original scientific paper UD C 593.1:591.13(262.3-16) NUTRIENT-ENRICHMEN T EFFECT O N PLANKTO N COMPOSITIO N Patri ci ja MOZETIČ, Valentina TURK, A/en/ca MALE] Marine Biofogicai Station Piran, Si-6330 Piran, Fornače 41 ABSTRACT Three enrichment experiments were conducted in order to analyze the development of plankton biomass and species composition as a response to different nutrient regimes. The additions of all nutrients had the most profound effect on phytoplankton biomass increase, whereas picoplankton abundance decreased continuously in all treat­ments. Initially dominating phytoplanktonic groups and species expanded significantly towards the end of expe­riments. Inputs of new nutrients not only stimulate phytoplankton growth in general but also affect the community structure. In such conditions the development of community structure seems to be controlled by the fast-growing opportunistic seasonally dominating species (mainly Ske 1 etonerna costatum and Chaetoceros spp.}. Key words: phytoplankton, bacteria, species composition, nutrients, enrichment experiment, Gulf of Trieste INTRODUCTION The Gulf of Trieste is a typical temperate coastal area where the dynamic of phytoplankton abundance and species/group composition usually follow seasonal and iriterannual fluctuations in relation to freshwater dis­charges and thereby nutrient inputs (Malej et ai, 1995). These seasonal fluctuations are characterized by a se­quence of events from a spring diatom bloom due to high nutrient concentrations supplied by rivers towards late-spring and summer microflageilate-dominated commu­nity, supported by regenerated nutrients typical for many coastal seas and estuaries (Garcia-Soto et a I., 1990; Ra­gueneau et a!., 1994). In nutrient-enriched environments diatoms seem to be more successful in taking up nutrients due to their "luxury consumption" (Sakshaug & Olsen, 1986) and higher growth rates (Furnas, 1990) compared to smaller cells like microflagei lates and picoplanktonic cyanobacteria. On the other hand, microti age! lates and especially cyanobacteria dominate in nutrient-depleted environments (iriarte, 1993; Fuks, 1995), where low nu­trient concentrations prevent development of diatoms. However, some studies have recently indicated that this sequence might be altered by a silicate limitation of dia­tom blooms in coastal waters (Smayda, 1990; Conley & Malone, 1992). Namely, in many industrialized coastal areas anthropogenic enrichment by nitrogen and phos­phorus compounds induced a decline in Si/N and Si/P ra­tios, causing modifications of phytoplankton succession (controlled not only by.nutrients' amount but also by their ratios) (Fisher et ai, 1988; Egge & Aksnes, 1992). As a consequence, diatom-dominated spring blooms are re­placed with flagellate blooms (microflagellates, din-of I a gel lates), among which many harmful and toxic spe­cies are found (Smayda, 1990). The aim of this work was to analyze the development of phytoplankton biomass and species composition in nutrient-enrichment experiments. These controlled ex­periments were integrated in a three-year EU/Environ­ment project PALOMA that had a principal goal to esti­mate the effects of different nutrient concentrations and ratios on coastal plankton dynamics and the production of organic matter (Cauwet, 1996; Malej etal., 1997a). In the present contribution we focused on development of dominant phytoplankton species and groups in different nutrient regimes and tried to evaluate their dynamics relative to control conditions without nutrient additions. Pa trie !!a MOZETlC et aL: NUTRIENT-ENRICHMENT EFFECT O N PLANKTON COMPOSITION, 3!-42 MATERIALS AN D METHODS Materials Three enrichment experiments were carried out: 27 June - 2 July 1994 (PALEX 1), 3-12 April 1995 (PALF.X 2), and 22 March - 3 April 1996 {PALEX 3). Six to seven treatment regimes were set up using the natural plank­ton, collected in the south-eastern part of the Gulf of Trieste at the subsurface as experimental assemblage. In the first two experiments we applied the following treat­ments: control without any addition (A), single additions of phosphate (B: 0.6 pM I"5), nitrogen as nitrate + am­monium (C: 5.1 + 1.8 pM I-'), silicate (D: 10,6 pM I"5), a mixture of all nutrients (C ; 0. 6 pM P0 4 3 " F , 5.1 JJM N0 3 - I-' , 1. 8 JJ M NH 4 + I"5 , 10. 