Estuarine, Coastal and Shelf Science 68 (2006) 547e556 www.elsevier.com/locate/ecss Microphytobenthos vertical migratory photoresponse as characterised by light-response curves of surface biomass Jo~ao Serôdio*, Helena Coelho, Sónia Vieira, Sónia Cruz Departamento de Biologia, Universidade de Aveiro, Campus de Santiago, 3810-193 Aveiro, Portugal Received 30 November 2005; accepted 8 March 2006 Available online 22 May 2006 Abstract The migratory response of intertidal microphytobenthos to changes in irradiance was studied on undisturbed estuarine sediments. Two nondestructive optical techniques were used to trace variations in vivo of surface biomass: PAM fluorometry, for measuring the minimum fluorescence level (Fo); and spectral reflectance analysis, for quantifying the normalized difference vegetation index (NDVI). Following the formation of a dense biofilm at the surface, replicated sediment samples were simultaneously exposed to six different irradiance levels, ranging from 50 to 1500 mmol m
2 s
1, during a period of 120 min. The migratory photoresponse of the biofilms was characterised by constructing biomass vs. light curves (BLC), relating the accumulation of microalgal biomass after that period (estimated by Fo or NDVI) to the irradiance level incident on the surface. BLCs allow characterising the main features of the migratory photoresponse of intact biofilms. Typical BLC showed a clear biphasic pattern, with an increase in microalgal accumulation under irradiances below 100 mmol m
2 s
1, maximum values under 100e250 mmol m
2 s
1, and a gradual decrease of surface biomass under higher irradiances, indicating a strong photophobic downward migratory response. Similar BLC patterns were obtained when measuring Fo or NDVI. The construction of BLCs for biofilms from intertidal sites with distinctive sediment characteristics and diatom taxonomic composition allowed to detected significant differences in the migratory photoresponse. Biofilms from a muddy sediment exhibited considerably larger amplitude in the migratory photoresponse than the biofilms from a sandy mud site, especially under high irradiances. The photophobic migratory response to high light was found to vary among diatom species, particularly in the case of the biofilms from the muddy sediments. Ó 2006 Elsevier Ltd. All rights reserved. Keywords: biomass; chlorophyll fluorescence; microphytobenthos; phototaxis; reflectance; vertical migrations 1. Introduction The migratory behaviour of sediment-inhabiting microalgae is a long known phenomenon, particularly well documented for estuarine intertidal microphytobenthos (Consalvey et al., 2004b). Many species of diatoms, euglenophytes and cyanobacteria are capable of moving vertically within the upper layers of the sediment, in synchronization with dayenight and tidal cycles. These rhythmic movements are not limited to the photic zone of the sediment (Pinckney et al., 1994), and are known to be partially endogenously controlled (Round and Palmer, 1966; Serôdio et al., 1997). In recent years, this * Corresponding author. E-mail address: jserodio@bio.ua.pt (J. Serôdio). 0272-7714/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.ecss.2006.03.005 phenomenon has been increasingly studied, largely because of its recognized ecological importance as a key controlling factor of short-term variability in community-level microphytobenthic productivity (Pinckney and Zingmark, 1991; Serôdio et al., 2001). The rhythmic vertical migration of large numbers of microalgae causes the periodic accumulation of large amounts of microalgal biomass in the photic zone of the sediment. By considerably affecting the fraction of solar irradiance that can be absorbed and used for photosynthesis by the biofilm, large fluctuations in depth-integrated production rates are thus caused (Serôdio et al., 2001). Accordingly, this source of short-term variability has been formally included in the formulation of primary production models (Pinckney and Zingmark, 1993; Guarini et al., 2000; Serôdio and Catarino, 2000). 