Depth distribution of photosynthetic pigments and diatoms in the sediments of a

microtidal fjord

Angela Wulff1,*, Sirje Vilbaste2 & Jaak Truu31Department of Marine Ecology, Marine Botany, Goteborg University, P.O. Box 461, SE 405 30 Goteborg, Sweden2Institute of Zoology and Botany, Estonian Agricultural University, Riia 181, 51014 Tartu, Estonia3Environmental Protection Institute, Estonian Agricultural University, Kreutzwaldi 5, 51014 Tartu, Estonia(*Author for correspondence: E-mail: angela.wulff@marbot.gu.se)

Received 8 December 2003; in revised form 8 July 2004; accepted 15 July 2004

Key words: benthic, diatoms, photosynthetic pigments, sediment, HPLC

Abstract

The depth distribution of photosynthetic pigments and benthic marine diatoms was investigated in latespring at three different sites on the Swedish west coast. At each site, sediment cores were taken at sixdepths (7–35 m) by scuba divers. It was hypothesized that (1) living benthic diatoms constitute a substantialpart of the benthic microflora even at depths where the light levels are <1% of the surface irradiance, and(2) the changing light environment along the depth gradient will be reflected in (a) the composition ofdiatom assemblages, and (b) different pigment ratios. Sediment microalgal communities were analysedusing epifluorescence microscopy (to study live cells), light microscopy and scanning electron microscopy(diatom preparations), and HPLC (photosynthetic pigments). Pigments were calculated as concentrations(mg m)2) and as ratios relative to chlorophyll a. Hypothesis (1) was accepted. At 20 m, the irradiance was0.2% of surface irradiance and at 7 m, 1%. Living (epifluorescent) benthic diatoms were found down to20 m at all sites. The cell counts corroborated the diatom pigment concentrations, decreasing with depthfrom 7 to 25 m, levelling out between 25 and 35 m. There were significant positive correlations betweenchlorophyll a and living (epifluorescent) benthic diatoms and between the diatom pigment fucoxanthin andchlorophyll a. Hypothesis (2) was only partly accepted because it could not be shown that light was themain environmental factor. A principal component analysis on diatom species showed that pelagic formscharacterized the deeper locations (25–35 m), and epipelic–epipsammic taxa the shallower sites (7–20 m).Redundancy analyses showed a significant relationship between diatom taxa and environmental factors –temperature, salinity, and light intensities explained 57% of diatom taxa variations.

Introduction

Benthic microalgae play an important role in thecarbon budget of coastal ecosystems, a fact thathas been known for several decades (e.g. Teal,1962; Cadee & Hegeman, 1974); and in estuaries,microphytobenthic communities can account for asubstantial part (50%) of the total primary pro-ductivity (Underwood & Kromkamp, 1999). Fur-thermore, subtidal benthic microalgae on the

continental shelves can account for 42% of thetotal areal primary productivity (Nelson et al.,1999). Although microbenthic communities havegained increased attention (reflected for example,in special sessions at international conferences),most studies have been conducted on tidal flats orin non-tidal shallow-water habitats (water depth<5 m). Very little, however, is known about thedepth distribution of these communities, especiallyin the marine habitat (reviewed in Cahoon, 1999;

Hydrobiologia (2005) 534: 117–130 � Springer 2005

Totti, 2003). Under favourable conditions, benthicmicroalgae often form cohesive microbial mats onthe surface of shallow-water sediments (Stal &Caumette, 1994; Sundback et al., 1996a), anddiatom-dominated mats are the most commontype of microbial mats in marine northern-tem-perate areas. The present study was inspired byobservations of a brown diatom mat at 17 m depthwhile scuba diving on the Swedish west coast. Themat consisted mainly of the diatoms Gyrosigmasp., Pleurosigma sp., Haslea sp., and Nitzschia cf.sigmoidea (200–300 lm).

Gullmar Fjord has been extensively studied formore than 100 years, through studies of bottomstratigraphy, chemical components, macroalgae,foraminifera, and macrofauna. Little attention hasso far been paid to the microphytobenthic part ofthe community. Because benthic diatoms areknown to withstand low light conditions (Steven-son & Stoermer, 1981; Sundback & Jonsson, 1988;Cahoon, 1999), and some species even tolerateanoxia (Admiraal et al., 1982), it was hypothesizedthat (1) living benthic diatoms constitute a sub-stantial part of the benthic flora even at depthswhere the light levels <1% of the surface irradi-ance, and (2) the changing light environment alongthe depth gradient will be reflected in (a) thecomposition of diatom assemblages, and (b) dif-ferent pigment ratios.