6 p M S i H) , an d additions of rain water (E) and river water (F) as natural nutrient sources (15% v/v dilutions). In PALE X 3 only control (Al , A16), and treatments with the addition of phosphate (B1, B16) and all nutrients (Gl , G16) were set up, using plankton assemblage from the subsurface (1 m) and from the depth of fluorescence maximum (16 m). Experimental plankton assemblages were enclosed in Nalgene containers (8 and 20 I) and incubated in situ at 2 m depth. All sampling procedure and set-up of the enrichment experiments are described in detail in Mozetic etal. (1997) and Male} eta!. (1997a). Samples were withdrawn daily or every second day and inorganic nutrients, particulate and dissolved or­ganic nitrogen and carbon, particulate and dissolved carbohydrates, phytoplankton structure and pigment biomarkers, bacterioplankton abundance, and primary and bacterial production were measured. For the pur­pose of this work we present the initial nutrient status, and the development of phytoplankton biomass, cell abundance (phyto- and bacterioplankton) and taxo­nornic composition, while other data are presented in Cauwet etal. (1998, in press). Methods Nutrients were analyzed in filtered (NO2", NO3-, PO43-) and unfiltered (NH4+, Si) samples using standard procedures (Grasshoff, 1983). Phytoplankton biomass was determined fluorometri­cally as chlorophyll a (Chi a) concentration (Hoim-Hansen ef al., 1965). Subsamples of seawater (25 ml) were filtered onto 0.22 pm Millipore filters, extracted in 90% acetone and the fluorescence of extracts measured on a Turner fiuorometer 112. Cell counts. Samples for enumerating plankton were preserved with neutralized formalin (1.5-2% final con­centration). Phytoplankton abundance (micro- and nanoplankton) and taxonomic composition were de­termined on an inverted microscope using the technique of Utermohl (1958). Organisms identified on a species or genus level belonged to three algal classes (Bacil­lariophyceae - diatoms, Dinophyceae - dinoflageilates, Prymnesiophyceae - coccolithophores) and to one non­taxonornic group (microflagellates). Bacterioplankton (cyanobacteria and heterotrophic bacteria) and picoplanktonic eucaryotes (2-3 pm) were counted with an epifluorescence microscope (1250x magnification), Cyanobacteria and small eucaryotic cells were counted in green excitation light according to the protocol of Takahashi ef al. (1985) and heterotrophic bacteria in UV light using DAPI according to Porter & Feig (1980). To study the effect of different nutrient additions on species composition, we compared species abundances in different treatments relative to control conditions at the time of biomass maximum. Firstly, we chose the most abundant species and/or groups (> 1% of total abundance) in the initial phytoplankton assemblages as well as on the day of chlorophyll maximum. Afterwards, the relative increase or decrease of the most abundant species/groups was expressed as the ratio between the abundance in different treatments and control treatment (X/Control, where X stands for single treatment). During PALEX 3 epifluorescence micrographs of phytoplankton and bacterioplankton stained with pri­muline and DAPI, respectively, were taken with Olym­pus camera in UV light. Tab. 1: Nutrient conditions and basic biological pa­rameters of inoculated assemblages during enrichment experiments PALEX 1, 2 and 3, Tab. 1: Hranilne razmere h osnovni biološki parametri naravne združbe /ta začetku obogatitvenib poizkusov PALEX h 2 in 3. Parameter PALEX 1 PALEX 2 PALEX 3 1 m 16 m nitratefyjMS"1) 3.44 3.62 1.01 0.93 phosphate ipM I-1) 0.1 0.01 0.03 0.06 silicate ((JM f-') 8.11 1.15 1.85 0.13 N/P 60 > 100 40 33 N/Si < 1 4 < 1 15 Si/P 80 > 100 62 2 Chi a (pg F ) 0.68 33 9 0.94 1.23 phytoplank. {ceîls F') 0.4 x 106 7.3 x tO6 0.7 x 106 0.8 x 10& diafoms (% phyto.) 39 88 52 49 ^flagellates (% phyto.) 55 11 41 42 cyanobacteria (cells F1} 2.9 x 10? 1.3 x tO7 9.5 x 10* 7.9 x tO5 beîero. bacteria {ceîls F ) 9.3 x 10s 4.8 x 10 s 1.5 X 108 3.4 X T0s RESULTS Characteristics of inoculated assemblages Nutrient concentrations and their ratios measured in field samples from which the three initial assemblages Patricija MOZETIČ eta/.: NUTRIENT-ENRICHMENT EFFECT O N PLANKTO N COMPOSITION, 31-42 were taken (Table 1) indicated P-limitation of phyto­pfankton growth (N/P >30). Initiaf assemblages were also Si-limited in PALEX 2, and especially in PALEX 3 phytoplankton collected from 16 m. initial phytoplank­ton biomass was the highest in PALEX 2 and the lowest in PALEX 1, so was phytoplankton abundance. The dominant phytoplankton groups were microflagellates in PALEX 1 and diatoms in PALEX 2, while proportions of these two groups were rather similar in PALEX 3. Bacte­rial component (autotrophic cyanobacteria and heterot­rophic bacteria) was higher in june/July experiment compared to spring experiments (PALEX 2 and 3). Piankton standing stock The duration of experiments was different for the PALEX 1, 2, 3 experiments: we finished the experiment when phytoplankton reached the stationary phase in PALEX 2 and 3. The exception was PALEX 1, where the experiment stopped when the population was still growing, in all three experiments the largest increase of the phytoplankton biomass (Chi a) was observed in treatment with the addition of all nutrients (G treatment), although stimulating effect of other nutrients, especially phosphate, was also notable (Fig. 1). In PALEX 1 the maximal biomass in G treatment was measured at the end of the experiment (day 6: 4.31 pg Chl a H) , while during PALEX 2 and 3 peak values amounting to 20.38 and 22.47 pg Chl a H were measured after 5-6 days, re­spectively. Likewise the Chl a biomass, we observed a similar pattern of phytoplankton abundance during the three experiments (data not shown). The highest cell densities were counted in treatments with the addition of all nu­trients, reaching values from 1.8x107 and 3.6x107 cells H in PALEX 1 and 3, respectively, and up to 7.2x10» cells H in PALEX 2. The common feature of all three experiments was the absolute and relative growth of the dominant groups in the inoculated assemblages (see Table 7} in all treatments. At the end of the experiments the average relative proportion (determined from ail treatments) of microflagellates increased up to 98% in PALEX 1, while diatoms represented 93 and 72% of total phytoplankton in PALEX 2 and 3, respectively. Din­oflagellates and coccolithophores were less important, especially in PALEX 2, where their proportions ac­counted for less than 1% of total phytoplankton at the beginning as well at the end of the experiment. The relative contribution of these two groups was higher in PALEX 1 and 3, with dinoflageliates being more impor­tant in PALEX 1 (4.5% at the beginning) and cocco­lithophores in PALEX 3 (4.7-5.4% at the beginning). Compared to eucaryotic micro- and nanoplankton, cyanobacteria were not very successful (Fig. 2a), In PALEX 1 cyanobacteria were completely outcompeted by larger size-classes and picoplanktonic eucaryotes (da- Fig. 1: Chlorophyll a biomass in different nutrient treat­ments during PALEX experiments 1 (27 June - 2 July 1994), 2 (3-12 April 1995) and 3 (22 March - 3 April 1996). Si. 1: Klorofilna biomasa v različnih hranilnih razmerah v obogatitvenih poizkusih PALEX 1 (27.6. - 2.7.1994), 2 (3. - 12.4.1995) in 3 (22.3. -3.4.1996). ta not shown), while in PALEX 2 their abundance de­creased steadily in all treatments. The same was ob­served in PALEX 3 except for control conditions (AT.6). Bacterial abundance increased initially and had al­ready reached maximal values on days 2-4 in all three PALEX experiments, preceding the autotrophic compo­nent of the system (Fig. 2b, Fig. 4a). Species composition in relation to nutrient additions Species composition was quite similar in the two spring experiments (PALEX 2 and 3), In both cases we collected a population during the late phase of a diatom bloom with the highest species diversity among the dia­toms. The dominant diatom species were Pseudonitzsc­hia pseudodelicatissima, Skeletonema costatum and Chaetoceros sp. 2 in PALEX 2, and Chaetoceros spp. (mainly C. compressus, C. decipiens and other larger species), P. pseudodelicatissima, Bacteriastrum sp. and Palricijä MOZETIČ et ah NUTRIENT-ENRICHMENT EFFECT O N PLANKTON COMPOSITION , 3 T -42 3-OË+7 3.0E+7 ­ 8 . ,2 2.0E+7 ­IMV O« O cs « U 1-0E+7 0.0E+0 1 2 3 4 S é O.OE+O 2 4 i 8 50 2.0E+7 ­1.0E+7 ­O.OE+O 2 4 6 8 10 12 2.0E+9 ­ 2.0E+9 ­ 2.0E+? ­ -!3 « 1.0E+9 'C a U M t.OE+9 1.0E+9 ­ o.oe+o O.OE+O O.OE+O 4!0 4 6 8 10 12 6 t days days PALEX 1 PALEX3 PALEX 2 Fig. 2: Cyanobaclerial (a) and bacterial (b) abundance in different nutrient treatments during PALEX experiments 1, 2 and 3. Only data for assemblage collected af the depth of fluorescence maximum (16 m) are shown for PALEX 3. SI. 2: Gostota cijanobakterij (a) in bakterij (b) v različnih hranilnih razmerah v obogatitvenih poizkusih PALEX 1, 2 in 3. V PALEX 3 poizkusu so prikazani rezultati le za združbo, zajeto na globini fluorescentnega viška (16 m). Cylindrotheca closterium in PALEX 3 (Figs. 4b-f). Among dinoflagellates, Gymnodiniurn sp., Prorocentrum min­imum and Protoperidinium minusculum were the most abundant, while Emiliania huxleyi and a non-identified species were the