548 J. Serôdio et al. / Estuarine, Coastal and Shelf Science 68 (2006) 547e556 Benthic microalgal motility is known to be affected by various environmental factors, such as light intensity (Perkins, 1960; Paterson, 1986; Kingston, 1999a), light spectrum (Cohn et al., 1999), sediment physical disturbance (Hopkins, 1966), water cover (Pinckney et al., 1994; Mitbavkar and Anil, 2004), wave action (Kingston, 1999b), temperature (Cohn et al., 2003), and sub-surface nutrients (Kingston, 2002). Migratory responses to irradiance are particularly interesting due to the large variability of this parameter under in situ intertidal conditions and because it directly affects the functioning of the photosynthetic apparatus, triggering photoprotective mechanisms, and causing photoinhibition (Serôdio et al., 2005a). The influence of irradiance on the migratory behaviour is also important for the modelling of primary productivity and for the characterisation of the phototactic responses under high light, in the context of the often suggested hypothesis that the downward vertical migration may represent a form of behavioural photoacclimation or photoprotection (Admiraal, 1984; Kromkamp et al., 1998; Serôdio et al., 2001). Most manipulative studies of benthic microalgal motility as a response to environmental stimuli have been carried out on single-species microalgal populations grown in culture, under conditions markedly different from the natural microenvironment inhabited by natural assemblages (Cohn and Disparti, 1994; Cohn et al., 1999, 2004). On undisturbed sediments, such investigations have been limited to euglenophytes (Kingston, 1999a). To our knowledge, no systematic studies have been carried out explicitly on the migratory photoresponse of sediment-inhabiting diatoms while comprising natural biofilms. This work addresses the characterisation of the migratory response of natural microphytobenthic biofilms to irradiance. The phototactic response was studied by measuring the variation with incident irradiance of the surface biomass, here defined as the biomass present in the photic zone of the sediment and that is detectable by the optical techniques used in vivo to non-destructively estimate it: pulse amplitude modulation (PAM) fluorometry (for measuring the minimum fluorescence level (Fo)) and spectral reflectance analysis (for measuring the normalized difference vegetation index (NDVI)). The migratory photoresponse of undisturbed microphytobenthos was quantitatively characterised by constructing biomass vs. light curves, plots that relate the photoaccumulation of biomass (estimated by Fo or NDVI) to the irradiance level incident on the sediment surface. This approach was applied to the comparative study of the phototactic response of microphytobenthic biofilms growing on sediments of different granulometries and with different species composition. 2. Materials and methods 2.1. Sampling Undisturbed sediment samples were collected on two intertidal flats in the Ria de Aveiro, a mesotidal estuary of the west coast of Portugal: Vista Alegre (VA; 40  350 N, 8  410 W), on the west margin of the Canal de Ílhavo, and Gafanha da Encarnaç~ao (GE; 40  380 N, 8  440 W), on the east margin of the Canal de Mira. Sampling sites had different granulometric characteristics. The VA sampling site composed of fine muddy sediments (97% particles below 63 mm) while the GE site composed of sandy mud (45.3% particles between 63 and 125 mm; 42.7% below 63 mm). Sampling was carried out from November 2004 to April 2005. Sediment samples were collected using plastic corers (3.6 cm diameter) and taken to the laboratory where all the measurements were carried out. Samples were sectioned with minimum disturbance into plastic rings of 5 mm deep (same diameter of sampling corers), and maintained in a Petri dish containing water collected at the sampling site. When necessary, slurries were prepared by resuspending repeatedly the top 2 mm of sediment in filtered site water using a syringe. Microalgal suspensions were prepared by placing two pieces of lens tissue on the surface of illuminated sediment cores during daytime low tide. Microalgae were collected by resuspending the upper piece of lens tissue in filtered site water (Eaton and Moss, 1966). 2.2. Chlorophyll fluorescence Variable chlorophyll a fluorescence was measured using a PAM fluorometer comprising a computer-operated PAMControl Unit (Walz, Effeltrich, Germany) and a WATER-EDFUniversal emitter-detector unit (Gademann Instruments GmbH, Würzburg, Germany). This instrument uses a modulated blue light (LED-lamp peaking at 450 nm, half-bandwidth of 20 nm; further details in Serôdio et al., 2005b) as source for measuring, actinic and saturating light, emitted at a frequency of 18 Hz when measuring Fo. When using sediment samples (undisturbed samples or slurries), fluorescence was measured using a 6-mm diameter Fluid Light Guide fiberoptics bundle that delivered the measuring and saturating light provided by the fluorometer. Measurements were taken by positioning the fiberoptics perpendicularly to the sediment surface, at a fixed distance of 1 mm. The relative position of the fiberoptics and the sediment surface was controlled using a micromanipulator (MM33, Märtzhäuher, Germany). When measuring on microalgal suspensions, the fiberoptics bundle was connected to a fluorescence cuvette (KS-101, Walz). Unless stated otherwise, samples were dark-adapted for 2 min before the fluorescence level (Fo) was measured. On each occasion, Fo was measured in three separate, non-overlapping areas within each sample. 