Short-term changes of the mesozooplankton community and copepod gut pigment in the Chukchi Sea in autumn

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changes with SWE.In terms of abundance, calanoid copepods constituted the most dominated taxa (mean: 57 %), followed by barnacle larvae (31 %).Within the calanoid copepods, small-sized Pseudocalanus spp.(65 %) and large-sized Calanus glacialis (30 %) dominated.In the population structure of C. glacialis, copepodid stage 5 (C5) dominated, and the mean copepodid stage did not vary with SWE.The dominance of accumulated lipids in C5 and C6 females with immature gonads indicated that they were preparing for seasonal diapause.The gut pigment of C. glacialis C5 was higher at night and was correlated with ambient chl a, and a significant increase was observed after SWE (2.6 vs. 4.5 ng pigment ind. −1 ).Assuming C : Chl a ratio, the grazing impact by C. glacialis C5 was estimated to be 4.14 mg C m −2 day −1 , which corresponded to 0.5-4.6 % of the standing stock of micro-size phytoplankton.Compared with the metabolic food requirement, their feeding on phytoplankton accounted for 12.6 % of their total food requirement.These facts suggest that C. glacialis could not maintain their population on solely phytoplankton food, and other food sources (i.e., microzooplankton) are important in autumn.As observed for the increase in gut pigment, temporal phytoplankton bloom, which is enhanced by the atmospheric turbulence (SWE) in autumn, may have a positive effect on copepod nutrition.However, because of the

Introduction
In marine ecosystems of the western Arctic Ocean, mesozooplankton is a secondary producer and an important food resource for pelagic fishes and whales (Lowry et al., 2004;Ashjian et al., 2010).In terms of biomass, mesozooplankton in the western Arctic Ocean is dominated by Arctic copepods: i.e., Calanus glacialis, Calanus hyperboreus, Metridia longa and Pseudocalanus spp.In the southern Chukchi Sea, large Pacific copepods (e.g., Calanus marshallae, Neocalanus cristatus, Neocalanus flemingeri, Neocalanus plumchrus, Eucalanus bungii and Metridia pacifica) dominated in biomass because of the inflow of Pacific water.Thus, the zooplankton community in this region is greatly governed by water mass formation (Hopcroft et al., 2010;Matsuno et al., 2011Matsuno et al., , 2012)).Concerning seasonal change, a comparison of the zooplankton community between summer and autumn (Llinás et al., 2009) and year-round changes based on the two-week sampling interval at a drifting ice station in the Arctic basin were reported (Ashjian et al., 2003).However, short-term changes in the zooplankton community based on high-frequent sampling (two to four times per day) have not been reported.
Recently, a drastic reduction in the area of sea ice has been observed in the Arctic Ocean during summer, and the sea ice reduction was greatest in the western Arctic Ocean (Stroeve et al., 2007;Comiso et al., 2008;Markus et al., 2009).Furthermore, increases in frequency and magnitude of cyclones and a northward shift of their track during summer have been reported in recent years (Serreze et al., 2000;Cabe et al., 2001;Zhang et al., 2004;Sepp and Jaagus, 2011).While the importance of such changes is clear, little information is available regarding their effect on atmospheric turbulence in marine ecosystems in the western Arctic Ocean.From 10 to 25 Introduction

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Full September 2013, high-frequent sampling/observation was made at a fixed station in the Chukchi Sea, and the occurrence of strong wind events (SWE), vertical flux of nutrients and changes in primary production and microplankton communities were reported (Nishino et al., 2015;Yokoi et al., 2015).However, no information is available regarding how the mesozooplankton community responds to such atmospheric turbulence and oceanic environmental changes.
In the present study, we evaluated short-term changes of the mesozooplankton community in the Chukchi Sea during autumn based on the high-frequent samplings made simultaneously by Nishino et al. (2015) and Yokoi et al. (2015).We estimated the grazing impact of dominant copepods based on their gut pigments and evaluated the effect of SWE (Nishino et al., 2015) and short-term changes of microplankton assemblages (Yokoi et al., 2015) on the mesozooplankton community in the Chukchi Sea in autumn.