dominant coccolithophores. in PALEX 1 species diversity of dinoflagellates was the same as of diatoms, although the abundance of the former group was lower. The dominant species were small Chae­toceros sp.1, Navicula sp., Cylindrotheca closterium and a non-identified species for diatoms, and Ceratium furca, Gymnodiniurn sp. and various species of the genus Prorocentrum for dinoflagellates. O n the day of chlorophyll maximum we compared the most abundant species/groups in different nutrient regimes relative to control conditions. In all three ex­periments maximal Chi a was reached on sixth day and diatom species prevailed in PALEX 2 and 3. Microflagel­lates which dominated in PALEX 1 were taken as a group, likewise dinoflagellates in PALEX 1 and 2 due to very low abundance of single dinoflagellate species (<1% of total abundance). O n two occasions cocco­lithophores and non-identified, naked, Gymnodinium­like dinoflagellates were also chosen. The highest increase of species abundance relative to control we observed in treatment G (Fig. 3) along with interesting differences between species and/or groups. The highest ceil densities were counted for mi­croflagelfates (up to 1.8x107 ceils H) , P. pseudodeiica­tissima (up to 7.6x1 Q8 cells H) , and Chaetoceros sp. 3 (up toi.3x10 7 cells H ) on day 6 (Table 2) in PALEX 1, 2 and 3, respectively. However, the most marked respon­ses to addition of all nutrients were observed for other species: diatoms Chaetoceros sp.1 and Navicula sp. in PALEX 1 (fig. 3a), and 5. costatum in other two experi­ments. The relative increase of S. costatum was higher in PALEX 3, when a high growth was observed also for Cylindrotheca closterium (Fig. 3b & 3c, Figs. 4a, c). In PALEX 1 a clear increase relative to control was observed also in other treatments, especially with the additions of phosphate and rain water. However, the stimulating effect of nutrient additions was much lower for other phytoplankton groups compared to diatom species (Fig. 3a). Similarly, a higher response was ob­served for coccolithophores compared to microflagel­lates and dinoflagellates. In this experiment the addition of inorganic nitrogen alone also stimulated the growth especially of coccolithophores with Emiliania huxleyi being the predominant species. A similar situation was found also in PALEX 3, where among non-diatom species/groups only the coccolitho­ ?31ricii3 MOZETIČ el a i.-. NUTRIENT-ENRICHMENT EFFECT O N PlftNKTON COMPOSITION, 31-42 420 95 O IH •4-» S Uo a) C h act I Navic dino cocc micro 40 37 E3 c 15­ m D tm E L r~i F -M 10 s © y ..maTTCIh A -neat d Jkiu j / I T W ...HiOrttar Chaet 2 Cylin Pseu Skel dino micro 132 140 120. RI I i BI6 100: / ZZ3Q16 40 I» 30 O p 20 10 c) .•JI , Bact Chaet 3 Cylin Pseu Skei dino n.id.E. hux micro Fig, 3: Ratios between the abundance of dominant pbytopiankton species/groups in different treatments (X) and the respective abundance in control conditions on day 6 during PALEX experiments 1 (a), 2 (b) and 3 (c). Only data for assemblage collected at the depth of fluorescence maximum (16 m) are shown for PALEX 3. Legend: Chaet (1, 2, 3) = Cbaetoceros (sp. 1, spp. 2, spp. 3); Navic = Navicuia sp.; Cylin = Cylindrotheca closterium; Pseu ~ Pseudonitzschia pseudodelicatissima; Skel ~ Skeletonema costatum; fiacf = Bacteriastrum sp.; E. hux s Emiliania huxleyi; dino (n.id.) = dinoflagellates (non identified); cocc = coccolithophores; micro = microflagellates. SI. 3: Razmerja med gostoto prevladujočih fitoplanktonskih vrst/skupin v posameznih hranilnih razmerah (X) in gostoto v kontroli 6. dne obogatitvenih poizkusov PALEX 1 (a), 2 (b) in 3 (c). V poizkusu PALEX 3 so prikazani rezultati le za združbo, zajeto na globini fluorescentnega viška (16 m). Legenda: Chaet (1, 2, 3) = Cbaetoceros (sp. 1, sp. 2, sp. 3); Navic » Navicuia sp.; Cylin = Cylindrotheca closterium; Pseu = Pseudonitzschia pseudodelicatissima; Skel = Skeletonema costatum; Bact = Bacteriastrum sp.; E. hux = Emiliania huxleyi; dino (n.id.) -dinoflagelati (nedoločeni); cocc = kokolitoforidi; micro = mikroflagelati. Palricija MOZETIČ et al.: NUTRIENT-ENRICHMENT EFFECT O N PLANKTO N COMPOSITION, 31-42 phore E. huxleyi responded significantly to nutrient additions compared to control (Fig. 3c). The addition of phosphate alone had stimulating effect only on 5. costa­tum and to lesser extent to other diatoms and E. huxleyi. The highest relative increase in PALEX 2 (37x) was much lower than those in PALEX 1 (420x) and PALEX 3 (132x). Only the addition of all nutrients provoked a clear