The recognised importance of the primary productiv-ity of benthic microalgae in littoral zones (MacIntyre etal. 1996, Serôdio & Catarino 2000) has led to an in-creased focus on understanding the dynamics of thesecommunities (MacIntyre et al. 1996, Underwood &Kromkamp 1999). Distinct assemblages colonise rocks,
sediments and coral reefs, and inhabit intertidal andsubtidal areas in almost all latitudes (MacIntyre et al.1996, Barranguet 1997, Serôdio et al. 1997).
The primary production of microphytobenthos(MPB) is particularly high in intertidal mudflats (Mac-Intyre et al. 1996 and references therein, Barranguet1997) and shallow subtidal sites (Miles & Sundbäck2000, Glud et al. 2002), representing a significant frac-
Discovery of microphytobenthos migration in thesubtidal zone
Sorcha Ni Longphuirt1,*, Aude Leynaert1, Jean-Marc Guarini2, Laurent Chauvaud1, Pascal Claquin3, Olivier Herlory4, Erwan Amice1, Pierre Huonnic1, Olivier Ragueneau1
1LEMAR, Laboratoire des Sciences de l’Environnement Marin, UMR 6539 CNRS, Institut Universitaire Européen de la Mer, Technopôle Brest-Iroise, Place Nicolas Copernic, 29280 Plouzané, France
2Observatoire Océanologique Laboratoire Arago, BP 44, 66654 Banyuls/Mer, France3LBBM, Laboratoire de Biologie et de Biotechnologies Marines, Université de Caen Basse-Normandie, Esplanade de la paix,
14032 Caen, France4LBEM, Laboratoire de Biologie et Environnement Marins, FRE 2727 CNRS, Université de La Rochelle, Avenue M. Crépeau,
17042 La Rochelle, France
ABSTRACT: Microphytobenthos (MPB) contribute significantly to primary production in many estu-arine and coastal waters. Although the vertical migration of microphytobenthos is well integratedinto benthic studies of intertidal areas, the presence and importance of such migration has not yetbeen investigated in subtidal areas. In situ measurements and sampling in the Bay of Brest, FrenchAtlantic coast, in November 2003 and May, July and September 2004 and subsequent laboratoryexperiments showed that pulse amplitude modulated (PAM) fluorometry can be employed to followvertical migration of subtidal MPB. Steady-state fluorescence (F ) was highly correlated with chloro-phyll a (chl a) concentrations in the top 200 μm of sediments and could be used to detect in situchanges in MPB biomass. When (as in the present study) subtidal variations in light and temperatureare negligible compared to those in intertidal areas, variations in fluorescence parameters due tochanges in chl a concentrations are much greater than those due to photochemical and non-photochemical quenching. In situ surveys during 3 seasons showed a daily bell-like periodicity in F ,regardless of tidal oscillations. Sediment cores incubated at constant light and temperature displayedsimilar day/night fluorescence variations, indicating an endogenous rhythm, and that the diel cycleis the main factor triggering migration. Our results revealed a difference in the functioning of subtidaland intertidal MPB, in which migration is linked not only to diel but also to tidal cycles. In future stud-ies of subtidal benthic primary production, care should be taken in sampling strategies, particularlyin selecting sampling times, as the migratory behaviour of the MPB may greatly alter estimates ofavailable or actively photosynthesising biomass.
Resale or republication not permitted without written consent of the publisher
Mar Ecol Prog Ser 328: 143–154, 2006
tion of overall primary productivity in estuarineregions (MacIntyre et al. 1996, Underwood & Krom-kamp 1999). In addition, subtidal MPB regulate nutri-ent fluxes at the sediment–water interface (Sundbäcket al. 1991), forming an active filter and reducing theflow of nutrients into the pelagic zone (Facca et al.2002).
Within intertidal sediments light is rapidly attenu-ated, and the photic depth is shallow (Guarini et al.2000); thus photosynthesis and primary productionoccur when the MPB migrate to the surface of muddysediments during daytime emersions (Paterson &Crawford 1986, Janssen et al. 1999, Serôdio & Catarino2000). As tidal waters recede, microalgae quicklymigrate to the sediment surface where they receivethe maximum incident light energy available. Thisendogenous migratory rhythm is maintained whenexternal physical synchronisers are removed (Palmer& Round 1965, Serôdio et al. 1997). The migrationresults in the formation of a biofilm, which consists of adense layer of cells at the mud surface (Paterson &Crawford 1986). The composition of this biofilm maychange during the course of the diel period due to thesequential micro-cycling of different MPB species(Underwood et al. 2005). Cells migrate into deepersediments before the rising tide or at nightfall (Serôdioet al. 1997, Guarini et al. 2000). A portion of the biofilmhas also been shown to remain on the surface duringthe rising tide (Paterson & Crawford 1986, Janssen etal. 1999), resulting in the resuspension of cells into thewater column (de Jonge & van Beusekom 1995, Blan-chard et al. 1997). In the pioneering work of Palmer &Round (1965) on the migration of Euglena obtusa, theauthors postulated that the MPB migrate under theinfluence of a permanent positive geotactic responsewhich is overlaid by a rhythmic variation in phototaxis.In this and subsequent work (Round & Palmer 1966)they concluded that the migration was fundamentallydiurnal but that a superimposed tidal influence led toan alteration in this rhythm.