Field sampling
Zooplankton samplings were conducted at a fixed station in the Chukchi Sea (72 • 45 N, 168 • 15 W, depth 56 m) from 10 to 25 September 2013 (Fig. 1) (Nishino et al., 2015).Zooplankton samples were collected by vertical hauls with a NORPAC net (mouth diameter 45 cm, mesh size 335 µm; Motoda, 1957) and ring net (mouth diameter 80 cm, mesh size 335 µm) from 49 m depth to the sea surface two to four times per day (total of 47 times) including both day and night.Zooplankton samples collected by NORPAC nets were preserved with 5 % buffered formalin immediately on board.The ring net samples were used for copepod gut pigment measurement.For evaluation of the diel vertical migration (DVM) of copepods, day and night vertical stratified hauls with closing PCP nets (mouth diameter 45 cm, mesh size 62 µm; Kawamura, 1989) from two layers (0-20 m and 20-49 m) were made on 14 and 22 September.The samples from the PCP net were preserved with 5 % buffered formalin.Introduction

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Full At the fixed station, temperature, salinity and chlorophyll a (chl a) fluorescence were measured by CTD (Sea-Bird Electronics Inc., SBE911Plus) casts at a frequency of two to four times per day.To evaluate size fractionated chl a, water samples from the sea surface and the maximum fluorescence layer (16.8-27.7 m depth) were collected by a bucket and rosette multi-sampler mounted on CTD, respectively.Water samples were filtered through 20, 10 and 2 µm pore-size membranes and GF/F filters, and chl a was extracted with N,N-dimethylformamide and measured with a fluorometer (Turner Designs, Inc., 10-AU-005).

Gut pigment
For fresh samples collected by ring nets, 10 % v/v soda (saturated CO 2 in water) was added to avoid copepod grazing, gut evacuation and decomposition of gut pigment.Fresh specimens of C. glacialis copepodid stage 5 (C5) were sorted under a stereomicroscope.Batches of fifteen specimens were immersed in 6 mL N,N-dimethylformamide and stored in dark, cold conditions overnight to extract chlorophyll and phaeopigments.After the extraction of pigment, the chlorophyll and phaeopigments were measured with a fluorometer (Turner Designs, Inc., 10-AU-005).Chlorophyll and phaeopigments were summed and expressed as gut pigments (ng pigment ind. −1 ) (cf.Mackas and Bohrer, 1976).
The gut pigment of C. glacialis C5 was higher at nighttime than daytime.Assuming their grazing at night, the grazing rate (GR ind., mg pigment ind. −1 day −1 ) of C. glacialis C5 was calculated from following equation: where GP is the individual gut pigment at night (ng pigment ind.following equation: where N is the abundance of C. glacialis C5 (ind.m −2 ), and Int.chl a is standing stock of large-sized (> 10 µm) chl a (mg m −2 ).

Zooplankton community
In the land laboratory, identification and enumeration with taxa were performed on zooplankton samples collected by NORPAC nets under a stereomicroscope.For dominant taxa (calanoid copepods), identification was made with species and copepodid stage levels.For species identification of calanoid copepods, we referred mainly to Brodsky (1967) and Frost (1974) for Calanus spp., Miller (1988) for Neocalanus spp.and Frost (1989) for Pseudocalanus spp.For Pseudocalanus spp., species identification was made only for late copepodid stage C5 females/males (C5F/M) and C6F/M, and their early copepodid stages (C1-C4) were treated as Pseudocalanus spp.
For the evaluation of the DVM of large dominant copepods, we enumerated C. glacialis from PCP net samples.For C. glacialis, the lipid accumulation of C5 was scored into three categories: I (the oil droplet length (ODL) was 0-4 % of the prosome length (PL)), II (ODL was 4-40 % of PL) and III (ODL was > 40 % of PL).Gonad maturation of C. glacialis C6F was also scored into three categories: I (immature), II (small oocytes in the ovary or oviduct) and III (large eggs or distended opaque filled-in oviduct).For this gonad maturation index, we cited those for C. hyperboreus (Hirche and Niehoff, 1996).
From NORPAC net samples, the mean copepodid stage (MCS) of C. glacialis was calculated from the following equation:

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Full where i is the number of the copepodid stage (1-6 indicates C1-C6), and Ai (ind.m −2 ) is the abundance of the i th copepodid stage (cf.Marin, 1987).
During the study period, SWE was observed from 18 to 19 September (Nishino et al., 2015).To evaluate the effect of SWE, the abundances of each zooplankton taxon and species were compared between "before SWE (10-18 September)" and "after SWE (19-25 September)" using the U test.This statistical analysis was performed with Stat View.

