Seasonal distribution of photosynthetically active phytoplankton using pulse amplitude modulated fluorometry in the large monomictic Lake Biwa, Japan

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1 Seasonal distribution of photosynthetically active phytoplankton using pulse amplitude modulated fluorometry in the large monomictic Lake Biwa, Japan NAOSHIGE GOTO 1 *, MASAKI KIHIRA 1 AND NORIKO ISHIDA 2 1 SCHOOL OF ENVIRONMENTAL SCIENCE, THE UNIVERSITY OF SHIGA PREFECTURE, 2500 HASSAKA-CHO, HIKONE, SHIGA , JAPAN AND 2 NAGOYA WOMAN S UNIVERSITY, 1302 TAKAMIYA-CHO, TENPAKU-KU, NAGOYA , JAPAN *CORRESPONDING AUTHOR: gotonao@ses.usp.ac.jp Received March 30, 2008; accepted in principle June 29, 2008; accepted for publication July 4, 2008; published online July 8, 2008 Corresponding editor: William Li The detailed seasonal distribution of photosynthetically active phytoplankton was measured using the pulse amplitude modulated fluorescence technique in the large monomictic Lake Biwa, Japan, throughout the year. At the same time, the biological and physicochemical factors (water temperature, irradiance, nutrients and phytoplankton species) affecting the primary production of phytoplankton were also measured in order to estimate the relationship between the distribution of the active phytoplankton and environmental factors. The potential maximum quantum yield (F v /F m ) of photosystem II was used as an index of the degree of potential photosynthetic competence of phytoplankton. As a result, the simultaneous measurement of F v /F m and algal biomass showed clearly the seasonal and spatial distribution patterns of the photosynthetically active phytoplankton community, and the distributions were strongly dependent on the seasonal variation in the thermally stratified structure. In addition, the value of F v /F m in the euphotic zone was found to be influenced mainly by the irradiance and to be affected indirectly by nutrient concentration and the species composition of the phytoplankton community. Greater understanding of the detailed distribution of photosynthetically active phytoplankton coupled with their biomass will encourage new developments in studies of aquatic ecosystems. INTRODUCTION Phytoplankton play an important role as a major supplier of organic matter for heterotrophic organisms, and in order to understand aquatic ecosystems, it is necessary to properly understand phytoplankton biomass and productivity. The chlorophyll a concentration is widely used and conveniently measured index of phytoplankton biomass. The seasonal and spatial distributions of phytoplankton have thereby been measured in detail. In contrast, it is difficult to measure the photosynthetic competence and/or photosynthetic physiological states of phytoplankton in detailed spatiotemporal distribution, because they have traditionally been measured using oxygen (Gaarder and Gran, 1927) and/or isotope tracers (Steemann-Nielsen, 1952; Hama et al., 1983) methods which require complex incubation procedures that are both laborious and time-consuming. However, it is very important to understand in detail the varying distribution of metabolic or photosynthetically active phytoplankton in the study of organic matter production and/or cycling in aquatic ecosystems. doi: /plankt/fbn073, available online at # The Author Published by Oxford University Press. All rights reserved. For permissions, please journals.permissions@oxfordjournals.org

2 Chlorophyll fluorescence has come to be used as a powerful probe of photosynthetic function for the study of phytoplankton photosynthesis instead of the abovementioned conventional incubation methods. A method utilizing pulse amplitude modulated (PAM) chlorophyll fluorometry has spread quickly and widely (Schreiber et al., 1986, 1995a). The advantages of the PAM fluorescence technique include its ability to capture a great deal of information related to the photosynthesis of phytoplankton in a convenient, rapid and non-invasive manner, and to assure high spatiotemporal resolution measurements in the study of aquatic photosynthesis. PAM fluorometry can estimate the potential maximum quantum yield (F v /F m ) of photosystem II (PSII), and the F v /F m value is one of the parameters most often used in the study of aquatic photosynthesis. F v /F m is a robust indicator of the degree of potential photosynthetic competence, nutrient stress and photoinhibition of phytoplankton, and the measurement of F v /F m in phytoplankton has contributed greatly to the developing field of the study of aquatic photosynthesis and ecosystems (Bergmann et al., 2002; Cavender-Bares and