2.3. Spectral reflectance Reflectance spectra were measured using a fiber optic spectrometer (USB2000-VIS-NIR, grating #3, Ocean Optics, Duiven, The Netherlands). Reflectance was recorded over the 350e1000 nm bandwidth with a sampling spectral resolution of 0.38 nm, using a 400-mm diameter fiber optic (model QP400-2-VIS/NIR-BX, Ocean Optics), positioned perpendicularly to the sediment surface at a fixed distance of 2.2 cm, set to determine a view field coincident with the area monitored by the PAM fiberoptics. Reflectance was determined from the light spectrum reflected from the undisturbed J. Serôdio et al. / Estuarine, Coastal and Shelf Science 68 (2006) 547e556 sediment sample, normalized to spectrum reflected from a reference white panel. A reflectance spectrum measured in the dark was subtracted with both the sample and the white reference spectra to account for the dark current noise of the spectrometer. Spectrum measurement was carried out by exposing the samples (and the reference panel) to a constant irradiance of 200 mmol m
2 s
1, provided by the same light source that was used to study the migratory photoresponses (see below). Three replicated spectra were measured on each occasion, and the mean spectrum was used for the subsequent calculations. Reflectance measurements were used to estimate the microphytobenthos surface biomass by calculating the normalized difference vegetation index (NDVI; Rouse et al., 1973): NDVI ¼ R750
R675 R750 þ R675 ð1Þ where R750 and R675 represent the reflectance measured at 750 and 675 nm, respectively. R750 and R675 were calculated by averaging the values obtained in the intervals of 749.62e 750.38 nm and 674.62e675.38 nm, respectively. 2.4. Fo, NDVI vs. sediment Chl a content To test the possibility to use the fluorescence parameter Fo and the reflectance-based index NDVI to trace changes in the microalgal concentration in the upper layers of the sediment, Fo and NDVI were measured in vertically homogenous sediment samples of increasing Chl a content, prepared by adding increasing amounts of microalgae to sterilized sediment (Serôdio et al., 1997). Microalgae used in these slurries were collected by using lens tissue on sediment samples exposed to low light (100e150 mmol m
2 s
1) for several hours in the morning after the day of collection. Fo was determined after the slurries were dark-adapted for 30 min. Chl a was extracted in 90% aqueous acetone and quantified spectrophotometrically following the method of Lorenzen (1967). Because the fluorescence parameter Fo may be affected by physiological effects determined by light exposure prior to dark-adaptation, the relationship between Fo and Chl a was studied in samples that were previously exposed to a range of different irradiance levels. To isolate the physiological response of Fo and to avoid the confounding effects of migratory responses to incident irradiance, experiments were carried out using microalgal suspensions. Microalgae were collected as described above and were resuspended in filtered site water. Suspensions were exposed to 100, 500 and 1500 mmol m
2 s
1 during 90 min, after which they were dark-adapted for 2 min and Fo was determined. The suspensions were filtered on GF/F filters and Chl a was determined spectrophotometrically. 549 Fo and NDVI. The results of these experiments were used to construct biomass vs. light curves, which by summarizing the migratory response to irradiance allow to characterise the phototactic responses of intact assemblages along a range of irradiances. Coinciding with the beginning of the low tide in the sampling site, samples were exposed to 150 mmol m
2 s