Materials and methods

Study site and collection of material

The study was carried out 8–13 May, 1997, inGullmar Fjord, on the Swedish west coast (58�15¢ N, 11� 27¢ E). The area has a maximum tidalamplitude of 20–30 cm. The weather was calm andsunny on all sampling days. Sediment cores weresampled by scuba diving without disturbing thesediment surface. Three sites were sampled ca.5 km apart. At each site, four sediment cores (i.d.90 mm) were randomly taken at six differentdepths: 7, 15, 20, 25, 30, and 35 m, giving a totalnumber of 72 cores. Because the aim was to studythe depth distribution of benthic diatoms at lowlight conditions, no samples were taken at waterdepths <7 m. Subsamples were taken of the upper5 mm of sediment using cut-off plastic syringes

(i.d. 8.7 mm). Temperature and salinity weremeasured at each sampling depth. At each sam-pling site, a light (PAR) gradient (1 m resolution)was measured under clear sky conditions at noonusing a quantum scalar irradiance meter. At eachsampling depth, 50 ml of seawater was taken foranalysis of inorganic nutrients (Table 1). At site A,only three depths were analysed. Inorganic nutri-ents (NO3 + NO2, PO4, and Si(OH)4) were anal-ysed on a TRAACS 800 autoanalyser (Braun &Lubbe).

Photosynthetic pigments

Two samples (i.d. 8.7 mm) were taken from eachcore (2 · 4 subsamples from each depth, giving atotal of 144 pigment samples), frozen in liquidnitrogen, transferred to a )80 �C freezer, andstored up to 2 months before analysis. Forextraction, 100% methanol was added to thesamples. Extraction proceeded at )18 �C for 48 h,and the samples were ultrasonicated and filtered(0.5 lm). The vials were kept on a cooled auto-sampler until analysis, which occurred within 12 h.Pigments were analysed by HPLC (Wright & Jef-frey, 1997) using a diode-array detector connectedin series to a fluorescence detector (both Spectra-physics). Absorbance detection was 436 nm. Thefluorescence detector was used to confirm theidentity of chlorophyll degradation products, assome of them co-elute and interfere with carote-noids. Chl a, chl c1c2, fucoxanthin, and diadino-xanthin were quantified as mg l)1 according toWright & Jeffrey (1997) and converted to mg m)2.

Epifluorescing microalgae

Two randomly chosen cores from each depth weresubsampled, and two subsamples (i.d. 8.7 mm)from both cores (i.e. 3 sites · 6 depths · 2cores · 2 subsamples) were diluted with glutaral-dehyde (final concentration 2.5%) to keep thechloroplasts intact until analysed. Algal cells weredetached from sediment particles through ultra-sonication. The sample was shaken for 30 s; afterca. 5 s (to allow sand grains to settle) severalindividual subsamples (40 ll) of suspensions werepipetted onto a microscope slide, and using anepifluorescence microscope (500· magnification),epifluorescing cells were counted.

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Diatom preparation

One sample (i.d. 8.7 mm) was taken from each core(four subsamples from each depth) and dilutedwith glutaraldehyde as above. Three of these foursubsamples were randomly chosen, washed fromthe glutaraldehyde and treated with hot sulphuricacid (Vilbaste et al., 2000). The diatom suspensionwas pipetted onto ethanol-cleaned cover slips thatwere left to dry in the air. Naphrax (refractive index1.7) was used as mounting medium. Diatom com-position and diversity were examined at fourdepths (7, 15, 20, 25 m) at each site. On each slide, aminimum of 500 valves were identified and countedalong a transect using interference microscopy(Zeiss Axiovert 135) using a 100· oil immersion

objective. The basic counting unit was a singlevalve, a complete frustule being counted as twounits. The absolute numbers of the counted taxawere converted to relative abundance (RA, %).Scanning electron microscopy (SEM) was used tocheck the identity of difficult small taxa.

Statistical analyses

Multivariate analysis was used to identify the maingradients in the diatom community compositionusing the program CANOCO 3.1 (ter Braak, 1994;ter Braak & Verdonschot, 1995) and ADE-4(Thioulouse et al., 1997). In order to study therelationship between diatom species and environ-mental factors, a redundancy analysis (RDA) was

Table 1. Different abiotic parameters at the different sites and depths

Site Light (%) Kd (m)1) Temp. (�C) Salinity NO3+NO2 (lM) PO4 (lM) Si(OH)4 (lM)

Site A

1 m 14.8 1.91

5 m 1.9 0.79

7 m 1.2 0.64 6 29 3.5 0.2 2.6

15 m 0.2 0.41 3 30

20 m 0.2 0.31 2 33

25 m 0.1 0.27 3 35 7.2 0.3 6.4

30 m 0.05 0.25 2 35 6.3 0.2 5.0

35 m 0 2 35

Site B

1 m 37 0.99

5 m 7.4 0.52

7 m 4.4 0.44 5 27 1.7 0.1 7.8

15 m 0.8 0.32 4.5 31 1.2 0.1 3.3

20 m 0.4 0.27 3 34 2.2 0.2 3.2

25 m 0.2 0.25 3 34 2.4 0.2 3.1

30 m 0.1 0.21 3 35 3.8 0.2 4.5

35 m 0 2.5 35 3.9 0.3 4.2

Site C

1 m 16.6 1.80

5 m 2.4 0.75

7 m 1.2 0.63 5 27 0.5 0.2 3.1

15 m 0.2 0.41 4.5 27 0.8 0.1 3.1

20 m 0.2 0.31 2 29 2.0 0.1 4.1

25 m 0.1 0.27 2 33 4.1 0.2 4.1

30 m 0.05 0.25 2 34 4.9 0.3 4.4

35 m 0 2 33 7.2 0.4 6.6

Light measurements are presented as percentages of surface PAR irradiance.