A substantial number of studies and reviews havebeen published on subtidal MPB and their role in thecoastal system (Cahoon 1999 and references within),but the presence of a migratory dynamic in subtidalcommunities is virtually undocumented. Underwood etal. (1999) reported a visual change in the biomass ofGyrosigma balticum subtidal mats. Cells wereobserved on the sediment surface in the morning andevening but seemed to burrow into the sediment atmidday when higher light irradiances reached thesediment surface. Glud et al. (2002), studying the MPBof an arctic fjord using pulse amplitude modulated(PAM) fluorometry, found no migratory movement fora range of irradiances applied in the laboratory,although motile genera were dominant. In their pri-
mary production calculations, migration was notaccounted for, although they did not exclude the possi-bility of such migration in situ.
If present, vertical migration of subtidal MPB, maydiffer from that of intertidal MPB as physical con-straints in the 2 environments are completely different.Subtidal MPB are not subject to aerial exposure or tothe drying out of sediments, and thus variations in lightand temperature are much smaller and factors such astidal stage and responses to available ambient lightlevels (Pinckey & Zingmark 1991, Perkins et al. 2003)may have a different influence on their functioningthan in intertidal MPB.
In the present study, our objectives were (1) to tracein situ variations in PAM fluorescence at the surface ofsubtidal sediment to assess if a migratory rhythm ispresent in benthic diatoms and, if so, (2) to determinethe main driving factor behind this, based on experi-ments performed in controlled conditions in the labo-ratory.
MATERIALS AND METHODS
Study site. The Bay of Brest is a semi-enclosedmarine ecosystem on the French Atlantic coast. Theecosystem is connected to coastal waters (Iroise Sea)by a narrow (2 km wide) and shallow (40 m) strait. It isa shallow-water ecosystem (average depth at the low-est sea level ca. 8 m) with about half of its total area(ca. 180 km2) shallower than 5 m. Main freshwaterinputs come from 2 rivers, the Aulne (southern) and theElorn (northern). Tidal oscillations induce short-termvariability in hydrological parameters and increasewater mixing (Chauvaud et al. 1996). Maximum tidalamplitude reaches 8 m during spring tides, which rep-resents an oscillation of 40% of the high tide volumeover the tidal cycle.
In situ measurements and sampling for laboratoryexperiments were undertaken at the Saint Anne site(48° 21’ 610’’ N, 4° 33’ 000’’ W), Bay of Brest, in Augustand November 2003 and May, July and September2004. During the study periods, the tidally controlledwater depth varied between ca. 4 and 11 m. Themedian grain size of the muddy sediments at the sitewas 100 μm, with 29% of the sediment being <63 μm.The site is sheltered on 3 sides, hence resuspension ofsediment and effects of currents are minimal.
Fluorescence measurements. An underwater fluo-rometer ‘Diving PAM’ (Heinz Walz) was used to recordthe fluorescence at the mud surface, in the field and incontrolled conditions in the laboratory. PAM fluoro-metry has previously been used to study MPB verticalmigration (Serôdio et al. 1997, Kromkamp et al. 1998,Serôdio 2003). A modulated (multiple turnover) red
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light from a light-emitting diode (LED) (650 nm, pulsewidth 3 μs, frequency 0.6 kHz) is applied to the cellsand a sensor then detects the returned fluorescencethat is emitted by the chl a present (see Maxwell &Johnson 2000 for review). Several parameters can bemeasured: steady-state fluorescence in ambient light(F ), minimal fluorescence after a period of dark adap-tation (F 0), maximum fluorescence after a multipleturnover, saturating flash applied following dark adap-tation (F m), and maximum fluorescence after a multi-ple turnover saturating flash applied in ambient lightwithout dark adaptation (F m’), (see Kromkamp &Forster [2003] for definitions and explications of all flu-orescence parameters). The effective photochemicalefficiency of Photosystem II (PSII) in light adapted cellscan be calculated as (Genty et al. 1989):
ΔF /F m’= (F m’ – F )/F m’ (1)
F 0 can be used as a proxy for the phototrophic bio-mass as it is well correlated with the chl a concentra-tion present (Serôdio et al. 1997, Barranguet & Krom-kamp 2000, Honeywill et al. 2002). It was howevertechnically impossible to manipulate the systemunderwater to induce dark adaptation on a repetitive,short, time scale; therefore, F was used in this study totrack vertical migration of MPB at the sediment surface(Kromkamp et al. 1998, Perkins et al. 2001, Serôdio2003). In all experiments, PAM fluorometer settingsand the distance between the probe and the sedimentsurface (10 mm) were maintained constant. In order tolimit the background fluorescence signal of the sedi-ment, for each experiment, the auto-zero function ofthe PAM was performed for sediment samples taken atdepths of 10 cm. The following settings were appliedfor the duration of the experiment: light saturationintensity, 8; saturation width, 0.8 s, out-gain, 8; lightgain, 1; actinic intensity, 5; damp, 2; measuringintensity, 8.
Three experiments were conducted:Chlorophyll a (chl a) concentrations and F mea-
surements at sediment surface: This first set of experi-ments consisted of measuring simultaneously the chl aconcentration at the sediment surface (0 to 200 μm)and the steady-state fluorescence, F, of undisturbedcores, to determine whether in situ PAM fluorometermeasurements could be used as a proxy for MPB bio-mass measurements. Sampling at the study site wascarried out by SCUBA divers at sunrise, on 17 Novem-ber 2003 (32 cores: 19 cores of 7 cm ∅ and 13 of 5 cm ∅)and on 7 May 2004 (9 cores, 5 cm ∅). Cores wereplaced immediately in an outdoor incubation bath andwere maintained at sea temperature (±0.5°C) by a con-tinuous flow of seawater. Neutral nickel screens atten-uating 50% of the incident light were placed over thecores to replicate in situ luminosity. Photosynthetically
active radiation (PAR) was recorded in the incubationbath.