1 for 1 h to induce the upward migration of motile microalgae (determined in previous tests). After this period, the samples were simultaneously exposed to a range of different irradiances for a period of 120 min. Each sample was exposed separately to a constant irradiance level during this period. Every 20 min, spectral reflectance was measured on each sample, after which was darkened for 2 min before Fo was determined. NDVI and Fo were determined for three matching nonoverlapping areas of each sample. Samples were exposed to actinic cold white light, provided by a halogen lamp (Quartzline lamp DDL 150W, General Electric, USA) emitting light with a continuous colour spectrum similar to natural sunlight. Light was delivered to the sample by a fiberoptics bundle connected to a 5-cm diameter light ring (Standard Ringlight and Volpi Intralux 5000-1, Volpi, Switzerland) which provided a homogeneous shade-free light field. Samples were placed below the centre of the light ring, at a fixed vertical distance from it (ca. 2 cm). Fo and NDVI were measured by passing the respective fiberoptics through the centre of the light ring. Photon irradiance incident on the sample surface was measured using a PAR micro-sensor (Spherical MicroQuantum Sensor, US-SQS/W, Walz). A total of six illumination systems were used to separately and simultaneously expose each of six sediment samples to each of the following irradiance levels: 50, 100, 250, 500, 1000 and 1500 mmol m
2 s
1. One additional sample was transferred to darkness and maintained in the dark during the measuring period. 2.6. Species identification After the construction of biomass vs. light curves, the upper 1 mm of the sediment samples exposed to 100, 500 and 1500 mmol m
2 s
1 was sectioned and diluted in a known volume of filtered sea water for determination of taxonomic composition. Sub-samples of the diluted slurries were fixed in 1% v/v formaldehyde and viewed under bright-field microscope for determination of the relative abundance of major taxonomic groups (diatoms, euglenophytes and cyanobacteria), by counting a minimum of 400 cells on three replicated sub-samples. Diatom identification was done on sub-samples oxidized using nitric acid and potassium dichromate, and digested in concentrated HCl. 3. Results 2.5. Migratory photoresponse: biomass vs. light curves 3.1. Fo, NDVI vs. sediment Chl a content The migratory photoresponse of microphytobenthic assemblages was studied by simultaneously exposing replicate sediment samples to different irradiance levels and by following the induced changes in surface biomass through variations in Both Fo and NDVI were found to vary linearly with the sediment Chl a content (Fig. 1). Highly significant correlations were obtained between Chl a content and Fo (r ¼ 0.965, p < 0.001) and NDVI (r ¼ 0.938, p < 0.001). Linear 550 J. Serôdio et al. / Estuarine, Coastal and Shelf Science 68 (2006) 547e556 600 A B y = 902.3 x + 37.3 r = 0.965 500 y = 0.59 x + 0.16 0.5 r = 0.938 Fo (a.u.) 300 0.3 NDVI 0.4 400 200 0.2 100 0 0.1 0.0 0.1 0.2 0.3 0.4 0.5 0.0 0.1 0.2 0.3 0.4 0.5 0.6 Chl a (mg g-1) Fig. 1. Linear relationship between sediment Chl a content and the fluorescence parameter Fo (A) and the spectral reflectance index NDVI (B). relationships were also found between Fo and Chl a concentration when microalgae were previously exposed to actinic irradiance levels prior to the 2-min dark-adaptation ( p < 0.001 in all cases; Fig. 2). The Fo vs. Chl a relationship was not substantially affected by the previous light exposure, as no significant differences were found among the slopes or the intercepts of the linear regression equations ( p ¼ 0.474 and p ¼ 0.827, respectively; ANCOVA). 3.2. Migratory photoresponse The exposure of microalgal biofilms to different irradiance levels resulted in a large range of migratory responses. The 350 y = 0.141 x + 0.88 r2 = 0.921 300 Fo (a.u.) 250 200 150 100 100 50 500 1500 0 0.3 0.6 0.9 1.2 1.5 1.8 2.1 Chl a (mg L-1) Fig. 2. Effects of previous light exposure on the relationship between minimum fluorescence level (Fo) and the Chl a concentration in a microphytobenthos suspension. Linear regression equation based on all the data. Numbers represent the irradiance level to which the samples were exposed prior to the measurement of Fo (mmol m
2 s
1). typical variation in the migratory response with incident irradiance, as monitored by Fo, is illustrated in Fig. 3A. Under 50e100 mmol m
2 s
1, an increase in Fo was consistently observed, denoting the upward migration and accumulation of microalgae at the surface. In contrast, the transition to darkness induced a downward migratory response, causing a decrease of ca.
20% of Fo initial values after 60 min. A maximum increase in Fo was typically observed for 100e250 mmol m
2 s
1. Under higher irradiances, Fo decreased in all cases, with the rate of decrease increasing with the irradiance level applied. For 1500 mmol m
2 s
1, Fo was reduced by ca.
60% of the initial value, and by ca.