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carried out. RDA can be described as a multipleregression followed by a PCA on the fitted values.The arc sine square root transformed values of theRA of taxa (the mean of three replicate cores) wereused in the multivariate analysis. If a taxon wasnot present in at least four samples out of 36 with anumerical RA of at least 1% in a single sample, itwas excluded. As a result, 106 taxa were includedin the analyses. The following environmental fac-tors were used in the RDA as explanatory vari-ables: water temperature, salinity, and light(Table 1). The significance of correlations betweenRDA axes and environmental variables as well ascanonical coefficients were assessed on the bases ofapproximate t-test values (t>|2.1|). The effect ofsampling location as a grouping factor was esti-mated using the multivariate randomization test(Manly, 1997). The relationships between thediatom taxa and the pigment data table wereanalysed with co-inertia analysis (Doledec &Chessel, 1994); the method allows one to relatetwo data tables and extract information commonto both tables.

The pigment data and the epifluorescing cellsdata were analysed by two-factor ANOVA withdepth and place as the independent variables,n ¼ 4. Differences were accepted as significant atp < 0.05. Cochran’s test was used to checkhomogeneity of variances and data with hetero-geneous variances were transformed according toUnderwood (1997).

Results

Study area

Sampling sites A and C had similar bathymetry,whereas the slope was considerably steeper at siteB. At all sites, the sediment appeared more floc-culent at depths >20 m, probably due to accu-mulation of organic material. A clearly visiblebrown mat, indicating the presence of diatoms,was found down to 7 m depth, and a mat-likestructure was observed down to ca. 17 m. Molluscshells were present at all depths.

Salinity increased with depth down to 25 m andlevelled out between 25 and 35 m (salinity 34–35).The temperature decreased from 5 to 6 �C at 7 m,levelling out at 20 m (2–3 �C) (Table 1). During

the sampling period, the weather conditions werestable and sunny with a clear sky. The surfacePAR irradiance was around 1600 lmol pho-tons m)2 s)1 at all sites and on all samplingoccasions. The light penetration depth was similarat sites A and C, with ca. 15% of the incomingradiation left at 1 m depth, and the 1% level wasreached at 7 m (Table 1). At site B, light pene-trated deeper, and the 1% level was reached at14 m. The attenuation coefficients (Kd; m

)1) (Ta-ble 1) for sites A and C were 0.25 m)1, and for siteB, 0.21 m)1 (0–30 m). In general, the concentra-tions of all inorganic nutrients analysed increasedwith depth (Table 1). The purpose of the inorganicnutrient analysis was to find a tendency of differ-ences between sites; since no differences werefound it will not be further discussed.

Photosynthetic pigments

The pigments were analysed as concentrations(mg m)2) and ratios relative to chl a. At all siteschl a showed a pattern of a significant decreasewith increasing depth (Fig. 1). These patternsagreed well with those found for microalgalcounts, and the highest significant correlation be-tween chl a and epifluorescent cells was foundfor benthic diatoms (r ¼ 0.97–0.99, site A, B,p < 0.05) and for benthic plus pelagic diatoms(r ¼ 0.84, site C, p < 0.05). The chl a concentra-tions varied between 34 and 42 mg m)2 at 7 m,decreased to 17–20 mg m)2 at 20 m; and at 25–35 m depth, the amounts varied between 3.6 and

07 15 20 25 30 35

10

20

30

40

50

Depth, m

Chl

orop

hyll

a, m

g m

-2 Site ASite BSite C

Figure 1. Chlorophyll a concentrations in surface sediment

samples (0–5 mm) from different sites and water depths. Sym-

bols represent mean values of four replicate cores ±SE.

120

7.1 mg m)2 (Fig. 1). The concentrations of chlo-rophyll breakdown products showed a patchydistribution, and no significant differences betweendepths were found. For example, small peaks ofpheophytin a and ca. six different peaks of pheo-phorbides or pheophorbide-like pigments wereusually observed, especially at depths deeper than15 m. The concentrations of the diatom pigmentschl c1c2, fucoxanthin, and diadinoxanthin all de-creased significantly with increasing depth, but the

magnitude of declines varied between sites (Fig. 2).The overall best correlation between pigments wasfound between fucoxanthin and chl a (r ¼ 0.95–0.99, p < 0.05). The carotenoid betacaroteneshowed a patchy distribution, and no significantdifferences between depths were found. It shouldbe noted, however, that betacarotene was oftenmixed with breakdown products. Other pigmentsfound were lutein, zeaxanthin, and chl b. Gener-ally, lutein and chl b correlated very well, but

Figure 2. Concentrations (mg m)2) and pigment ratios (relative to chlorophyll a) (weight:weight) of different pigments in surface

sediment samples (0–5 mm) from different sites and water depths. Symbols represent mean values of four replicate cores ±SE.