response of specific species or groups compared to con­trol (Fig. 3b). However, a slight increase was observed also for 5. costatum and Chaetoceros sp.2 in treatments with the addition of phosphate (B) and river water (F). DISCUSSION AN D CONCLUSION S Response of plankton assemblages to nutrient additions In all three enrichment experiments the most marked response was observed in the plankton assemblages re­ceiving all nutrients (G treatment), followed by the addi­tion of phosphate (B treatment), river (F treatment) and rain (E treatment) water. The additions of the latter two in approx. natural dilutions enhanced biomass accumu­lation especially in PALEX 1 (early summer), suggesting the importance of these freshwater sources of nutrients in the northern Adriatic. Similar responses as in experi­mental conditions were observed also in the field (Malej etal., 1995; 1997a; 1997b). However, the development of autotrophic and het­erotrophic components of planktonic assemblage was quite different. Phytoplanktonic groups, which domi­nated absolutely and relatively in the initial assem­blages, expanded significantly towards the end of the experiments. Growth of microflagellates in PALEX 1 and diatoms in PALEX 2 and 3 was highly stimulated by the addition of nutrients, especially in the mixed treatments. This reflected not only in a large increase of their abun­dance (86- to 105-fold) but also in their relative propor­tions (for example, from 49% at the beginning to 94% at the biomass peak in G treatments). On the other hand, cyanobacteria were less success­ful than larger cells (nano- and microplankton) being outcompeted in all three experiments (Fig. 2a). This was especially evident in G treatments in all PALEX experi­ments, where cyanobacterial abundance decreased sharply and was the lowest compared to other treat­ments. In areas with high nutrient supply larger cells, mainly diatoms, seem to respond faster to nutrients' in­put (Sakshaug & Olsen, 1986; Stolte & Riegman, 1995), whereas in oligotrophic areas smaller cells are respon­sible for most of the primary production. This phenome­non is in part due to different nutrient uptake strategies adopted by phytoplankton species of different size classes as algal uptake activity is finally limited by available surface area (Stolte & Riegman, 1995). How­ever, in a recent laboratory experiment performed on the cosmopolitan cyanobacterial genus Synechococcus and diatom Thalassiosira weisflogii Donald et al. (1997) pointed out that differences in nutrient uptake might have originated also from different taxonomic as well as evolutionary position of the two organisms: the first be­ing procaryotic and the second one eucaryotic. This re­fers to the enzymes involved in nutrient uptake or up­take kinetics that are related to taxonomic groups and not to cell size (Stolte & Riegman, 1995). As in our ex­periment Donald et al. (1997) found out that cyanobac­teria Synechococcus grew better in P-limited conditions (A16 treatment in PALEX 3 experiment), while under P-replete conditions larger diatom species posses higher rates of nutrient uptake and a better ability to incorpo­rate phosphate than cyanobacteria. In all PALEX experiments the response of heterotro­phic bacteria to nutrient addition (Fig. 2b) preceded the response of phytoplankton indicating the importance of competition for inorganic nutrients. In fact many authors observed, in freshwater systems mainly, this competitive character of bacteria for inorganic nutrients, especially when concentrations of dissolved organic matter are low (Toolan et al., 1991; Coveney & Wetzel, 1992; Wehr et al., 1994). Bacterial growth can be directly limited by the phosphate availability (Toolan et al., 1991), regard­less the phytoplankton response (Le et al., 1994), sug­gesting that the nutrient uptake of these microorganisms can at times become a sink for nutrients within the mi- Fig. 4a: Mixed plankton community dominated by dia­tom Cylindrotheca closterium and blue-stained hetero­trophic bacteria in PALEX 3 experiment (400x). Epi­fluorescence microscopy, DAPI stained, UV light. SI. 4a: Mešana planktonska združba s prevladujočo dia­tomejsko vrsto Cylindrotheca closterium in modro obar­vanimi heterotrofnimi bakterijami v obogatitvenem po­izkusu PALEX 3 (400x). Epifluorescentna mikroskopija, barvano z DAPI-jem, UV svetloba. Figs. 4b-f: Dominating diatoms of the phytoplankton as­semblage and bacterioplankton during PALEX 3 ex­periment: day 6, G treatment (mixed nutrients). Epi­fluorescence microscopy, primuline