For each measurement, cores were removed fromthe incubation bath. Subsamples (2 from each 7 cm ∅core and 1 from each 5 cm ∅ core) were taken usingpolycarbonate tubes of 3 cm ∅. In November a total of51 subsamples were acquired and in May, 9 subsam-ples were extracted. The tubes were first placed at thesurface of the sediment to mark the area of sampling,and F measurements were then made within eachmark. The tubes were subsequently pushed into thesediment and the surface of each subsample wasfrozen with liquid nitrogen (Wiltshire et al. 1997). Thefrozen subsamples were stored at –80°C until treat-ment and were freeze-dried within the following 48 h.The topmost 200 μm (considered as the sediment depthto which fluorescence is detectable by the Diving PAMfluorometer; Kromkamp et al. 1998, Honeywill et al.2002) was cut using a freezing cryotome (LEICA, CM1900). As a result of the extremely difficult and finetechnique involved in the handling and cutting of sucha thin layer of sediment, only 35 out of the 51 cores inNovember and 7 out of the 9 cores in May were used inthe experiment. Reasons for exclusion of samplesincluded disintegration of surface layers during freeze-drying, breakage of sediment surface layers whilecutting, and uneven surface that made precise cuttingimpossible.
The chl a in the cut layer was estimated using themethod of Lorenzen (1966); 10 ml of 90% acetone wasadded to each sample that was then kept in the darkand in constant agitation, at 4°C, for approximately18 h. Subsequently, samples were centrifuged for5 min at 2000 rpm (402 × g). Chl a and phaeopigmentconcentrations were measured in the supernatantbefore and after acidification, respectively, with aKONTRON fluorometer (Kontron Instruments).
In situ fluorescence measurements: In the secondset of experiments, the diving PAM fluorometer waspositioned at the sediment surface by SCUBA diversand programmed to record automatically. In situ fluo-rescence parameters (F , Fm’) were measured over adaylight photoperiod in November 2003 and two 48 hperiods in July and September 2004, respectively.ΔF /Fm’ (Eq. 1) was calculated from the collected data.Environmental variables at the sediment surface (PAR,temperature, water depth, salinity) were recordedsimultaneously. Throughout each manipulation, thePAM measuring probe was positioned and maintainedby a frame at 10 ± 1 mm from the sediment surface.Measurements were programmed at 15 min intervals,to prevent undesirable stress effects of repeated satu-ration pulses on the MPB such as modification ofmigration, or lack of relaxation of the primary electronacceptors after excitation. At the end of the observa-
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tion period the PAM fluorometer was brought to thesurface and the data were downloaded. Results arepresented as average hourly values.
PAR was recorded at the sediment surface with aLicor quantameter (LI-192SA, Li-COR) connected to aLicor 1400 data logger. Water depth (m), salinity andtemperature (°C) were recorded at the study site usingthe YSI 6920 multi-parameter logging system.
On 28 July a core was sampled and incubated out-side the laboratory to record the visual changes in sur-face colour and thus surface cover of MPB. Seawater,pumped directly from the site, was circulated gentlyaround the core to avoid disruption of the sediment–water interface. The combination of neutral nickelscreens (as used in the first experiment) and the con-stantly flowing water ensured that the core was kept asclose to in situ conditions as possible. Over a 24 hperiod (29 July) the core surface was photographed at2 h intervals.
Laboratory experiments on cores exposed to con-stant light and temperature: The third series of experi-ments was undertaken in the laboratory and consistedof measuring continuously the surface fluorescence(F and Fm’) of cores maintained in controlled and con-stant conditions of light and temperature, in order todetermine the presence of an endogenous rhythmwithin the MPB. On 8 August and 14 November 2003,a core was extracted from the Saint-Anne site andtaken immediately to the laboratory and placed underconstant light (17 μmol m–2 s–1) and temperature(13°C). After adjusting the level of overlying seawaterto 10 cm, the PAM was set to record fluorescence val-ues every 15 min during a 48 h period. On 14 Novem-ber, after a dark adaptation period of 15 min, F0 wasmeasured every 2 h from 11:00 to 19:00 h.
On 13 November, 1 additional core was taken for aqualitative determination of MPB species at the sedi-ment surface. The cells, resuspended in filtered sea-water, were examined using an inverted microscopy(Leica DM IRB).
RESULTS
Relationship between chl a concentrations and F
The variations in chl a concentrations (mg chl a m–2) inthe topmost 200 μm, as a function of F measurements, inMay 2004 and November 2003 are shown in Fig. 1. ThePAM settings and distance of the probe from the sedi-ment surface were identical, enabling comparisonsbetween the 2 seasons. Chl a concentrations over the 2seasons ranged between 0.3 and 16.9 mg m–2, but con-centrations in November (0.3 to 1.4 mg chl a m–2) werebelow those in May (4.9 to 16.9 mg chl a m–2).