121

zeaxanthin differed, indicating a zeaxanthin sourceother than green algae. Both lutein and zeaxanthinwere patchily distributed and decreased withdepth.

For pigment ratios relative to chl a, the out-come differed from the distribution pattern of theconcentrations. Many pigment ratios varied be-tween depths with no apparent decrease (Fig. 2). Asignificant increase in chlorophyll degradationproducts with depth (ratios to chl a) was observedat all sites (not shown). When comparing ratios ofthe diatom pigments fucoxanthin and diadino-xanthin to chl a, only fucoxanthin showed a sta-tistically significant depth effect (Fig. 2).

Microalgae

The epifluoresence counting of cells was donemainly to corroborate the pigment analysis and togive an idea of what sediment taxa were present aslive cells (i.e. with intact epifluorescing chloro-plasts). Sites A and B closely resembled each other,with a maximum number of sediment diatoms at7 m, 52–93 · 106 cells m)2, decreasing to 27–31 · 106 cells m)2 at 20 m depth, levelling outfrom 25 m (Fig. 3). Site C differed, with similarcell numbers down to 20 m depth, decreasing to25 m and levelling out (Fig. 3). Note, that cellnumbers from deeper than 20 m are based on fewcounts (<50 cells), and these results should beconsidered with caution. Dominating taxa be-longed to the genera Amphora, Nitzschia, Gyro-sigma, Pleurosigma, Navicula, Cocconeis,Achnanthes, and Diploneis. In addition, planktoniccells were present in the sediment and unidentified

centric taxa. Single coccoid cyanobacteria werefound at all sites at depths shallower than 20 m.Unidentified cysts were rare but occasionally seenat all sites. The proportion of epifluorescingplanktonic species was on average 17% for thedepth range 7–20 m, and 53% for 25–35 m. At25 m at site C, however, the proportion ofplanktonic cells was 24%.

Dominance and diversity of diatom species

Altogether 198 diatom taxa were recorded fromthe sediment samples. Almost 45% of all countedcells were planktonic species. The dominating taxa(mean RA >3%) were Skeletonema costatum (pe-lagic and benthic; mean RA 34%), Achnanthes cf.delicatula (epipsammic; mean RA 4.0%), Opephoraolsenii (epipsammic and epiphytic; mean RA3.8%), Martyana atomus (epipsammic; meanRA 3.3%), and Aulacoseira spp. (pelagic; meanRA 3.1%). Skeletonema costatum is generallyconsidered a pelagic species but it thrives in thebenthic habitat for periods long enough not to beconsidered only a consequence of sedimentationfollowing a pelagic bloom. Therefore we havechosen to treat S. costatum as a pelagic species inthe statistical analyses but we discuss the possi-bility of it also being benthic. There were someother taxa that occurred in almost all samples andsporadically showed rather high RA values (>5%).Among them were representatives from differenthabitats: Achnanthes delicatula (epipsammic andepilithic), Fallacia cryptolyra (epipelic), Naviculagregaria (epipelic and epilithic), Navicula permin-uta (epilithic), and Chaetoceros spp. (pelagic).

The relative abundance of the nine commonestdiatom taxa along the depth gradient is shown inFigure 4. The RA of Achnanthes delicatula,Martyana atomus, Opephora olsenii, Navicula gre-garia, and N. perminuta was negatively related todepth; in contrast with the pelagic taxa Aulacoseiraspp., Chaetoceros spp., and Skeletonema costatum,whose presence increased with increasing depth.An epipelic diatom, Fallacia cryptolyra, showed aninteresting feature in this respect – the RA of it wasrelatively low at 7 m, achieved the maximum at15 m, and then started to decrease again (Fig. 4).

The Shannon–Weaver diversity index (H¢) washigh (mean 4.38), varying between 2.57 and 5.39,and correlated (r ¼ 0.84, p < 0.01) with species

020406080

100120

7 m 15 m 20 m 25 m 30 m 35 m Depth, m

Cel

l num

ber *

106 m

-2

Site ASite BSite C

Figure 3. Cell numbers of fluorescing benthic diatoms in sur-

face sediment samples (0–5 mm) from different sites and water

depths. Symbols represent mean values of two replicate cores

±SE.

122

richness, which varied from 51 to 90, and evenness(J¢) (r ¼ 0.98, p < 0.01), which ranged from 0.45to 0.85 (Fig. 5). The median diversity of the dia-tom community was lower in site A compared withB and C. The diatom community showed a sig-nificant lower diversity with increasing depth(r ¼ )0.61, p < 0.01).