and DAPI stained, UV light. SI. 4b-f: Prevladujoče diatomeje v fitoplanktonski združ­bi in bakterije 6. dne obogatitvenega poizkusa PALEX 3, mešane hranilne razmere (G). Epifluorescentna mikro­skopija, barvano s primulinom in DAPI-jem, UV svetloba. b) Pseudonitzschia sp., Chaetoceros spp. (200x) c) Skeletonema costatum (400x) d) Skeletonema costatum, Chaetoceros spp., Pseudo­nitzschia pseudodelicatissima (200x) e) Chaetoceros decipiens, Chaetoceros spp. (200x) f) DAPI stained bacteria (filamentous, rods and coccal forms) around autotrophic diatom cell (400x). DAPI obarvane bakterije (nitaste, paličaste in okrogle oblike) okoli avtotrofne diatomejske celice (400x). ANNALES 13/'98 Patricija MOZETIČ et al.: NUTRIENT-ENRICHMENT EFFECT O N PLANKTON COMPOSITION, 31-42 Patrie tja MOZETI Č «ai. : NUTRIENT-ENRICHMEN T EFFECT O N PLANKTO N COMPOSITION , 31-42 crobiai loop (Toolan et al., 1991; Coveney & Wetzel, 1992). Although these observations arise from freshwa­ter environment we can find some similarities to PALEX experiments, initial conditions in our experiments were always P-Iimited and a fast response of bacteria to nutri­ent addition followed by a sharp decrease was observed. Cyanobacterial as well as bacterial decrease could also be due to grazing by heterotrophic nanoflagellates (HNAN). In fact HNAN abundance in PALEX 1 in­creased sharply (Malej eta/., 1997a), while for PALEX 2 and 3 data are not available. Wehr et al. (1994) also suggested that more rapid mineralization of organic matter occur with herbivory on autotrophic cyanobac­teiia, indicating that HNAN may perhaps prefer cyanob­acteria to heterotrophic bacteria as a food source. Species composition and succession of phytoplankton Phytoplankton growth in all three enrichment ex­periments was limited by the availability of nutrients and unbalanced ratios. All three initial (field) assemblages were P-limited (N/P>30). With the addition of nutrients high N/P ratios changed close to the optimal Redfieid ratio (N/P=16) in the mixed treatment (C bottles) while in B treatment (phosphate addition) optimal nutrient ra­tios were achieved only in PALEX 1. In PALEX 2 and 3, N/P ratios in B treatments were much lower than 16, suggesting nitrogen co-limitation of phytoplankton growth. Consequently, the highest response of phyto­plankton community was achieved in mixed treatments. Among other treatments only during PALEX 1 phyto­plankton was significantly stimulated following phos­phate addition alone (see Fig. 1). However, phosphate and nitrogen alone are not suf­ficient for phytoplankton growth, especially in diatom-dominated assemblages. Besides microflagellates, dia­toms were the most abundant group during our experi­ments, suggesting silicate as crucial nutrient as well, if we consider 2 pM silicate per liter as the limiting con­centration (Fisher et a!., 1988; Egge & Aksnes, 1992) we can presume that silicate was co-limiting diatoms most of the time during PALEX experiments. High silicate concentrations (5-21 pM I"1) were found in all enclo­sures of PALEX 1 experiment, where microflagellates were the most abundant, and predominant group (Table 2). However, the addition of all nutrients stimulated diatom growth much more than flagellates (Fig. 3a) as indicated by treatment/control ratios (up to 420 for dia­toms and 16 for microflagellates). Especially in mixed treatment and with the addition of phosphate Chaetoc­eros sp.1 and Navicula sp. abundance increased ex­tremely (420- and 95-times in C enclosure, respec­tively), while in control their abundance decreased or did not change (Table 2). A high relative increase of two diatom species was observed also in the treatment with the addition of rain water (E), as previously reported also from the field after heavy summer storms (Male; ef a!., 1997b). In PALEX 2 and 3 experiments/dominated by diatoms, the most evident feature was the highest rela­tive increase of Skeletonema costatum, followed by the increase of Chaetoceros spp. and Cylindrotheca closte­rium (Figs. 3b, c). Although these species were not the most abundant (except Chaetoceros sp. 3 in PALEX 3; Table 2) their increase was the greaiest. This was par­ticularly true of 5. costatum increasing up to 37- and 132-times in PALEX 2 and 3, respectively. In both ex­periments the most numerous species was Pseu­donitzschia pseudodelicatissima, in all enclosures in­cluding control, but for this species a slight increase was observed only when all nutrients were added (G, G16). Beside the greatest response of selected species