Hydrography
During the sampling period, the temperature ranged from −1.5 to 3.3 • C, and thermocline was observed at a depth of approximately 25 m (Fig. 2a).Cold water below 0 • C continuously occurred below thermocline, whereas the temperature above thermocline decreased from 3.3 to 1.5 • C during the study period (Fig. 2a).Salinity ranged from 31.1 to 32.8, and halocline was observed at approximately 25 m, which was parallel to thermocline (Fig. 2b).Salinity below halocline was constant ca.> 32, whereas salinity in the upper layer increased from 31.1 to 31.6 throughout the study period.Chl a ranged from 0.08 to 3.25 mg m −3 and increased after the SWE (Fig. 2c).The T -S diagram showed that the hydrographic conditions in the upper layer changed temporally; thus, temperature decreased while salinity increased during the study period (Fig. 2d).
For calanoid copepods, 15 species belonging to nine genera were identified.Within them, Pseudocalanus spp.and C. glacialis dominated and composed 65 and 30 % of Introduction

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Full the total copepods, respectively.According to a comparison of time periods before and after the SWE, the total zooplankton abundance did not change, whereas one calanoid copepod Centropages abdominalis and cyclopoid copepods increased after the SWE (Table 1).On the other hand, one calanoid copepod Metridia pacifica, appendicularians, barnacle larvae, euphausiids and gymnosomes (Clione limacina) decreased after the SWE.

Calanus glacialis
On both 14 and 22 September, the C1-C4 and C5 populations of C. glacialis occurred mainly at lower layers (20-49 m) during the daytime, and they migrated to upper layers (0-20 m) at night (Fig. 3).It should be noted that nearly half of C5 remained in the lower layer both day and night.Lipid accumulation was higher for the C5 population residing in the lower layer.C6F occurred at a lower layer throughout the day.Throughout the study period, the standing stock of C. glacialis ranged from 1990 to 14 554 ind.m −2 , and no significant changes were detected with the SWE (Fig. 4a, Table 1).For the population structure, all copepodid stages (C1 to C6F/M) occurred, and C5 was the most dominant stage (36 %) of their population (Fig. 4).The MCS did not vary with SWE (Fig. 4a).Throughout the study period, the lipid accumulation of C5 was high (Fig. 4b), and the gonad maturation of C6F was dominated by immature specimens (Fig. 4c).These parameters exhibited no significant change with the SWE (U test, p = 0.285-0.303).
The gut pigment of C. glacialis C5 ranged from 0.6 to 12.3 ng pigment ind.−1 and showed a significant increase after the SWE (mean values: 2.6 vs. 4.5 ng pigment ind. −1 , U test, p < 0.01) (Fig. 5a).In a comparison between day and night, most dates except 18 and 23 September showed high gut pigment levels at night by a factor of two to five times those of the daytime.In both day and night, the gut pigments were correlated with a standing stock of chl a (p < 0.05) (Fig. 5b).

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Full During the study period, chl a peaked at 18 September, and micro-size (> 10 µm) dominated (54 % of total chl a), especially after the SWE (66 %) (Fig. 6b).The grazing impact of C. glacialis C5 on micro-size chl a ranged from 0.5-4.6 % on chl a standing stock day −1 and was high before the SWE from 10 to 15 September (Fig. 6c).