Multivariate analyses

The PCA of pigment data was performed as acorrelation matrix, because values for fucoxanthinand chl a were dominating and together made upca. 90% of total pigment weight. These two pig-ments, together with diadinoxanthin and zeaxan-

Figure 4. Relative abundance (RA the mean values of 9 cores ±SE) of nine commonest taxa at four different depths shown, (a)

epipsammic, (b) epipelic or epilithic, (c) pelagic taxa. Achnanthes delicatula and A. cf. delicatula are treated as one taxon. Note: RA

scale is different in different graphs.

123

thin, explained most of the data variation in thePCA (Fig. 6). The first axis of the PCA reflects thedepth gradient, and the second axis can be related

to differences between sites. The multivariate ran-domization test indicated significant differencesbetween three sampling locations ( p < 0.01) aswell as between sampling depth ( p < 0.001). Thedifference between sampling locations was biggerin comparisons involving shallower depths.

The ordination of the RA of 106 diatom speciesby PCA arranged the sites along gradients ofdepth and sampling area (Fig. 7). The first axiswere related to the depth gradient and explains43% of the variation; the second axis reflects thedifferences between the three sampling sites. Thelater was not statistically significant. Again, as forthe PCA of pigments, the differences betweensampling areas were larger at shallower depths.The most important species for defining the firstPCA axis was S. costatum – it characterized thedeeper location. For shallow sites, the typical taxawere Achnanthes cf. delicatula, Martyana atomus,and Achnanthes sp. (all epipsammic taxa).

In the ordination based on the RDA (Fig. 8),the length and direction of the variable arrowsindicate their importance and their approximatecorrelation to the ordination axis, respectively.There was a significant relationship between dia-tom data and environmental variables, whereintemperature, salinity, and light explained 49.9% of

Figure 5. Differences in Shannon–Weaver diversity index (H¢)values shown as boxplots by (a) location and (b) depth.

1

234

5

67

8

910

11

-0.8

1.6-2.1 2.9

A7

A15

A20

A25

B7B15

B20B25 C7C15

C20

F1

F2

Figure 6. Position of sampling location according to PCA

analysis of pigment data. Each location is represented by a

centroid of four replicate measurements. Lines join samples

taken along the same transect (depth gradient).

-0.4

0.4-0.5 0.5

A7m

A15mA20m

A25m

B7m B15m B20m B25mC7m

C15m

C20m

C25m

F2(1

5.5%

)

F1 (42.6%)

Figure 7. Ordination of diatom samples based on PCA of the

relative abundance of 106 diatom taxa. Lines join samples ta-

ken along the same transect (depth gradient).

124

diatom taxa variations. The location of speciesscores relative the arrows indicate the environ-mental preferences of each species. The two most

important variables contributing significantly tothe RDA axes were salinity (r ¼ )0.85, t ¼ )3.1)and light (r ¼ 0.68, t ¼ 3.2). The first RDA axiswas statistically significant (p < 0.01). The eigen-values of the first three axes were 0.352, 0.104, and0.043, respectively.

Four distribution patterns of taxa can be dis-cerned from the RDA. (1) The species with scoressituated in the left-hand part near the salinity ar-row of the ordination plot (Fig. 8) were moreabundant in deeper water. Odontella aurita,Cocconeis costata, Thalassionema nitzschiodes, andRhabdonema arcuatum are all marine species. (2)The species with scores in the upper right quadrantof the diagram prefer lower salinity and arebrackish species (e.g. Navicula phyllepta, Nitzschiadissipata, Pinnularia quadratarea). (3) The diatomsrecorded near the arrow for light occurred mainlyin shallow water where the light conditions werebetter. These are typical epipsammic taxa, such asAchnanthes sp., Opephora olsenii, and Naviculagermanopolonica, and epipelic ones like Stauro-phora salina and Amphora staurophora. (4) Thetaxa situated on the upper left-hand corner of thegraph (negatively correlated with light) are mostlyplanktonic forms (Cyclostephanus dubius, Chae-toceros spp., Thalassiosira spp., Detonula sp.).

According to co-inertia analysis (explained as acorrelation between two PCA analyses), the rela-tionship between diatom taxa and pigment datawas significant ( p < 0.01) (not shown). Thedominant trend was related to depth. Epipelic andepipsammic diatom species Achnanthes sp., Am-phora abludens, A. luciae, A. staurophora, andStaurophora salina were positively correlated withe.g. fucoxanthin and chl a. Pelagic taxa such asChaetoceros spp., a small centric diatom, Detonulasp., and Thalassiosira angulata had a negativerelationship with these pigments. In fact, most ofthe later named taxa were dead or inactive (cysts)on the bottom.

Discussion

Our experimental design was well replicated andsuccessful on a spatial scale but can only be con-sidered a snapshot in time. However, the abioticvalues fall within the range found by others in thearea, and therefore we believe our results can be

Figure 8. Ordination biplot based on RDA of diatom species

data with respect to light measurements, water temperature

(Temp), and salinity. The displayed species are selected on the

basis that more than 30% of their variance is accounted for by

the diagram. Achnanthes cf. delicatula (ACH CFDE); Achnan-

thes lemmermanni (ACH LEMM) Achnanthes sp. (ACH SP);

Amphora beaufortiana (AMP BEAU); Amphora copulata (AMP

COPU); Amphora staurophora (AMP STAU); Aulacoseira spp.