40% of the maximum value (Fig. 3A). It was also often observed that the downward migratory response under high light was more rapid than the upward migratory response under low light: under 1500 mmol m
2 s
1, most of Fo variation occurred in less than 40 min, while under 100 mmol m
2 s
1, Fo continued to increase after 100 min. These biofilms were dominated by diatoms (relative abundance > 99%), with the species Navicula phyllepta Kützing, Nitzschia frustulum (Kützing) Grunow, Parlibellus crucicula (W. Smith) Witkowski, Lange-Bertalot and Metzeltim, and Gyrosigma fasciola (Ehrenberg) Griffth and Henfrey accounting for 61.7% of cells counts after exposure to 100 mmol m
2 s
1. The decrease in Fo under high light was matched with a proportional decrease in cell concentration in the upper 1 mm of sediment, from 2.90 to 2.60  106 cells ml
1 (for 100 and 1500 mmol m
2 s
1, respectively). This decrease in cell concentration was clearly detectable by a substantial change in the colour of the sediment surface. The variation in the migratory response with irradiance can be summarized by plotting Fo against E and by constructing photoaccumulation or biomass vs. light curves (BLC; Fig. 3B). These curves allow to quantitatively characterise the main features of the migratory photoresponse of the biofilm: a clear biphasic pattern, with a steep variation in biomass accumulation under low irradiances, an almost symmetrical peak 551 J. Serôdio et al. / Estuarine, Coastal and Shelf Science 68 (2006) 547e556 B A 100 1.4 Fo (rel. units) 60 min 120 min 1.6 1.4 1.2 1.2 50 250 500 0 1.0 0.8 1.0 0.8 Fo (rel. units) 1.6 1000 0.6 0.6 1500 0.4 0.4 0.2 0.2 0 20 40 60 80 100 120 140 0 250 500 750 1000 1250 1500 Irradiance (µmol quanta m-2 s-1) Time (min) Fig. 3. (A) Typical migratory light response of undisturbed microphytobenthos biofilms, as determined by measuring fluorescence parameter Fo on samples preexposed to constant irradiance of 150 mmol m
2 s
1 for 120 min. Numbers represent the irradiance level prior to the measurement of Fo (mmol m
2 s
1). Fo values normalized to initial values. (B) Photoaccumulation or biomass vs. light curves (BLC), constructed from the data in panel (A). Examples of BLCs obtained after 60 and 120 min of light exposure. Mean values of three measurements. 3.3. Application to the comparison of biofilms The construction of BLCs for microphytobenthos from intertidal sites with distinctive sediment granulometry revealed a significant difference in the migratory photoresponse (Fig. 4). Although the same general pattern was found in both cases, similar to the one described above (Fig. 3), the microalgal biofilm from the site with finer sediment (VA) exhibited a larger amplitude in the migratory response to changes in irradiance, with Fo varying by more than 120% between maximum and minimum values (under 100 and 1500 mmol m
2 s
1, respectively). In contrast, the migratory response of the biofilm from the sandier sediment (GE) was less accentuated, both under low and high irradiances, with the maximum range of Fo variation being less than 40%. The difference in the migratory response of the two types of biofilms is particularly evident regarding the decrease of surface biomass under high light. While for VA, Fo was reduced by more than
20% (relatively to initial values) under 500 mmol m
2 s
1 and by ca.
65% under 1500 mmol m
2 s
1, in the case of GE, values below the initial value were found only under 1000 mmol m
2 s
1, and the maximum decrease reached less than
17%. The two types of biofilms presented important differences regarding species composition and relative abundance. The biofilms from the muddy site VA were dominated by epipelic diatoms Navicula gregaria, Navicula phyllepta, Parlibellus crucicula and Gyrosigma fasciola. The assemblages from the sandy mud site GE were dominated by diatoms Achnanthes delicatula Kützing, Achnanthes minutissima Kützing and N. gregaria Donkin. The relative abundance of euglenophytes (Euglena sp.) and cyanobacteria (Oscillatoria sp., Merismopedia sp.) was in all cases <1%. Despite the difference in species composition, in both types of biofilms the decrease in Fo was followed by a parallel decrease in microalgal cell concentration in the upper 1 mm of sediment 1.8 VA GE 1.6 1.4 Fo (rel. units) under E  100 mmol m
2 s
1, and a continuous decrease under higher irradiances, almost linear for E  500 mmol m
2 s
1. Although some variability was found among days, the same overall pattern was consistently observed in all the experiments, which was usually formed and stabilized after 1 h of light exposure (Fig. 3B). The biphasic pattern observed when measuring Fo was confirmed to represent changes in microalgal biomass accumulation at the surface as very similar results were obtained when measuring NDVI. Highly significant correlations ( p < 0.001) were obtained between Fo and NDVI for all the BLC constructed. This general pattern of photoresponse was observed in all the experiments that were carried out during a period of several months, both when measuring Fo and NDVI. 1.2 1.0 0.8 0.6 0.4 0.2 0 250 500 750 1000 1250 1500 Irradiance (µmol quanta m-2 s-1) Fig. 4. Biomass vs. light curves for microphytobenthos biofilms from two intertidal sites with distinctive sediment characteristics (VA: mud; GE: sandy mud). Mean values of three measurements. Vertical bars represent 1 standard deviation. 