As F and chl a both showed lower values in Novem-ber than in May, and the 2 data sets did not overlap,they were analysed separately. The relationships be-tween chl a concentrations and F values were investi-gated using linear regression models (Statgraph statis-tical computer program). For both sets of experiments Fwas significantly correlated with the chl a concentra-tion (p < 0.01). The resultant model equations were:
chl a (mg m–2) = 0.04(± 0.01) × F – 0.89(± 1.39)(r2 = 0.89) for May
(2)
chl a (mg m–2) = 0.01(± 0.01) × F – 0.01(± 0.10)(r2 = 0.67) for November
(3)
Unilateral Fisher F-tests showed a significant differ-ence between the slopes for the 2 seasons and theirrespective intercepts at the origin (Proba{F<F0.01/H0}< 0.01): the slope of the May data was steeper than thatof the November data, and the intercept at the originwas higher.
Taxonomic identification of cells at the sediment sur-face revealed that the genus Gyrosigma representedthe largest number of cells. G. attenuatum and G. fas-ciola were identified along with other benthic diatomssuch as Pleurosigma sp., Melosira sp., Navicula spp.,Berkeleya spp., Amphora sp. and Cocconeis spp.
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Ni Longphuirt et al.: Subtidal microphytobenthic migration
Variations of in situ fluorescence parameters
Variations in F, Fm’ and ΔF/Fm’ (Eq. 1) as a function oflight and tidally controlled water depth, were followedin summer (July and September) and in autumn(November) (Fig. 2). Between the 2 seasons, the aver-age daily PAR decreased by a factor of 5 (from 57 to12 μmol quanta m–2 s–1). Average water temperaturedecreased from 18.08 ± 0.35 to 13.08 ± 0.12°C, andsalinity fluctuated slightly between 35.33 ± 0.28 and34.24 ± 0.04‰ (data not shown).
Measurements on 28 and 29 July (Fig. 2A,B) werecarried out continuously throughout 2 full diurnalcycles. The tidal cycle on 28 July resulted in anincrease in the depth of water over the study site from5.5 to 8.9 m from the beginning of the observations tohigh tide at 14:47 h. A similar tidal cycle was observedfor the second day with high tide at 15:53 h (waterdepth 9.3 m). F increased immediately after sunrise(around 07:00 h) and then followed a bell-shapedcurve. On 28 July the maximum F value (731 arbitraryunits, a.u.) was reached at 16:49 h, approximately 2 hafter local noon (mid-point between dawn and dusk),when the tide was receding. During the second dailycycle, although the daily peak is difficult to see on thepresented graph (Fig. 2A), the data showed that fluo-rescence peaked about the same time as on the pre-vious day, with similar amplitude, despite a 1 h shift inthe time of high tide.
A stable minimum value was reached on the first dayafter midnight (00:37 h) with F values remaining closeto 200 a.u. for the rest of the night. PAR at the sedimentsurface had reached its minimum value (<10 μmolquanta m–2 s–1) approximately 2 h earlier at 22:00 h. Onthe second day, recordings of fluorescence and PARwere stopped at 21:25 h (F = 292 a.u.). At this time, Fvalues were similar to those for the same period on theprevious day.
The expected cyclical change in PAR for both daysshowed several irregularities. Fluorescence was unaf-fected by the anomalies in the curve with the exceptionof the second day when a large increase in PAR from58 to 224 μmol quanta m–2 s–1 between 09:00 and11:00 h, coincided with a noticeable increase in F from433 to 947 a.u.
Fig. 2B illustrates the variation in Fm’ and ΔF/Fm’throughout the 2 d period in July. Both parametersshowed persistent oscillations synchronised with theday–night cycle. Fm’ demonstrated a rapid and large in-crease concomitant with a rise in F. Fm’ reached values of2105 and 2549, respectively on Days 1 and 2, corre-sponding to an increase in Fm’ of 350 to 486% from theinitial value and from the previous night’s measure-ments. Fm’ then declined at the same time as the PAR andremained constant (around 500 a.u.) during the night.The PAR anomaly on the second day also influenced Fm’.
The effective photochemical efficiency of PSII(ΔF/Fm’) showed a 30% variation throughout the day:
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from 0.47 to 0.68. As for Fm’, a sharp increase wasobserved at the night–day transition. During the day,the efficiency remained relatively constant, with onlyslight variability, until a steady decrease began at20:00 h. Night time observations revealed continuoussmall amplitude fluctuations in the efficiency. Changesin F and Fm’ arising from the peak in measured PAR onthe morning of the second day resulted in a very slightdip in efficiency.
In September, fluorescence parameters and PARwere recorded over 2 full days (Fig. 2C,D). Tidal condi-tions were the reverse of those in the previous experi-ment, with low tide occurring at 13:00 h (4.74 m) and13:54 h (4.87 m) on 29 and 30 September, respectively.Variations in F were in a narrower range (from 105 to300 a.u.) than in July (max. 730 a.u.), whereas PAR wasonly slightly lower and did not exceed 155 μmol quantam–2 s–1. Highest PAR values were around 13:00 h onboth days; local noon was at 15:30 h. However, despitethe reversal of tidal oscillations, the shape of the Fcurve was identical to that in July. F increased at sun-rise (08:50 h), reached a maximum approximately 2 hafter local noon (17:00 h), and decreased until itreached a stable base value (F = 102 a.u.) at dusk.
Fm’ and ΔF/Fm’ displayed large changes throughoutthe photoperiod (Fig. 2D). Fm’ increased from 155 a.u.during the hours of darkness to a maximum of 720 a.u.on 29 and 30 September. Similarly, ΔF/Fm’ increasedconsiderably, increasing from 0.36 to 0.61 a.u. andfrom 0.43 to 0.60 a.u. between night and day for Days 1and 2, respectively. It then remained fairly constantthroughout the light period until decreasing in corre-spondence to the drop in F and Fm’.