(AUL SP); Berkeleya spp. (BER SP); Biremis lucens (BIR

LUCE); Chaetoceros spp. + cysts (CHA SPP); Centric sp.

(CEN SP); Cocconeis costata (COC COST); Cyclostephanus

dubius (CYCS DUB); Cylindrotheca closterium (CYL CLOS);

Detonula sp. (DET SP); Diatoma moniliformis (DIA MONI);

Diploneis smithii (DIP SMIT); Entomoneis sp.( ENT SP); Fal-

lacia cryptolyra (FAL CRYP); Fallacia forcipata (FAL FORC);

Fallacia litoricola (FAL LITO); Fallacia tenera (FAL TENE);

Gomphonema parvulum (GOM PARV); Gyrosigma peisonis

(GYR PEIS); Gyrosigma tenuissima (GYR TENU); Navicula

bipustulata (NAV BIPU); Navicula capitata (NAV CAPI);

Navicula clamans (NAV CLAM); Navicula directa (NAV

DIRE); Navicula cf. fauta (NAV FAUT); Navicula flanatica

(NAV FLAN); Navicula germanopolonica (NAV GERM);

Navicula gregaria (NAV GREG); Navicula phyllepta (NAV

PHYL); Navicula portnova (NAV PORT); Navicula ramosiss-

ima (NAV RAMO); Nitzschia dissipata (NIT DISS); Nitzschia

cf. distans (NIT DIST); Nitzschia hybrida (NIT HYBR);

Odontella aurita (ODO AURI); Opephora olsenii (OPE OLSE);

Pinnularia quadratarea (PIN QUAD); Pleurosigma aestuarii

(PLE AEST); Psammodictyon panduriforme (PSA PAND);

Rhabdonema arcuatum (RHA ARCU); Staurophora salina

(STA SALI); Tabularia fasciculata (TABU FAS); Tabularia

investiens (TABU INV); Thalassionema nitzschoides (THAL

NIT); Thalassiosira angulata (THA ANGU); Thalassiosira ec-

centrica (THA ECCE).

125

considered representative for the season in the areastudied. We like to mention some methodologicalconsiderations. Small epipsammic species werefound in the diatom analysis, but very few werefound when counting epifluorescent cells, and it islikely they were overlooked or did not detach fromthe sand grains. The chl a concentration per cellfurther support this conclusion, because chl a percell was up to 10 times too high, even for a shade-adapted community. In dead cells, chl a degradesover hours (Cahoon et al., 1994), and it is notlikely the chl a concentrations were overestimated.Because we found these small cells at all sites anddepths we do not expect them to have made animpact on the statistical analyses. Furthermore, itcan be argued that the sediment sampling depth(5 mm) was too deep, because the euphotic zone ina sandy sediment usually is <3 mm (MacIntyreet al., 1996). However, as pointed out by MacIn-tyre & Cullen (1995), photosynthetically compe-tent diatoms are present well below the sedimentlight penetration depth.

In contrast to the turbid water column in tidalareas during high tide, in non-tidal or microtidalareas the water column can be clear all day, andthere is a good potential for a photosyntheticallyactive microphytobenthic community. However,occasionally the water column also in non-tidalwaters can be turbid due to river discharge duringlong periods of rain. Although the weather con-ditions during this study, and 2 weeks preceding,were stable and without rainfall, the higherattenuation coefficients found in the upper waterlayer might be explained by river discharge. This isfurther indicated by the different salinities amongthe sites. In a study in the same area (Site C) byEngelsen (unpublished), the salinity was 17, 23, 27,and 33 at depths 1, 5, 10, and 15 m. At a nearbysite in the fjord, salinity has been reported to bebetween 15 and 28 in the upper meter during May,1995–1998.

Hypothesis 1

Intact epifluorescing cells were found at depthswell below the euphotic zone, and thereforeHypothesis 1 can be accepted.

Further support for accepting the hypothesiswas obtained from the pigment analysis. Ourassumption that fucoxanthin and chl a mostly

originated from living benthic diatoms was sup-ported by the co-inertia analyses, where fucoxan-thin and chl a were positively related to typicalepipelic–epipsammic diatoms, and the oppositewas found for pelagic taxa. The chl a concentra-tions at 7 m depth (34–42 mg m)2) were wellwithin the range previously found for shallow-water sandy sediments (<1 m) on the Swedishwest coast (Wulff et al., 1997; Odmark et al., 1998;Wulff et al., 1999), when using the same HPLCtechnique as in the present study. In this study, it islikely that chl a concentrations (as well as the otherphotosynthetic pigments) from 25 m depths anddeeper represent background values – pigments indifferent stages of degradation, originating fromsedimented planktonic species or possibly frommicroalgal resting stages (cf. Cahoon et al., 1994).The low numbers of epifluorescent cells as well asthe relative dominance of cysts and planktonicspecies support this assumption. One can arguethat such a background value also exists at shal-lower depths, but if the chl a concentrations from25–35 m depth are subtracted from pigment con-centrations at 7–20 m depth the chl a concentra-tions are still within the range 8–37 mg m)2;furthermore, the cell counts confirmed a highproportion of epifluorescing benthic diatoms.