552 J. Serôdio et al. / Estuarine, Coastal and Shelf Science 68 (2006) 547e556 VA GE 3.0 2.5 Cell concentration (x 106 ml-1) A B 2.9 2.4 2.8 2.3 2.7 2.2 2.6 2.1 2.5 2.0 100 100 Relative abundance (%) C D 80 80 60 60 40 40 20 20 0 100 500 1500 100 500 1500 0 Irradiance (µmol quanta m-2 s-1) Navicula phyllepta Navicula gregaria Parlibellus crucicula Gyrosigma fasciola Cocconeis scutellum Nitzschia frustulum Other Achnantes delicatula Achnantes minutissima Navicula gregaria Nitzschia sp. Amphora sp. Navicula phyllepta Other Fig. 5. Microalgal concentration (A and B) and mean relative abundance of the main diatom taxa (C and D) in the upper 1 mm of sediment of sampling sites VA (mud) and GE (sandy mud) after exposure to different irradiances. Mean values of three measurements. Vertical bars represent 1 standard error. Only relative abundances > 5% are presented. Relative abundance of euglenophytes and cyanobacteria always <1%. (Fig. 5A, B), which decreased significantly with irradiance (one-way ANOVA, p < 0.001). However, the downward migratory response to high irradiance was found to vary among species, and this species-specific variability was found to be larger for the assemblages from VA than from GE. In the case of VA, while the relative abundance of some species was found to decrease with irradiance (N. gregaria and P. crucicula; Fig. 5C), indicating a particularly active migratory photoresponse, others showed an opposite trend (Cocconeis scutellum, Nitzschia frustulum; Fig. 5C), reflecting a weaker photophobic response to high light. In the case of GE, most species did not exhibit a marked variation in relative abundance with increasing light level (Fig. 5D). The exception was A. minutissima, which showed an increase in relative abundance as irradiance increased, which indicates a less pronounced photophobic response than most other species. 4. Discussion 4.1. Monitoring microphytobenthos biomass through optical methods Due to significant operational advantages over previously applied methods, PAM fluorometry became widely used for study of the migratory behaviour of sediment-inhabiting microalgae. Through the measurement of the minimum fluorescence yield, Fo, this method allows the rapid and nondestructive monitoring of variations in the microphytobenthic biomass in the vicinity of the sediment surface. Fo was shown to allow the estimation of the ‘productive biomass’ of microphytobenthos, defined as the microalgal biomass present in the photic zone, weighted by its contribution to depth-integrated photosynthesis (Serôdio et al., 2001). Fo was also shown to correlate well with the ‘photosynthetically active biomass’, J. Serôdio et al. / Estuarine, Coastal and Shelf Science 68 (2006) 547e556 defined simply as the microalgal biomass present in the photic zone of the sediment (Guarini et al., 2000; Honeywill et al., 2002). Nevertheless, the use of Fo as a proxy for microphytobenthos biomass has potentially important shortcomings. Because the fluorescence emission per unit Chl a depends on the physiological status of the microalgae, Fo has been preferred to other fluorescence parameters as it was shown to be the least affected by environmental factors (Serôdio et al., 1997, 2001; Consalvey et al., 2004a). However, the measurement of Fo requires the darkening of the sample which, in the case of diatoms, should ideally be prolonged for a period time of tens of minutes (Ting and Owens, 1993). As the results of this study confirmed, the exposure to darkness, even during the early part of the light period, can induce a confounding decrease in surface biomass. As a compromise between minimizing the physiological effects of recent light history and the artifactual migratory responses to darkness, Fo has been measured by applying relatively short dark-adaptation periods, such as 2 min (Serôdio et al., 2005b), 5 min (Serôdio et al., 2001) or 15 min (Honeywill et al., 2002; Consalvey et al., 2004a). The results of this study indicate that variations in Fo, measured after only 2 min of dark-adaptation can be mostly attributed to changes in the Chl a content of the sediment. Physiological effects causing the quenching of Fo under high light may be expected not to confound significantly the changes in Fo resulting from variations in microalgal biomass, because its effects can be assumed as much smaller that the typical changes in microalgal biomass associated to vertical migrations. Indices based on spectral reflectance, like NDVI, have been increasingly used as an alternative optical method for noninvasively monitoring changes in microphytobenthos biomass (Paterson et al., 1998; Decho et al., 2003; Carrère et al., 2004; Murphy et al., 2004). The use of NDVI avoids most problems associated to the use of Fo, as it is not affected by the physiological status of the microalgae and thus its determination does not require dark-adaptation of the sample. A possible cause for discrepancies between Fo and NDVI-based estimates of surface Chl a content is the different depth interval below the sediment surface which is monitored by two types of methods. This depth interval is determined by the optical properties of the sediment, which control the penetration of downwelling measuring light (of the PAM fluorometer) and of ambient light, and the attenuation of upwelling fluorescence and reflected light (Serôdio et al., 1997, 2001; Kromkamp et al., 1998). In this study, the estimation of the depth interval correspondent to reflectance signals was not attempted. However, because reflectance was measured under a constant irradiance level, the vertical zone monitored by NDVI and Fo may be expected to be proportional. Accordingly, similar migratory responses were obtained using the two methods. 