The November observation (Fig. 2E,F) demonstratedsimilar curves with, however, smaller amplitudes thanin July and September. Lower light intensities andshorter daylight exposure were experienced, and localnoon was close to 13:00 h. These seasonal factors wereclearly reflected in the fluorescence curves by lowervalues and a shorter curve period. The peak in F,although lower than in the summer and early autumnperiods, occurred approximately 1 h after local noon(13:44 h on 21 November; F = 457 a.u.) while tidal oscil-lations resulted in a high tide at 14:38 h. Nightfalloccurred at 18:00 h and final data logging was at19:00 h with a recorded fluorescence of 132 a.u.
Fm’ rose steadily from 08:00 h, until a maximal valueat 11:00 h of 903 a.u. (Fig. 2F). A steady state was main-tained until the descent began at 14:00 h and the finalrecording showed a value of 349 a.u. at 19:00 h. Theslopes of both the Fm’ and F curves governed the shapeof the ΔF/Fm’ curve, which rose with a relatively steepslope until 09:00 h and then continued to increasemore slowly until 12:00 h (ΔF/Fm’= 0.60), decliningafter this point to 0.42 at 19:00 h.
Visible changes in surface biofilm concentration
Cores taken from the site and photographed over thecourse of 1 d showed a visible change in surface colourover the photoperiod (Fig. 3). At 07:00 h the sedimentsurface was an homogenous pale brown. At 13:00 hdarker brown areas had appeared at the surface. Vari-ability was evidenced by the lack of colour homogene-ity at the sediment surface. As the day advanced achange in the surface colour patchiness was seen(15:00 h) with a slight but visible reduction in the dia-meter and intensity of the dark brown areas. At 19:00 hthe colour had changed as the sediment surfacebecame more visible, and there was a clear contrast tothe early afternoon situation.
Diel surface fluorescence changes under constantlight and temperature
Variations in surface F values observed in constantconditions (Fig. 4) showed that when cores wereplaced in constant light and temperature, F remainedsynchronised with the light/dark cycle. On 7 August2003 (Fig. 4A) fluorescence measurements began at15:00 h. The signal decreased to a minimum (F = 222a.u. at 00:30 h) during the rising tide. The followingday fluorescence started to increase at 05:30 h. Themaximum on 8 August (F = 778 a.u.) was observed 2 hafter local noon, similar to in situ observations for thesame season. High tide on 8 August was at 15:46 h. Onthe evening of 8 to 9 August, F values did not appear toattain a steady base line but rather an inflection wasnoted, with F dropping to a minimum of 462 a.u. at02:30 h and then beginning to rise again. A similaroverall trend in F and Fm’ was evident, with an increasein peak measurements from Days 1 to 2 resulting in a100% increase. ΔF/Fm’ varied from morning to eveningfrom 0.36 to 0.61, and from 0.43 to 0.60, on Days 1 and2, respectively. Calculated ΔF/Fm’ was similar for allphotoperiods and the efficiency peak appeared to beconsistent between days.
The replicate experiment on 14, 15 and 16 Novem-ber (Fig. 4B) showed a similar pattern, despite a con-verse in situ tidal situation: low tide coinciding thistime with local noon. Although laboratory light andtemperature conditions were identical to those inAugust, F values in November remained in a lowerrange. After reaching a maximal value of 282 a.u., flu-orescence began to decrease at 14:25 h, about 1 h30 min after local noon. A baseline of F was reached at19:24 h (F = 128 a.u.), similar to in situ measurements,and subsequently rose again at 05:30 h. The peak forthe second day similarly took place at 14:30 h with avalue of 321 a.u. On this day, minimum stable values
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were reached at 21:30 h. Maximum and minimum Fvalues increased in both seasons from the first to thesecond day of the experiment. F0, measured on the14 November, followed the F curve closely, with valuesremaining just below those of F. Light-adapted Fm’ andcalculated ΔF/Fm’ curves were comparable to thoserecorded on 29 and 30 September in situ, with a con-siderable elevation in both parameters during thedaytime.
DISCUSSION
F, a tool to estimate benthic chl a biomass in thesubtidal zone
The majority of previous studies of MPB biomassused a greater sampling depth than this study, and sodirect comparisons are somewhat difficult. However,the concentrations presented here are in the lower
range of MPB biomass estimates, using different tech-niques of collection and sampling depths, compiled inthe review by MacIntyre et al. (1996), who cited valuesranging from <1 to 560 mg chl a m–2. Comparisons ofthe results of previous studies using similar methods ofextraction and, more importantly, replicate sedimentdepths, show that the concentrations in this study are inthe range of values presented by Herlory et al. (2004)but are in the lower range of MPB biomass estimatesgiven by Honeywill et al. (2002) for the same sedimentsampling depth in 2 European intertidal sites.
Comparison with the biomass of phytoplankton inthe overlying waters shows that MPB is of great impor-tance to the ecosystem as it may represent as much asone-third of the maximum biomass observed in thewater column in the Bay of Brest during the springbloom (authors’ unpubl. data).