Bazzaz, 2004). Although the PAM fluorescence technique rapidly provides much useful information on the photosynthesis of phytoplankton, the technique has not been used much for the investigation of the detailed seasonal and spatial distribution pattern in active phytoplankton. The detailed distribution in the active phytoplankton community has not been investigated (Figueroa et al., 1997; Marchetti et al., 2006), though an enormous amount of data have been accumulated regarding the detailed distribution in the phytoplankton biomass. In particular, there is little knowledge regarding the annual cycle of the distribution pattern in photosynthetically active phytoplankton (Aiken et al., 2004). Therefore, we measured the detailed seasonal distribution of photosynthetically active phytoplankton using the PAM fluorescence technique in the large monomictic Lake Biwa, Japan, throughout the year. At the same time, we also measured the physicochemical and biological factors (water temperature, irradiance, nutrients and phytoplankton species) affecting the primary production of phytoplankton and have estimated the relationship between the distribution of the active phytoplankton and environmental factors based on the results obtained. METHOD Sampling Sampling of the lake water for measurements of F v /F m on the freshwater phytoplankton community was Fig. 1. Map of Lake Biwa, Japan showing location of sampling Station S1 (open circle) and T1 T11 (filled circles). Filled triangle indicates the location of the Hikone Local Meteorological Observatory. carried out at Sta. S1 in the north basin of Lake Biwa (surface area: 670 km 2 ; mean water depth: 43 m; maximum depth 104 m), Japan, approximately once a month from September 2004 to September 2005 (Fig. 1). Water samples were collected at 10 depths (0, 5, 10, 15, 20, 25, 30, 40, 50 and 60 m) from the surface to the bottom using a Van Dohrn water sampler throughout the sampling period. In addition, the spatial distribution of F v /F m was investigated in all areas (11 transect points: Stas T1 T11) of the lake on 1 and 2 November 2004 (Fig. 1) when water samples were taken at 2 9 layers from the surface to the lake bottom at each station also using a Van Dorn water sampler. Simultaneously, we collected physical data (water temperature and underwater irradiance) and water samples for the determination of nutrients [dissolved inorganic nitrogen (DIN: sum of ammonia, nitrite and nitrate), phosphate phosphorus (DIP) and dissolved silica (DSi)], chlorophyll a (Chl a) concentration and phytoplankton species. Chlorophyll fluorescence The parameter F v /F m was measured with a Heinz Walz GmbH Water-PAM chlorophyll fluorometer equipped 1170

3 N. GOTO ET AL. j SEASONAL DISTRIBUTION OF PHOTOSYNTHETICALLY ACTIVE PHYTOPLANKTON with a Water-ED Emitter-Detector Unit that featured a measuring chamber with a 15 mm diameter quartz cuvette after min of adaptation to the dark (Schreiber et al., 1995b). F v /F m provides an assessment of the maximum photochemical efficiency of PSII reaction centers of darkness-adapted algal cells. Physicochemical and biological factors Water temperature and underwater irradiance were measured using an ALEC ACL-220 CTD fitted with a LiCor LI-193 PAR sensor. Daily mean global solar radiation for each month was obtained from the Hikone Local Meteorological Observatory (Fig. 1). Water samples collected for chemical analysis of nutrients and Chl a were filtered immediately through Whatman GF/F glass fiber filters precombusted at 4508C. Filtrates for the determination of nutrients were stored at 2308C, whereas filters for the determination of Chl a were stored at 2808C until analysis in the laboratory. Ammonia was determined by the method of Sagi (1966), nitrite after Bendschneider and Robinson (1952), DSi after Mullin and Riley (1955) and DIP after Murphy and Riley (1962). Nitrate was analyzed by a Dionex DX-120 ion chromatographic analyzer. Chl a concentration was analyzed fluorometrically using a Turner Designs TD-700 fluorometer that was calibrated with pure Chl a (Sigma Chemical) after extraction in 90% acetone, and then ultrasonicated for 30 s (Holm-Hansen et al., 1965). Samples of phytoplankton were fixed using buffered formalin (0.4% final concentration), and species composition was determined using an Olympus BX-50 light microscope at magnification. Algal species were identified using permanent preparations, and at least 500 cells per sample were counted. To determine the actual biovolume of each algal species, the shape of each species was measured microscopically using a Mitani Mac Scope ver The numbers of