4.2. Biomass vs. light curves Microphytobenthos are probably unique among aquatic photoautotrophs in having the capacity of adjusting 553 photosynthetic activity not only through physiological regulation of the use of absorbed light energy by the photosynthetic apparatus, but also through active control of light absorption, by behaviourally exploiting environmental light gradients. Although the exposure to light is controlled at the individual level, by the movement of cells within the vertical light gradient, its effects are expressed at the community level through variations in the biofilm biomass present in the photic zone. As photosynthesis vs. light (PeI ) curves are used to characterise the physiological response to changes in absorbed light energy, the variable migratory response to changes in ambient irradiance can be adequately described by biomass vs. light curves. BLCs characterise the migratory photoresponse of the whole biofilm, thus providing a way to compare the behavioural photoresponse of microalgal assemblages along time, site characteristics, or species composition. Results on the migratory response to light of microphytobenthic assemblages were first reported in the form of BLCs by Kingston (1999a), summarizing the results of manipulative experiments on the phototactic response of Euglena proxima populations under in situ conditions. In this present study, to avoid the possibly confounding effects of temperature on the migratory photoresponse (Cohn et al., 2003), unavoidable when measurements are carried out in situ, cold light sources were used to ensure the characterisation of the response to incident irradiance independently of temperature. Nevertheless, it cannot be excluded that temperature, directly associated to variations in incident solar irradiance, may also play a role as an environmental clue in the determination of the migratory pattern of natural assemblages in situ. 4.3. Migratory photoresponse and behavioural photoprotection The construction of BLC on intact microphytobenthos samples revealed a consistent biphasic photoaccumulation pattern that resulted from the upward migration under low to moderate irradiances (50e250 mmol m
2 s
1) and the downward migration under very low (<50 mmol m
2 s
1) and high (>500 mmol m
2 s
1) irradiances. The downward migratory response to low light or darkness is likely to result from the fact that microalgae take the decrease in ambient light as an environmental clue indicating sunset or incoming high tide. Of particular interest is the downward migratory response under high light, consistently found in this study. This photophobic behaviour has often been hypothesized as a form of ‘behavioural photoprotection’, through which motile microalgae avoid exposure to potentially damaging irradiance levels (Admiraal, 1984; Kromkamp et al., 1998; Perkins et al., 2001; Serôdio et al., 2001; Underwood, 2002). This eventual photoprotective ability would be functionally equivalent to the well-known phenomenon of chloroplast avoidance movements in higher plants (Haupt and Scheuerlein, 1990) which effectively decreases photodamage caused by high light (Kasahara et al., 2002). Although the downward migration of sediment-inhabiting microalgae under high light is frequently mentioned in the literature, only a few studies have 554 J. Serôdio et al. / Estuarine, Coastal and Shelf Science 68 (2006) 547e556 shown experimental evidence of this behaviour, either directly, through manipulation of light levels (Perkins, 1960; Kingston, 1999a) or indirectly, through the analysis of the light response of depth-integrated estimates of photosystem II electron transport rates (Perkins et al., 2001). Nevertheless, the migratory photoresponse behaviour of diatoms has been described in detailed from studies carried out on unialgal cultures (Cohn et al., 1999, 2003, 2004). The biphasic response to light reported in this study is in close agreement with the results obtained with the diatom Cruticula cuspidata, that has shown the occurrence of step-down photophobic migratory responses under 50e100 mmol m
2 s
1 and step-up photophobic responses under irradiances above 500 mmol m
2 s
1 (Cohn et al., 2004). While the decrease in Fo or NDVI observed when samples are transferred from low to high irradiance is a clear indication of a photophobic migratory reaction to high light, the available data do not allow to conclude on the photoprotective role of this behaviour. Nevertheless, it is interesting to note that the irradiance levels under which the photophobic response is triggered (100e250 mmol m
2 s
1) generally coincide with levels found, on microphytobenthic assemblages from the same intertidal site, to induce the operation of reversible photoprotective nonphotochemical quenching (NPQ) mechanisms (Serôdio et al., 2005a). NPQ processes operate by reducing the transfer of absorbed light energy to the reaction centers and, while providing an effective protection against excessive light, result in the lowering of the rate of photosynthesis (Müller et al., 2001). This suggests that the negative phototaxis under high light may in fact reflect a way to regulate the light level to which the cells are exposed to, in order to minimize the triggering of physiological photoprotective mechanisms and to maintain the capacity for high photosynthetic rates. The peak in biomass accumulation observed under low light may simply reflect the fact that such irradiance levels are too low to cause photodamages and to induce physiological or behavioural photoprotective responses. Under such favourable light conditions, it may be expected the continuous accumulation of microalgae at the surface, only limited by space availability. This would explain the fact that under 100 mmol m