Our results demonstrate that under the experimentalconditions of this study the application of PAM fluo-rometry to subtidal sediments permits the detection of
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Fig. 3. Colour change in the surface of a sediment core throughout a day (29 July 2004)
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large fluctuations in F at the sediment surface, and thatsuch variations can be attributed to changes in chl aconcentrations in the upper layers of the sedimentcaused by vertical migration of cells. Regardless of itsslope and intercept, the significant linear correlation(r2 = 0.89, p < 0.01, in May) between F and chl a con-centrations allows estimation of the relative variation,or rates of variation, of chl a in this zone directly fromthe in situ measured value of F.
The correlation in November (Fig. 1B), although sta-tistically consistent, showed a larger part of the vari-ance unexplained by the linear regression model (r2 =0.67, p < 0.01). Significant seasonal differences wereobserved for the slopes of the 2 linear relationshipsbetween F and chl a concentration. These differencesmight be related to shifts in the average ecophysiolog-ical condition of the community, background fluores-cence, changes in taxonomic composition and/ortemperature (Kromkamp et al. 1998).
In cases of low chl a concentrations, such as those inFig. 1B, a deviation of the apparent fluorescence mea-surements from the actual concentration in the sedi-ments sampled can occur (Forster & Kromkamp 2004).The influence of cells positioned at deeper levelswithin the sediment can have an affect on the fluores-
cence measured, leading to inaccurate results andhence a low correlation with the chl a measured. How-ever, the results do suggest that F can be used to esti-mate the microalgal biomass, but care must be taken tovalidate measurements for each site and season.
Evidence of migration
The migratory pattern of benthic diatoms was firstevidenced by the evolution of the surface colour ofsediment cores (Fig. 3). Colouration of the sediment isa general indicator of the nature of the sediment sur-face, although it is dependent on subjective percep-tion. Algal cells accumulate at the surface layer of thesediment in sufficient density to impart a visible darkbrown colouration to the substratum. Areas where thesediment appears dark brown are generally dominatedby diatom assemblages (Paterson et al. 1998). Thiscolouration appeared to be maximal around local noonand disappeared in the afternoon (Fig. 3). Althoughthese visual observations of the evolution of the algalbiomass at the sediment surface are non-quantitative,they are consistent with in situ fluorescence measure-ments. During all our observations, F began to increaseshortly after sunrise, peaked 1 or 2 h after local noon,and then decreased down to a constant base value,reached a few hours after complete darkness (Fig. 2).The photographic evidence thus corroborates that thevariations observed in F are related to variations inchl a at the surface layer of the sediment.
However, a number of factors need to be consideredto interpret PAM fluorometry parameters; for exampleSerôdio et al. (2001) concluded that F and Fm’ areclearly inadequate for tracing variations of biomass inMPB samples because F can rise with light and poten-tially decrease with high light as various photochemi-cal and non-photochemical quenching mechanismscome into operation. Irradiance increases can result inthe closure of reaction centres, increasing fluorescencere-emitted by cells and thus augmenting F measure-ments. Conversely, non-photochemical quenching(NPQ) processes, often induced by high light, evokethe reversible de-epoxidation reaction which convertsthe xanthophyll cycle pigment diadinoxanthin (DD) tothe energy dissipating form diatoxanthin (DT), result-ing in the dissipation of excess energy in the form ofheat and hence a decrease in F and Fm’ (Kromkamp etal. 1998, Müller et al. 2001, Perkins et al. 2001, 2003).Serôdio et al. (2001) were however referring to thevariability in the relationship between fluorescenceintensity and chl a concentration as a function of largevariations in temperature (5 to 35°C) and light (200 to2200 μmol m–2 s1) that characterise the estuarine inter-tidal environment (Serôdio & Catarino 2000). In com-
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Fig. 4. F, Fm’ and ΔF/Fm’ changes measured in cores sampledfrom the Saint Anne site in (A) August and (B) November2003, and kept at constant temperature and light conditions.F0 was also measured on 14 November between 11:00 and19:00 h. Vertical shaded bars: high-tide periods; horizontal
black bars: night times (recorded in situ)
Ni Longphuirt et al.: Subtidal microphytobenthic migration
parison, variations in the subtidal environment of thepresent study are much less: temperature variationswere lower than 1°C and maximum light irradiancewas 250 μmol m2 s–1, and hence the MPB cells werepresumed not to be light-saturated. Thus, our resultsshow that there is a change in chl a concentrations atthe sediment surface, leading to variation in F that ismuch greater than any variation that could arise fromphysiological responses to light and temperature vari-ations. In order to obtain a more detailed conception ofthe physical movements of the community, Fm’ wasmeasured simultaneously with F and ΔF/Fm’ subse-quently calculated (Eq. 1).
Fm’ increased above Fm levels in all observationmonths (Fig. 2), a phenomenon previously describedfor diatom cultures (Jakob et al. 1999). Under low lightconditions, following a period of dark adaptation, theactivation of ATP synthase and carbon metabolism candissipate the proton gradient, leading to a gradualexpoxidation of DT to DD (Jakob et al. 1999). The over-all outcome is an increase in the light-capturing pig-ment (DD) and hence an increase in Fm’ above Fm. Theincreases in Fm’ in our study were however extremelyhigh (between 350 and 486% of nighttime values) andare thus considered too high to be exclusively a resultof epoxidation. A more probable reason for the rise inFm’ is an increase in the number of viable cells at thesediment surface arising from vertical migration.Decreasing light during the second part of the daywould rationally lead to an increase in Fm’ in stationarycells (Perkins et al. 2001) through the conversion of DTto DD and hence an increase in Fm’; however adecrease was noted in our observations.