Although pelagic forms constituted ca. 10–45%of total counts (diatom species), the dominatingtaxon was Skeletonema costatum, a pelagic speciesthat thrives in the benthic habitat for periods longenough not to be considered only a consequence ofsedimentation following a pelagic bloom (Sun-dback & Jonsson, 1988; Yap, 1991; Sundbacket al., 1996b; Sundback & Miles, 2002). Forexample, Sundback & Jonsson (1988) observed, ‘abloom of the spring bloom diatom S. costatum wasinduced on the sediment surface’. Furthermore,49% of the total cell numbers at 14–16 m depthwere S. costatum. At shallower depths (2–5 m),however, only 5% was attributed to this speciesor other centric diatoms. These findings agreewell with our results showing that epifluorescingcells of S. costatum were observed down to 20 mdepth. In another study, a 20% contribution tototal microalgal biomass was attributed toS. costatum at 4 m depth in April, and it wassuggested that this species contributes to thebenthic primary productivity (Sundback et al.,1996b).

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Despite the absence of data on planktonicspecies from the Gullmar Fjord, data from adja-cent areas and fjords show that in May 1997, theplanktonic flora was dominated by dinoflagellates;and diatoms were abundant at only one site ofeight. In early April, however, S. costatum was themost abundant species, but planktonic algal bio-mass was low at this time. In 1997, an unusualplanktonic bloom occurred during winter alongthe Swedish west coast, and again S. costatum wasthe dominating species. Based on these data, weconclude that the S. costatum that was found inthe upper 5 mm of sediment originated from aplanktonic bloom 1–3 months prior to this study,a time period long enough to assume that theepifluorescing cells of S. costatum must have con-tinued to photosynthesize there.

The observation that diatoms can grow (sur-vive) under very low light intensities (singlelmol photons m)2 s)1) is an ability already de-scribed by others, both from laboratory work(Peters, 1996; Jochem, 1999) and in situ (reviewedin Cahoon, 1999). A preference for mid-depthswas found along a depth gradient (6–27 m) inLake Michigan (Stevenson & Stoermer, 1981),where diatom abundance was found to be greaterat 9 and 15 m, compared with shallower depths.The diatom preference for mid-depths was prob-ably due to less physical disturbance such as waveaction (Stevenson & Stoermer, 1981), which in thepresent study might be applicable to site C, since asmall marina is located nearby. A similar trendwas found on the Swedish west coast, with higherbiomass (measured as chl a) found in the depthinterval 14–16 m, compared with shallower depths(Sundback & Jonsson, 1988). In our study, anegative correlation (r ¼ )0.61) was observed be-tween the diversity and depth (7–25 m). So far, wehave concluded that benthic diatoms have thecapability to survive and contribute to the benthicflora. But are they active primary producers? Onthe basis of two recent studies at Site C, the answeris yes. In the first study, Engelsen (unpublished)measured in situ primary productivity (14C-incu-bations) and concluded that, at 10 and 15 mdepth, the primary productivity was between 0.23and 0.09 mg C m)2 h)1 (May, 1.4–0.3 lmol pho-tons m)2 s)1) and from 1.32 to 0.7 mg C m)2 h)1

(September, 40–18 lmol photons m)2 s)1). Thecores were incubated in situ for 2.5–3.5 h. In the

second study, Sundback et al. (2004) found mi-crobenthic oxygen production down to 15 m depth(maximum depth studied).

Hypothesis 2

Hypothesis 2a (the changing light environmentalong the depth gradient will be reflected in thecomposition of diatom assemblages) is accepted. Achanged diatom assemblage with depth was found,however, it could not be related to light only. Of thenine most common diatom taxa, the epipelic taxawere negatively correlated with depth, and theopposite was found for the pelagic taxa. Thus, wehave a diatom assemblage dominated by epipelictaxa at depths 7–20 m, and deeper down the pela-gic influence increases (also supported by the pro-portion of epifluorescent planktonic cells). Weassume that the pelagic taxa, apart from S. costa-tum, make only a minor contribution to the benthicprimary productivity, because they epifluoresce fora short period when sedimented down from thewater column. In addition, Barranguet et al. (1996)concluded that, in deeper bottom waters, phyto-plankton production could be absent due to lightlimitation, but microphytobenthos could still beproductive. We cannot conclude that light was themost important environmental factor, since salinitywas found to be equally important in the RDA.Furthermore, among the 15 most abundant epip-elic species in a muddy estuary on the east coast ofthe UK, Underwood et al. (1998) identifiedNavicula phyllepta, N. gregaria, and Nitzschia dis-sipata, three species observed also in this study. Thetwo Navicula species were found to be positivelycorrelated to nutrients and negatively correlated tosalinity (Underwood et al., 1998). In our study, nosediment nutrients were measured, but a tendencyof decreasing abundance of these species with in-creased salinity was found. According to the RDA,some species seemed more affected by light thansalinity, and those were typically epipsammic–epipelic species. Pelagic species were negativelycorrelated to light, and several were found dead orinactive (cysts) on the bottom. The primary pro-ductivity data from Engelsen (unpublished) sup-ports our original theory that light was the mainenvironmental variable; more specifically, theyfound that primary productivity was positivelycorrelated to light intensity.