to mixed nutrients, the addition of phosphate slightly stimulated only S. costatum, and Bacteriastrum sp. in PALEX 3. In PALEX 2 the addition of river water (F) en­hanced Chaetoceros sp.2 growth. No significant re­sponse of dinoflagellates and microflagellates compared to control was observed in both experiments. Another group, which was significantly stimulated by the addition of all nutrients, rain water, as well as by the addition of inorganic nitrogen (C) and phosphate only, were coccolithophores with Emiliania huxleyi as the most abundant species, in PALEX 3 experiment the abundance of coccolithophore E. huxleyi itself ac­counted for more than 1% of total abundance, while in PALEX 2 coccolithophores exhibited a small increase relative to control only in G and C (nitrogen) treatments (data not shown; Mozetič, 1997). E. huxleyi-is known to form dense blooms in north­east Atlantic (Tyrrell & Taylor, 1996) and Norwegian fjords (Egge & Heimdal, 1994) usually in mid-summer when surface irradiances are high. Mesocosm experi­ments and modeling studies from these areas have sug­gested that E. huxleyi may have a competitive advan­tage over other phytoplankton, when phosphate is limit­ing but nitrate is abundant. E. huxleyi is believed to utilize better dissolved organic phosphorus than other phytoplankton (Aksnes et al., 1994) due to greater activ­ity of enzyme alkaline phosphatase. In similar experi­ments such as our PALEX (multi-species chemostat ex­periments; Riegman ef ai, 1992) higher numbers of L. huxleyi were obtained at high N/P ratios rather than at low N/P ratios. Besides specific nutritional conditions high surface irradiances were the most influential pa­rameter for bloom development (Egge & Heimdal, 1994). Our results from day 6 in PALEX 1 experiment showed indeed the highest concentrations of inorganic nitrogen in C (21.9 pM H ) and E (16.2 pM H) . in these treatments, besides G and B, the highest increase of coccolithophores compared to control were observed (Fig. ,3a). Due to low phosphate concentrations (around 0.05 pM H) , N/P ratios in C and E enclosures were the highest (313 and 540, respectively), interestingly, the in­ Patricija MOZETIČ el ah NUTRIENT-ENRICHMENT EFFECT O N PLANKTON COMPOSITION , 31-42 crease of coccolithophores/f. huxleyi in this early sum­mer experiment was greater compared to PALEX 3 (Fig, 3c), although the initial abundance was 10-times higher in the later one (see Table 2). Tab. 2: Abundances (ceils H) of dominant phyto­plankton species/groups (>1% of total abundance) at the beginning of the enrichment experiments and on the day of Chi a maximum in controlled (A) and mixed conditions (G). Tab. 2: Gostota (cel. H) prevladujočih fitoplanktonskih vrst/skupin na začetku obogatitvenih poizkusov in na dan klorofilnega viška v kontroli (A) ter ob dodatku mešanice hranil (G). PALEX 1 START DAY & Control (A) Mix (G) Chaetoceros s p. i 1.3x10s 0.5x103 2.2x10s Navicula sp. 2.2x103 2.2x10J 2.1x10s dinoflagellates 1.6x104 9.2x103 3.8x10" coccolithophores 2.7x103 0.5x103 2.7xl04 microti age Hates 1.9x10s 1.1 xlO6 1.8x107 PAf.DC 2 Chaetoceros s p. 2 1.3x10s 2.9x50s 3.7x10« Cylindrotheca ciosterium 6.5xl03 7.3x1C 2.3x10s Pseudonitzschia pseudodeiic. 5.6x10ft 1.6x10? 7.6x10? Skehtonema costatum 6.5xt03 7.5x10s 2.8x10? dinoflagellates 4.1x10" 3.5x104 1.3x105 m ic roil age If ate s 0.2x105 Î .4x106 4.9x106 PALEX 3 Baeteria$trum sp. 5.9x104 1.7x10s 2.6x10« Chaetoceros sp.3 1.9x1 OS 3.2xl05 1.3x107 Cylindrotheca ciosterium 1,8x104 1.6x104 . 7.9x10s Pseudonitzscbia pseudodelic. 1,1x10s 3.1x10s 7.7x10ft Skeletonema costatum 6.3x1 Q3 9.4x103 1,2x10& dinoflagell. non-i dent. 8.1x103 6.7x103 I.SxIO4 Emilia nia huxleyi 2.6x104 3.5x1 Q4 5.2x10s microti age H a tes 3.4x10s 2.9x10s 8.6x10s In chemostat enrichment experiments previously performed in the Gulf of Trieste MozetiC (1993) ob­tained similar results with S. c.ostatum being generally the most stimulated by nutrient additions. Its opportun­istic nature to adapt rapidly to changing nutrient regimes was observed many times in controlled conditions (Sanders et a!., 1987) as well as in natural environment (V.e. estuaries and coastal areas; Kenyan, 1976; Garcia-Soto et al., 1990; Borkman & Turner, 1993; Hama & Handa, 1994; Marshall & Nesius, 1996). Sanders et al. (1987) in a series of enrichment experiments over a nearly 2-year period in the Chesapeake Bay observed important changes in dominant species and patterns of species succession. Nutrient addition caused a shift from flagellate species to small centric