cells, colonies or filaments were converted to biovolume using the average biovolume for each taxon obtained by the abovementioned graphic-analysis system. RESULTS Seasonal variation in F v /F m of phytoplankton community Lake Biwa has been defined as a monomictic lake characterized by its circulation (February) and stratification period (April December) (Fig. 2). Water temperature in the winter circulation period was almost uniform in all layers ( C, mean + SD). On the other Fig. 2. Daily mean global solar radiation each month and vertical distribution of water temperature (open circles represent the euphotic zone depth), F v /F m and chlorophyll a concentration at Station S1 of Lake Biwa from September 2004 to September hand, during the stratification period, the lake was divided into three separate layers: epilimnion (0 20 m: C, mean + SD), thermocline (20 30 m) and hypolimnion (30 60 m: C, mean + SD). The daily mean global solar radiation for each month ranged from 6.7 (December 2004) to 20.2 (May 2005) MJ m 22 day 21 (Fig. 2). The euphotic zone depth (depth of 1% relative irradiance) at Sta. S1 ranged from 11.9 (October 2004) to 29.5 (September 2005) m ( m, mean + SD) throughout the year (Fig. 2). F v /F m values measured on the natural phytoplankton community ranged from 0.02 (40 m depth in November 2004) to 0.73 (5 m depth in December 2004), and showed a tendency to increase in the mixing period and to decrease in the stratification period (Fig. 2). In the mixing period, F v /F m ranged from 0.60 to 0.70 ( , mean + SD) and was distributed approximately uniformly in all layers. On the other hand, in the 1171

4 stratification period, F v /F m in the epilimnion varied from 0.47 to 0.73 ( , mean + SD), and below the thermocline, it decreased greatly with increasing water depth. In the stratification period, F v /F m in the hypolimnion ranged from 0.02 to 0.69 ( , mean + SD). In addition, F v /F m in the surface water (0 m depth), particularly in the summer season, was lower when compared with other layers in the epilimnion. The concentration of Chl a ranged from 0.07 to 7.21 mg Chl a L 21 and showed two peaks in December 2004 (25 m depth) and May to July 2005 (5 10 m depth) (Fig. 2). In the mixing period, the Chl a concentration was distributed approximately uniformly in all layers ( mg Chl a 21 L 21, mean+ SD). On the other hand, in the stratification period, the Chl a concentration was higher in the surface layers ( mg Chl a 21 L 21, mean + SD), and below the thermocline, it decreased greatly (the hypolimnion: mg Chl a 21 L 21, mean + SD). The concentrations of nutrients (DIN, DIP and DSi) in the stratification period were lower in the epilimnion and increased gradually with increasing water depth, distributing almost uniformly in all layers in the mixing period (Fig. 3). The mean DIN:DIP and DSi:DIP molar ratios were 736 (range: ) and 1004 (range: ), respectively. The dominant algal groups in the epilimnion (10 m depth) were diatoms ( %, mean + SD) and chlorophytes ( %, mean + SD) throughout the sampling period, except for September 2005 when chlorophytes and dinophytes dominated (Fig. 4). During the study period, large diatoms (Aulacoseira, Fragilaria and Stephanodiscus) and large chlorophytes (Staurastrum) frequently became dominant. Spatial distributions in F v /F m of phytoplankton community Lake Biwa on 1 and 2 November 2004 was in the latter period of stratification, and water temperature in the epilimnion (0 20 m) and the hypolimnion (30 60 m) layers was C (mean + SD) and C (mean + SD), respectively (Fig. 5). The vertical distributions of F v /F m of the phytoplankton community were almost similar at all stations, i.e. F v /F m was higher in the epilimnion but dropped abruptly around the thermocline, ranging from 0.44 to 0.68 ( , mean + SD) in the epilimnion and ( , mean + SD) in the hypolimnion (Fig. 5). The concentration of Chl a ranged from 0.68 to 4.16 mgchla 21 L 21 ( mgchla 21 L 21, mean + SD) in the epilimnion and from 0.07 to 0.32 mgchl Fig. 3. Vertical distribution of the concentrations of DIN, DIP and DSi at Station S1 from September 2004 to September Fig. 4. Relative biomass (biovolume) of dominant algal groups at a depth of 10 m at Station S1 of Lake Biwa from September 2004 to September a 21 L 21 ( mgchla 21 L 21, mean+ SD) in the hypolimnion throughout all stations (Fig. 5). The highest Chl a concentration was observed in the southern area of the south basin (Sta. T11). The vertical distributions of nutrients (DIN, DIP and DSi) were similar at all stations, i.e. those concentrations in the epilimnion were at a lower levels and in the hypolimnion increased gradually with water depth (only the 1172

