decrease in Fo,is consistent with the return of the microalgae to sub-surface layers of sediment, where oxygen is rapidly de-pleted. The dissipation of non-photochemical quenchingformed in the dark could also be responsible, at leastpartially, for the increases in a observed during transi-tions from darkness to low light (Figs. 1, 4, 5, 6).

Oscillations in light-saturated photosynthesis areusually attributed to the regulation of the activity ofphotosynthetic electron transport chain and/or Calvincycle enzymes (Behrenfeld et al. 2004), and endogenouscircadian rhythms in the transcriptional regulation ofthe Calvin cycle enzyme RUBISCO have been shown innatural phytoplankton populations (Pichard et al. 1996).In spite of the widely accepted idea that aP and Pm areuncoupled physiologically, covariance may occur in sit-uations when light-saturated electron transport is lim-ited by the functioning of PSII reaction centres(Behrenfeld et al. 1998, 2004). Similarly, covariation of aand ETRm during subjective light periods could be dueto the partial activation and de-activation of carbonmetabolism enzymes triggered by signalling betweenlight (photosynthetic electron flow between PSII andPSI) and dark reactions (Pichard et al. 1996). In con-clusion, a full explanation of these results cannot begiven with the presently available data. Further insightcould be obtained by examination of the behaviour of Fo

and Fm separately (Behrenfeld and Kolber 1999), but theanalysis of the patterns of variation of these parameters

in undisturbed samples is unavoidably encumbered bythe confounding effects of vertical migrations.

Acknowledgements We thank J. Marques da Silva for discussionand critical comments on the manuscript. We also thank A. Caladoand A. Tim-Tim for kindly providing unialgal cultures of C. clos-terium. This work was supported by project PDCTM/MAR/15318/99, funded by Fundacao para a Ciencia e a Tecnologia. We thankthree anonymous reviewers for critical comments on the manu-script.

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