diatoms (5. costatum, Cycioteila sp., Cylindrotheca ciosterium, Tbalassiosira spp.) in N-enriched cultures, but only during the sum­mer-autumn period. Their results can be compared with the situation in PALEX 1 with a significant increase of small Chaetoceros species and Navicula sp. over mi­croti age Hates, although comparison with other treat­ments indicated that the addition of phosphate rather than inorganic nitrogen stimulated their growth. This different pbytoplankton response to specific nutrient additions can be attributed to regional (and perhaps sea­sonal) differences in determining the limiting nutrient (northern Adriatic vs. Chesapeake Bay subestuary). Several authors (Turpin & Harrison, 1979; Harrison & Turpin, 1982; Kilham & Kilham, 1984) proposed that nutrient flux and nutrient ratios are important factors in­fluencing the dominance of various taxonomic groups (diatoms vs. flagellate species), while nutrient paichiness or chemical form of the nutrient may influence the suc­cess of one particular species over another. Our enrichment experiments confirmed that inputs of new nutrients not only stimulate phytoplankton growth in general but also affect community structure. Total phyto­plankton biomass expressed as Chi a augmented approx. 20-times over six days period following the addition of nutrients with optimal Redfield ratio. Majority of eucar­yotic phytoplankton increased their abundance; in con­trast, cyanobacteria decreased. Among taxonomic groups besides diatoms, coccolithophores seemed to be stimu­lated most (10-50 times increase in six days) by addition of nutrients. Diatoms that were growing most successfully were Skeletonema costatum (increasing its abundance from 40 up to 130 times in six days) during spring expe­riments, and small Chaetoceros species (augmenting its abundance up to 420 times) in early summer experiment. ACKNOWLEDGMENTS We thank Dr. Branko .ermelj for technical assistan­ce. The study was a part of Project PALOMA (Production and Accumulation of Labile Organic Matter in the Adri­atic) and was financially supported by the PECO Program (Contract No. CIPD-CT94-0106) and the Ministry of Sci­ence and Technology of the Republic of Slovenia. Patritij« MOZETI Č e! al.: NUTRIENT-ENRICHMENT EFf LCT O N PLANKTO N COMPOSITSON , 31-12 VPLI V VNOS A HRANILNI H SNOV ! N A SESTAV O PLANKTONSK E ZDRUŽB E Patricija MOZETIČ, Valentina TURK, Alenka MALE) Morska biološka postaja Piran, SI-6330 Piran, Fornače 41 POVZETEK V prispevku avtorice opisujejo odziv planktonske združbe na različne hranilne razmere z uporabo metode obogatitvenih poizkusov, V treh poizkusih, ki so potekali v juniju/juliju 1994, aprilu 1995 in marcu/aprilu 1996, so med drugim spremljale razvoj planktonske biomase in spremembe v vrstni sestavi kot odgovor na povečane koncentracije hranilnih snovi. Naravni planktonski združbi, inkubirani in situ, so dodale štiri umetne vire hranilnih snovi (fosfat, anorganski dušik kot nitrat in amonij, silikat in mešanico vseh hranil) ter naravna vira hranil v obliki deževnice in rečne vode. Inkubacijska posoda brez dodatka hranilnih snovi je ponazarjala kontrolne razmere. V vseh treh poizkusih so največji porast fitoplanktonske biomase opazile ob dodatku mešanice hranil, nekoliko manjši pa je bil odgovor ob dodatku fosfata. Prav nasprotno pa je gostota cijanobakterij in heterotrofnih bakterij s časom strmo upadala. Fitoplanktonske skupine in vrste, ki so prevladovale v začetnem vzorcu morske vode, so znatno narasle tudi ob koncu poizkusov (mikroflagelati v prvem poizkusu, diatomeje s prevladujočo vrsto Pseudonitzschia pseudodeiicatissima v obeh spomladanskih poizkusih). Navkljub temu so avtorice zabeležile največji porast števila v primerjavi s kontrolo pri drugih, v začetku neprevladujočih vrstah (Skeletonema costatum, Chaetoceros spp., Cyiindrotheca closterium, Navicula sp., Erniliania huxleyt). Absolutno največji porast je povzročil dodatek mešanice vseh hranil pri vrstah Skeletonema costatum v spomladanskem času (132-kratni porast v primerjavi s kontrolo) in Chaetoceros sp. v poletnem poizkusu (420-kratni porast). Edina nediatomejska vrsta, ki se je močneje odzvala na dodatke mešanice hranil, deževnice in anorganskega dušika, je bila kokolitoforida Erniliania huxleyi v poletnem obdobju. Pričujoča raziskava je potrdila, da obogatitev morske vode s hranilnimi snovmi v optimalnem koncentracijskem razmerju pospeši rast celotne fitoplanktonske združbe, pomembno pa vpliva tudi na vrstno sestavo. 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