2 s
1, Fo and NDVI increased continuously for a longer period than under other irradiance levels. A steep decrease in surface biomass under high light was also found by Kingston (1999a) on microphytobenthos dominated by Euglena proxima. However, a peak in biomass accumulation under intermediate light levels was not found, which may explained by the absence of data for the range 30e300 mmol m
2 s
1. Thus, the undetected presence of a photoaccumulation pattern similar to the one found in this study cannot be excluded. The construction of BLCs on microphytobenthos from sediments of distinct granulometry revealed a clear difference regarding the migratory photoresponse. The different species composition of the two assemblages suggests that the variation in the photoresponse pattern may be associated to a distinct capacity for behaviourally regulating light exposure. In fact, the biofilms from fine the muddy sediments (VA) were dominated by epipelic diatoms, for which longer and faster vertical migrations are expected. Also, the assemblages from sandier sediments (GE) were composed to a large extent by diatoms of the genera Achnanthes and Amphora, known to include non-migratory, epipsamic species which live attached to the larger sediment grains (Round, 1979; Admiraal, 1984). The results indicate that the light-induced migratory behaviour may have a higher relative importance as a photoregulatory and photoprotective strategy in the case of epipelic-dominated biofilms. And that in the case of microalgal assemblages growing on coarser sediments, the regulation of light absorption and photoprotection may have to rely to a larger extent on physiological processes. BLCs could be used together with measurements of NPQ to ascertain the relative importance of behavioural and physiological photoprotection in epipelic and epipsamic-dominated microphytobenthic biofilms. In this study, the migratory photoresponse of microphytobenthos was characterised on the basis of variations in surface biomass. However, the occurrence of changes in species composition in the photic zone cannot be excluded as there is wide evidence that overall migratory behaviour of the whole biofilm is in fact composed of different, species-specific behaviour patterns (Palmer and Round, 1965; Round and Palmer, 1966; Round, 1979; Paterson, 1986; Perkins et al., 2002; Underwood et al., 2005). Considerable diversity in the migratory photoresponse may be expected as different species may have different migratory capacities or may respond diversely to changes in light. Diatom motility has been shown to vary with species (Bertrand, 1990), type of mucilage produced while moving (Bellinger et al., 2005) and the presence of other species (Cohn et al., 2003). Furthermore, the replacement of cells in the photic zone may also have occurred (‘micromigration’; Kromkamp et al., 1998), particularly during prolonged periods under high light. Because the photic depth (<200 mm; Serôdio et al., 1997; Kromkamp et al., 1998) is certainly much smaller than relatively large thickness of the sediment sections (1 mm) that were used for species identification and counting, it is conceivable that more important changes in species composition occurring in this depth interval may have passed largely undetected. 4.4. Implications for the modelling of primary productivity Although BLCs relate quantitatively the surface biomass with incident irradiance, they cannot be used directly to predict the variation in surface biomass from irradiance data measured during daytime emersion periods. First, because biomass vs. light curves were constructed only after the biofilm was formed at the sediment surface (induced by the exposure to low light), the relationship between biomass and irradiance thus established may not represent the pattern of microalgae accumulation following the hourly variation in solar light intensity. Secondly, the biomass measurements were made over relatively short periods, therefore not allowing for the build-up of factors that in situ have cumulative effects on photosynthesis and may enhance photodamage, like high pH, high O2 concentrations, or carbon depletion. Nevertheless, the obtained results have potentially important implications for the J. Serôdio et al. / Estuarine, Coastal and Shelf Science 68 (2006) 547e556 modelling of short-term (intraday) variability of the photosynthetic rate of microphytobenthos and for the estimation of primary productivity budgets for estuarine intertidal areas. Recent models acknowledge the importance of migratory rhythms for the quantification of the instantaneous rates of depth-integrated photosynthesis, by modelling it as a function of productive biomass, either implicitly (Pinckney and Zingmark, 1993) or explicitly (Guarini et al., 2000; Serôdio and Catarino, 2000). 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