In all 3 observation months, a plateau was recordedin the efficiency of the MPB during the photoperiod,showing that light effects on fluorescence measure-ments were extremely low. High electron transferrates, caused by exposure to high PAR levels will nor-mally decrease efficiency because of the closure ofReaction Centre II and the stimulation of NPQ. Theabsence of a decrease in efficiency during the daytimesuggests a lack of these 2 processes. The similarity ofdaytime values between days indicates that the physi-ological state of the MPB was constant.
During nighttime periods, an enforced natural darkadaptation of the cells results in measurements of themaximal (Fv/Fm) rather than effective photochemicalefficiency of PSII (Kromkamp & Forster 2003). The factthat this value is lower than effective PSII efficiencymeasured during the day again shows that fluores-cence measured during the day and that measuredduring the night does not emanate from the same com-munity of cells. Measurements during the night mightoriginate from cells that stay at the sediment surface,are less viable and incapable of migration. These
nighttime values may also reflect degrading pelagicphytoplankton which sediment down through thewater column and are re-mineralised at the sedimentsurface; however in the present study verification ofthe presence of pelagic cells was not undertaken.
These results show that the evolution of fluorescencemeasurements resulting from vertical migration ismore important than temperature and irradianceeffects on the physiology of the community. They indi-cate that, under subtidal environmental conditions,variations in F and Fm’ represent primarily the evolu-tion of chl a concentrations due to the migration of cellsto the sediment surface, although care must be takento re-determine the relationship for each site andseason.
What drives migration in the subtidal area?
Our fluorescence measurements demonstrate amigratory cycle of subtidal MPB, with cells movingtowards the sediment surface at the beginning of thephotoperiod. Biomass reached a maximum 1 to 2 hafter local noon, and thereafter decreased to a mini-mum value that remained fairly constant until sunrisethe following day. Although fluorescence oscillationsclosely followed those of light, for unknown reasons,peaks in F lagged behind the highest daytime PAR val-ues. However, fluorescence did appear to be closelycorrelated with photoperiod length. This is highlightedby the progression of day length with seasons: FromJuly to November the photoperiod shortened as sun-rise changed from 07:00 to 09:00 h and sunset from23:30 to 17:00 h, leading to a reduction in day lengthfrom 16 to 8 h. Similarly, local noon changed from14:30 to 14:00 to 13:30 h from July to September toNovember. This progression in the daylight period isreflected in the fluorescence results as a shortening (bya factor of 2) of the time the MPB spend at the sedi-ment–water interface and a shift in the time when themaximum biomass is reached (from approximately16:30 h in July to 14:00 h in November). Hence, thebeginning of the photoperiod acts as a trigger for theinitiation of migration, and the evolution of this periodover time results in a concurrent temporal advance-ment in the starting point of migration and similaralterations in peak times.
The fluorescence data and photographic evidenceshow that at the subtidal mud surface of the study site,a constant evolution of the biomass occurs within thetime frame of the photoperiod. Dynamically, verticalmigration in some intertidal areas can be a fast anddensity-dependent process (Guarini et al. 2000), andbiomass can be highly concentrated after a short timeperiod (Herlory et al. 2004) resulting in a cohesive
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biofilm. In other intertidal sites the curve of migrationcan however be bell-shaped, similar to the results ofthis study (Serodio et al. 1997, 2000).
The existence of an endogenous rhythm within thecells is evidenced by the persistence of a bell-likemigration (Fig. 4) in cores incubated in the laboratoryin the absence of light variations. In both experiments(Fig. 4), F maxima were within in situ ranges andcurves were synchronised with the photoperiod at thesampling site. Intertidal communities also show aninternal regulation of migration when exposed to con-stant light (Round & Palmer 1966).
During the second day of observations, F valueswere higher than for the previous photoperiod, sug-gesting an increase or accumulation in the chl a con-centration. MPB are subject to a number of phenomenain situ that result in a reduction or control of their bio-mass, such as feeding of benthic fauna and processesof resuspension (Blanchard 1998). In the laboratorythere was an absence of these biomass-curtailing fac-tors and hence an increase in the stock of MPB biomassis possible. Alternatively, the persistent exposure ofthe cells to light may have resulted in the upwardmigration of an increasing number of cells from deeperwithin the sediment during the course of the study.
The primary source of this migration in intertidal areashas been considered to be the interaction of a permanentpositive geotaxis with a non-permanent rhythmicvariation in phototaxis that is modulated by the availablelight (Palmer & Round 1965, Round & Palmer 1966).Hence, intertidal MPB migration is governed largely bytidal stage (Pinckey & Zingmark 1991, Perkins et al.2003) and diurnal cycles, which collectively control theavailable PAR present. In our study fluorescence mea-surements show no concurrence with tidal oscillations,and notwithstanding a reversal of the phase of the cyclefrom July and November to September no migratorydeviation was recorded, confirming that in the sub-tidal area the diurnal rhythm alone prevails.
The vertical migration of MPB in most intertidalareas enables them to combat burial and erosionresulting from the often violent displacement of sedi-ment, and thus allows them access to light and subse-quent protection from resuspension during flood tides(Blanchard 1998). In the subtidal area, burying due tosedimentation of organic material from the pelagiczone may make the mobility of MPB essential, andwould explain the migration of MPB towards the sedi-ment surface with increasing daylight. The reason forthe initiation of their downward migration so soon aftermidday is not known, but perhaps this migration pat-tern allows the cells sufficient time on the sedimentsurface to replenish their intracellular organic carbonbefore migrating deeper into the sediment (Under-wood et al. 2005 and references therein) where they
have access to the high nutrient concentrations of theinterstitial waters (Saburova & Polikarpov 2003).