5 N. GOTO ET AL. j SEASONAL DISTRIBUTION OF PHOTOSYNTHETICALLY ACTIVE PHYTOPLANKTON Fig. 5. Water temperature, F v /F m, chlorophyll a and DIN at 11 transect points (Stas T1 T11) on 1 and 2 November distribution of DIN concentration is displayed in Fig. 5). The mean of DIN:DIP and DSi:DIP molar ratios were (range: ) and (range: ), respectively. The dominant algal groups in the epilimnion (10 m depth) were diatoms ( %, mean + SD) and chlorophytes (29 + 7%, mean + SD) over all stations. At this time, large diatoms (Aulacoseira, Fragilaria and Stephanodiscus) and large chlorophytes (Closterium and Staurastrum) dominated all stations. DISCUSSION Seasonal distribution of photosynthetically active phytoplankton community Estimation of the degree of potential photosynthetic competence and/or condition of cells is possible on the basis of the measured F v /F m of phytoplankton (Genty et al., 1989; Schreiber et al., 1995a). We confirmed in our laboratory that F v /F m of cultured alga Aulacoseira granulata (Bacillariophyceae) isolated from Lake Biwa was high (.0.6) in the growth phase when photosynthetic O 2 production per unit Chl a was high, whereas the F v /F m decreased abruptly (,0.5) in the stationary to declining phase when photosynthetic O 2 production per unit Chl a fell dramatically (unpublished). In this study, the simultaneous measurement of F v /F m and Chl a concentration clearly revealed the seasonal and spatial distribution patterns of the photosynthetically active phytoplankton community. Thus, from summer to autumn 2004, some phytoplankton communities which had lower photosynthetic competence were distributed in the epilimnion (Fig. 2), and they were observed to be distributed in all areas of Lake Biwa (Fig. 5). Thereafter, high abundances of phytoplankton which had higher photosynthetic competence were distributed from the depth of 0 to 40 m in wintertime (December 2004). In the mixing period (February 2005), the highly active phytoplankton community was distributed in all layers, though the phytoplankton biomass itself was relatively small. A relatively large amount of phytoplankton was thereafter confirmed to be distributed in the epilimnion, though photosynthetic competence declined from spring to summer. It also became clear that a phytoplankton community with high photosynthetic competence inhabited the lower part of the epilimnion throughout the year. The vertical distributions of F v /F m at Sta. S1 were almost similar to that of water temperature throughout the year. Namely, F v /F m in the stratification period was higher in the upper thermocline and was lower in the lower thermocline; on the contrary, F v /F m in the mixing period was distributed almost uniformly in all layers (Fig. 2). In addition, vertical distributions of F v /F m in the latter period of stratification (November 2004) were similar at all stations and approximated those of water temperature (Fig. 5). These results indicate that the vertical distribution of F v /F m is greatly dependent on the seasonal variation of stratified structure. This understanding is very significant in terms of the organic matter production and physiological ecology of phytoplankton in aquatic ecosystems and the estimation of the factors limiting algal photosynthesis and growth (Moore et al., 2005; Kaiblinger et al., 2007). Moreover, understanding how phytoplankton with metabolic functions settle to the lake bottom is very useful for investigating the supply and consumption of dissolved oxygen and/or material cycles in the surface and deeper layers. The simultaneous detailed observation of algal photosynthetic competence and biomass will bring 1173

6 about new developments in various studies of aquatic environments. Relationship between F v /F m and environmental factors In the present study, F v /F m decreased gradually in all layers from spring to summer (April September 2005) in proportion to the development of thermal stratification with increasing water temperature (Fig. 2). In addition, the vertical distribution of F v /F m in the epilimnion tended to a minimum in the superficial water (0 m depth) and high in the lower layer (10 20 m depth). Figure 6 shows the vertical distribution of F v /F m throughout the study period. These distributions of F v /F m can be attributed mainly to three factors. First, the depression of F v /F m is considered to be caused by the increase in the duration of exposure at high irradiance. High irradiance including ultraviolet (UV) radiation causes damage to PSII reaction center proteins (Neale et al., 