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Hypothesis 2b (the changing light environmentalong the depth gradient will be reflected in dif-ferent pigment ratios) holds for the fucoxan-thin:chl a ratio. The fucoxanthin:chl a ratioincreased, with a simultaneous decrease in fuco-xanthin concentrations to 20 m depth, indicating aphotoacclimation for benthic diatoms. A ratio of0.7 was found in a shade-adapted diatom mat(Sundback et al., 1996a), and this value was alsotypical of those reported from a diatom-domi-nated tidal flat (in spring) in SW Netherlands(Barranguet et al., 1997); in our study, we foundsimilar values also. Engelsen (unpublished) foundthat the microphytobenthos from 1 to 15 m depthwas shade adapted, because the microalgae re-quired a lower light intensity to reach a light-sat-urated photosynthetic rate. Thus, one can arguethat the community was shade-adapted already at7 m, and a change in the ratio should have re-flected not a photoacclimation but a larger pro-portion of diatoms compared with other algalgroups. The use of pigment ratios in naturalcommunities is complicated by the fact thatchanges in pigment ratios can reflect not only theacclimation to environmental conditions but canalso reflect a change in community composition(cf. Goericke & Montoya, 1998).

Theoretically, other fucoxanthin-containingmicroalgae such as Dinophyceae, Chrysophyceae,and Haptophyceae could have contributed to theamount of fucoxanthin present in the sediment.However, of these groups, only members of Din-ophyceae were likely to be present, and further-more, the benthic dinoflagellate taxa usually foundin the experimental areas contain not fucoxanthinbut peridinin as the major carotenoid (Wulffunpublished). Therefore, the fucoxanthin:chl aratio should have reflected a physiological adap-tation of the benthic diatoms. In addition, fuco-xanthin is among the most labile carotenoids, witha half-life of a few days if oxygen and light arepresent (Leavitt & Carpenter, 1990; Leavitt, 1993),and is therefore not usually preserved in the sedi-ment. This implies that in the present study, ‘old’fucoxanthin should not have interfered with pig-ments from living cells.

The occurrence of breakdown products orunidentified pigments complicated quantificationof carotenoids, and we could not exclude the pos-sibility that, in some cases, an overestimation oc-

curred. A terrestrial origin of typical green algalpigments (chl b, lutein, and zeaxanthin) is notlikely, because these pigments usually degrade be-fore deposition in the sediment (Leavitt, 1993).Furthermore, the sampling took place in late springwhere a terrestrial input was unlikely. The occur-rence of several different peaks of pheophorbidesor pheophorbide-like pigments is typical for mar-ine sediments and reported from several studieswhere they suggested to originate from grazingactivities (Cariou-Le Gall & Blanchard, 1995;Brotas & Plante-Cuny, 1998). Similar chl-like pig-ments have been observed also in lakes (Leavitt &Carpenter, 1990). In our study, the scuba diversobserved several small burrows; and small macro-fauna, e.g. snails and polychaetes, were occasion-ally observed in the sediment cores. The occurrenceof several chloropigments stresses the fact that, forpigment measurements in sediments, it is advan-tageous to use a fluorescence detector in connectionwith the absorbance detector in order to betteridentify chloropigments and avoid interferencefrom the non-fluorescent carotenoids.

Although differences between sites were found,they were not statistically significant in any vari-able other than pigments, where the multivariaterandomization test showed a statistically signifi-cant difference between the three sampling loca-tions. In the ANOVA, however, no statisticallysignificant differences were found. We thereforeconclude that our sampling design was successfulin allowing us to draw general conclusions aboutthe inner part of the fjord.

In conclusion, we have shown that benthicdiatoms constitute a substantial part of the mi-crobenthic flora and are active (epifluorescing)under very low light conditions (single lmol pho-tons m)2 s)1) in situ. Our findings stress the factthat, when using pigment analysis in sediments, itis not reliable to assume that benthic microalgalproduction can be excluded below the euphoticzone (1% of surface irradiance).

Acknowledgements

We thank K. Wulff for diving assistance and thelandowner W. Nilsson for letting us use his prop-erty. This work was financially supported by thefunds: ‘Birgit och Birger Wahlstrom’, the Swedish

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Institute, ‘Kapten Carl Stenholm’, ‘Uddenberg-Nordingska’, ‘Per Adolf Larsson’, ‘Kungliga ochHvitfeldtska’, and the SYS-RESOURCE Programat The Natural History Museum, London. Theyare all greatly acknowledged for their support.

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