Implication of migration for primary production estimates
The integration of migration processes into calcula-tions of global primary production rates in intertidalareas can greatly modify estimates (Serôdio & Catarino2000), leading to significant alterations in daily andfortnightly timescales. In the subtidal, the verticalmigration of MPB will also influence the fraction oftotal biomass at the sediment surface contributing tobenthic production over the photoperiod. Applicationof the linear equation model to the in situ F data allowstheoretical quantification of the change in MPB bio-mass at the sediment surface. If we consider our data(Fig. 2), surface chl a concentrations could vary bymore than a factor of 5 (from 5 to 28 mg chl a m–2) overa diel period. These values illustrate the large-scaledifferences in surface biomass possible during themigration process.
Approximations using fixed or migrating biomassconcentrations will generate variability in model out-puts of productivity. To illustrate this variability weintegrated fixed and evolving chl a values into a theo-retical production model over 1 photoperiod. A simpli-fied model of a pre-existing exponential function(Webb et al. 1974), in which photo-inhibition was con-sidered negligible, was used to calculate production:
(4)
where PB is the gross production rate (mg C mg–1 chl ah–1), Pmax is the rate of maximum production (mg Cmg–1 chl a h–1), Ek is the minimum saturating irradi-ance (μmol quanta m–2 h–1), E is the in situ irradianceover a diurnal period (μmol quanta m–2 h–1); measure-ments from July (Fig. 2A) were used for this latter para-meter. As Pmax and Ek were unavailable for our studysite, we used mean theoretical values from a study onbenthic microalgae in a temperate area (Sundbäck &Jönsson 1988) (Pmax 1.07 mg C mg–1 chl a h–1 and Ek
172 μmol quanta m–2 h–1). Gross primary production(mg C m–2 h–1) was then calculated by multiplying theproduction by the chl a concentration. Observed F datafrom in situ experiments on 28 July (Fig. 2A) were usedto obtain a hypothetical daily range of chl a concentra-tions. We tested 4 hypothetical scenarios, the firstwhereby migration of the MPB is integrated into calcu-lations, and the other 3 whereby production is calcu-lated using a constant biomass and sampling is consid-ered to have taken place at midday (highest biomass,Scenario 2), mid-morning (mean biomass, Scenario 3)and night (lowest biomass, Scenario 4) periods.
P PBE Ekexpmax
/= −( )−( )1
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Ni Longphuirt et al.: Subtidal microphytobenthic migration
Differences in theoretic production curves for the 4scenarios are apparent in Fig. 5. Accumulated dailyproduction estimates vary considerably between sce-narios; values of 148, 199, 103 and 38 mg C m–2 d–1
were calculated for Scenarios 1, 2, 3 and 4, respec-tively. There is a 25% increase in daily productionrates if the peak chl a concentration is consideredrather than a concentration that evolves with migra-tion. Moreover, a 200% increase in approximated pro-duction is seen if measurements are taken at highest asopposed to the lowest surface biomass times. Evenwhen considering mid-morning chl a concentrations,production is underestimated by one-third. Overall,this theoretical exercise underlines the importance ofcorrect biomass sampling strategies in subtidal studiesand illustrates that a consideration of migration is nec-essary to avoid large and important errors in primaryproduction estimates.
CONCLUSION
Although subtidal MPB have been the subject ofmany studies, and the presence of migration has beensuggested (Underwood & Kromkamp 1999, Glud et al.2002), to our knowledge this is the first study to verifyand quantify the presence of subtidal MPB migration.We can conclude that a migratory pattern exists in sub-tidal MPB that is closely linked to the diel cycle andcan lead to large variations in their surface biomass.
The variability in environmental conditions of inter-tidal areas influences the evolution of the migrationpattern; in subtidal sediments these environmentalconditions and their respective variability are differ-ent, resulting in a migration that is influenced more bythe photoperiod alone than by a combination of lightand physical regimes. Further study into this phenom-
enon is required to define processes previously provedin intertidal areas such as the stratification of cells insuperficial sediments during the migration process(Herlory et al. 2004), the possible presence of a micro-migration of different species at the sediment surface(Underwood et al. 2005) and the influence of internalrhythms governing the migration process. However, asin intertidal studies, this constant migration may affectestimates of available or actively photosynthesisingbiomass and, as illustrated by the theoretical modelpresented, care should be taken in selecting appropri-ate sampling times of surface sediments for accuratequantification of the role of this compartment in littoralecosystems.
Acknowledgements. This work was funded by the Si-Webs(HPRN-CT-2002-00218) and ECCO European projects. Fromthe LEMAR, the authors also thank J. Richard, S. Martin, G.Thouzeau and F. Jean for their help in core collection and thelatter for his statistical aid, A. Le Mercier for his technicalexpertise during the photographing of cores and R. Marc andM. Briand for their help with the figures. The authors alsothank J. Cloern (USGS, USA) for his careful reading, his fruit-ful comments and suggestions. Thanks are also due to thoseat the LBEM laboratory at the University of La Rochelle, fortheir assistance in sample analyses. This is contribution 1019to the IUEM, European Institute for Marine Studies (Brest,France).
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Editorial responsibility: Otto Kinne (Editor-in-Chief), Oldendorf/Luhe, Germany
Submitted: November 4, 2005; Accepted: May 9, 2006Proofs received from author(s): December 7, 2006