1991; Bergmann et al., 2002; Bouchard et al., 2005), and this photodamage significantly decreases F v /F m of phytoplankton. Moreover, phytoplankton can reduce light-utilization efficiency under high irradiance (the down-regulation of PSII reaction centers) for the purpose of preventing the photodamage; in contrast, phytoplankton can increase light-utilization efficiency at low irradiance (Bracher and Wiencke, 2000; Kaiblinger et al., 2007). In this study, it is assumed that the seasonal Fig. 6. Vertical distribution of F v /F m throughout the observation period. Open circles represent the mean values of F v /F m at each depth. distributions of F v /F m were most strongly affected by light intensity as described above. Secondly, it is also thought that the decrease in nutrient supply from the deeper to the surface layer (depletion of nutrients in the epilimnion) contributed to the depression of F v /F m from spring to summer. The growth of phytoplankton in Lake Biwa was severely limited by phosphorus (DIN:DIP molar ratios: ; DSi:DIP molar ratios: ) throughout the year (Fig. 3), and the phosphorus limitation reportedly intensified from spring to summer in Lake Biwa (Tezuka, 1984). The repair of damaged PSII centers in phosphorusstarved algal cell may be impaired by a reduction of ATP synthesis (Lippemeier et al., 2001). Thus, since the deterioration in the nutritional status of phytoplankton leads to a delay in recovery from photodamage, it may be assumed that the nutrient stress contributed to the decline of F v /F m from spring to summer. Thirdly, it is supposed that the progression in species composition of phytoplankton from large diatoms and large chlorophytes in winter to small chlorophytes and dinophytes in spring to summer also influenced the decline of F v /F m (Fig. 4). In addition, the ratio of picophytoplankton, mainly composed of picocyanobacteria, to the total phytoplankton biomass increases greatly in summer in Lake Biwa (Maeda et al., 1992; Kihira et al., 2005). It has been reported that the F v /F m of small-size phytoplankton tends to be lower than that of large-size phytoplankton (Cermeño et al., 2005), and F v /F m changes greatly depending on the phytoplankton species (Cermeño et al., 2005). In short, it is concluded that F v /F m of the phytoplankton community declined gradually in the stratification period mainly due to the three factors mentioned above, i.e. high irradiance, nutrient limitation and algal succession to small-size phytoplankton. However, there are some reports that show that no significant difference was observed among algal species and/or size when F v /F m was measured (Juneau and Harrison, 2005; Dijkman and Kromkamp, 2006). Care must be taken in estimating a variation in F v /F m among algal species. In this study, F v /F m in the epilimnion increased in autumn (October November 2004) when the decrease in thermal stratification started (decline of thermocline) (Fig. 2). Subsequently, F v /F m rose to 0.7 at the end of stratification (December 2004), and the higher F v /F m values were distributed to a depth of 40 m. In the relationship between mean F v /F m in the euphotic zone and daily global solar radiation on the observational day (Fig. 7), the F v /F m increased with decreasing daily global solar radiation. This increase in F v /F m can be attributed mainly to the reduction in photodamage and the increase in light-utilization efficiency with the weakening of light intensity. Moreover, this increase may be affected partly by the increase in nutrient supply from 1174

7 N. GOTO ET AL. j SEASONAL DISTRIBUTION OF PHOTOSYNTHETICALLY ACTIVE PHYTOPLANKTON Fig. 7. Relationship between the mean value of F v /F m in the euphotic zone and daily mean global solar radiation on the observational day. the deeper layer with the declining thermocline, and at this time of year, the shift of the dominant algal groups from small to large phytoplankton also may have contributed to the increase of F v /F m (Fig. 4). Particularly, in December 2004, when F v /F m was the highest throughout the observation period, large heavy diatoms such as Aulacoseira, Fragilaria and Stephanodiscus became dominant in Lake Biwa. Some research has indicated that F v /F m of large diatoms tends to be higher than that of the other algal groups due to the differences in photosynthetic characteristics (Müller, 2004; Cermeño et al., 2005; Kaiblinger et al., 2007). In the winter season, these large and heavy diatoms sink rapidly to the hypolimnion while maintaining a viable condition. This rapid sedimentation of diatoms caused the expansion of the distribution in high F v /F m to the deeper layer. However, Büchel and Wilhelm (1993) reported that F v /F m of diatoms tends to be lower than that of the other algal groups. It is necessary to make further studies of the F v /F m of diatoms. High F v /F m (.0.6) in the epilimnion was still sustained in the mixing period (February 2005) (Fig. 2). This mainly reflected the effects of the aforementioned factors, i.e. the weakening of irradiance, supply of nutrients and dominance of large phytoplankton (Figs 3 and 4). In addition, the area of high F v /F m extended to the lake bottom at a depth of 60 m. This distribution resulted from the fact that active large diatoms Aulacoseira, Stephanodiscus and chlorophytes Staurastrum sank so rapidly to the hypolimnion before losing their photosynthetic activity. Large quantities of large diatoms settle rapidly to the lake bottom in Lake Biwa during the mixing period (Goto et al., 2007), and our recent observations also have confirmed that the sinking flux of those diatoms at 70 m depths was at its highest in February (unpublished). These results suggest that the transport of active phytoplankton to the deeper layer during the winter will have a great effect on the material cycles in the hypolimnion due to their metabolic action. As described above, the measurement of F v /F m provides not only information on the physiological state of phytoplankton but also valuable information on aquatic ecosystems. However, the interpretation of measured F v /F m requires care, because F v /F m varies depending on the independent or combined action of various factors (light, water temperature, nutrients and phytoplankton species, etc.). Parkhill et al. (Parkhill et al., 2001) showed that the F v /F m of phytoplankton can be utilized as an indicator of nutrient (N, P and Si) stress during unbalanced growth, but that the relationship between F v /F m and nutrient stress breaks down when phytoplankton become acclimated to nutrient limitation. Holeton et al. (Holeton et al., 2005) pointed out the possibility that any inherent taxonomic variation in F v /F m may be mistaken for a change in F v /F m with nutrient stress. Moreover, F v /F m is also affected by various substances, e.g. iron, copper and/or herbicides (Suzuki et al., 2002; West et al., 2003; Devilla et al., 2005; Pérez et al., 2006). Besides, the diel periodicity in F v /F m as a result of the cell cycle further complicates the estimation of F v /F m (Bergmann et al., 2002; Bruyant et al., 2005; Ditullio et al., 2005), and F v /F m in waters with a high concentration of phaeophytin must be corrected to avoid misinterpretation (Fuchs et al., 2002). As mentioned above, limiting factors and/or physiological states of phytoplankton based on the value of F v /F m must be estimated carefully with these various factors in mind. In conclusion, the detailed seasonal distributions of photosynthetically active phytoplankton were rapidly and readily shown by utilizing PAM chlorophyll fluorometry from algal cells, and the distributions were greatly dependent on the seasonal variation of thermally stratified structure. In addition, it is assumed that F v /F m of phytoplankton was affected mainly by irradiance and secondly by nutrient supply and/or species composition of the phytoplankton community. Moreover, the chlorophyll fluorometry helped in the assessment of limiting factors and/or physiological status for photosynthesis of phytoplankton. It follows from what has been said that the detailed observation of the photosynthetic competence of phytoplankton along with phytoplankton biomass provides a wealth of information for various studies 1175

8 regarding the dynamics of active phytoplankton and/or material cycles in aquatic ecosystems. FUNDING This work was financially supported by the University of Shiga Prefecture (grant number 3040). ACKNOWLEDGEMENTS We are grateful to Captain B. Kaigai of the RV Hassaka, the University of Shiga Prefecture (USP), for his generous assistance during the sample collection. Thanks are also due to the staff of the Limnological Laboratory, USP, for their contributions to water sampling and chemical analysis. REFERENCES Aiken, J., Fishwick, J., Moore, G. et al. (2004) The annual cycle of phytoplankton photosynthetic quantum efficiency, pigment composition and optical properties in the western English Channel. J. Mar. Biol. Ass. UK, 84, Bendschneider, K. and Robinson, R. J. (1952) A new spectrophotometric method for the determination of nitrite in sea water. J. Mar. Res., 11, Bergmann, T., Richardson, T. L., Paerl, H. W. et al. 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