Zooplankton community
The zooplankton community in the Chukchi Sea is known to have greater spatial and temporal changes (Springer et al., 1989;Llinás et al., 2009;Matsuno et al., 2011).
According to Matsuno et al. (2012), the calanoid copepod community in the western Arctic Ocean during the autumn could be divided into three groups (Shelf, Slope and Basin) that vary with depth.According to their criteria, a shallower bottom depth and higher temperature and salinity indicate that the zooplankton community in this study corresponded to the Shelf group defined by Matsuno et al. (2012).As characteristics of the Shelf group, high zooplankton abundance (mean: 75 683 ind.m −2 ) and dominance of neritic (Pseudocalanus spp.and Centropages abdominalis) and Pacific copepods (Eucalanus bungii, Neocalanus cristatus and Metridia pacifica) were reported (Matsuno et al., 2012).
Comparing the above characteristics by Matsuno et al. (2012), the zooplankton abundance of this study was nearly half (mean: 34 059 ind.m −2 ), there was a low abundance of small copepod Pseudocalanus spp.and cyclopoid copepods, and no occurrence of Arctic copepod Metridia longa was remarkable (Table 1).For the hydrography of this station, Nishino et al. (2015) noted that the upper warm and less saline water was the Pacific Summer water, which was transported to the Arctic Ocean during summer, and the cold saline water of the lower layer was the remnant brine of the Pacific Winter water, which was transported to the Arctic Ocean during winter.Geographically, the Introduction

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Full present station was located at a main stream of the Pacific water (Weingartner et al., 2005).The high abundance of the Pacific copepod M. pacifica and Neocalanus spp.and the lack of the occurrence of the Arctic M. longa in this study was thought to be a reflection of the water mass covering the station.Seasonal characteristics during summer included the addition of holoplankton and the dominance of the meroplankton (barnacle and bivalve larvae), which composed 39 % of zooplankton abundance (Hopcroft et al., 2010).Dominance of the barnacle larvae also occurred in this study (Table 1).Benthic barnacle adults released their larvae when they met phytoplankton blooms (Crisp, 1962;Clare and Walker, 1986), and their larvae spent two to three weeks at water columns and then settled (Herz, 1933).The abundance of barnacle larvae in this study (mean 10 430 ind.m −2 ) was 13-55 % lower than that in summer (19 114-79 899 ind.m −2 , Matsuno et al., 2011).It also should be noted that the abundance of barnacle larvae decreased significantly during the study period (Table 1).These facts suggest that most of the barnacle larvae may have ended the planktonic phase and settled to the sea bottom during the study period (autumn).
Concerning the effect of the SWE, several limited taxa and species showed significant changes in abundance (Table 1).Within them, the decrease of barnacle larvae may have been caused by their seasonal settlement and was not related to the SWE.For dominant species, cyclopoid copepods were increased after the SWE (Table 1).The generation length of cyclopoid copepods was reported to be two to three months in the Arctic Ocean (Dvoretsky and Dvoretsky, 2009).In ambient temperature (-1.5 to 3.3 • C), the egg hatching of this taxon is estimated to be 11-41 days (Nielsen et al., 2002).These facts suggest that the increase of cyclopoid copepods would not be caused by their reproduction within the study period (16 days).As an alternative cause, the horizontal advection of the water mass during the study period, which was reported by Nishino et al. (2015), should be considered.These results suggest that the effect of SWE on zooplankton abundance was relatively small because of the longer generation length of mesozooplankton in this region.

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Population structure of C. glacialis
Calanus glacialis is a key species that dominates in the zooplankton biomass and commonly occurs in the continental shelf throughout the Arctic Ocean (Conover and Huntley, 1991;Lane et al., 2008).The generation length of this species is known to vary from one to three years (mostly two years) depending on annual primary productivity in each region (Falk-Petersen et al., 2009).For the life cycle of C. glacialis, they grow to C3-C4 at the epipelagic layer during the first summer, then descend to the deep layer and enter diapause, developing to C5 at the epipelagic layer in the second summer, then down to the deep layer and molting to the adult stage (C6) using stored lipid as energy.They reproduce at the epipelagic layer, using the grazed phytoplankton bloom as energy (Longhurst et al., 1984;Ashjian et al., 2003;Falk-Petersen et al., 2009).
Concerning the population structure, Ashjian et al. (2003) reported that C. glacialis around the Northwind Abyssal Plain was dominated by C5 and C6F in September.
In the present study, the population structure of C. glacialis was dominated by C5 (Fig. 4a), and their MCS (mean ± SD: 3.77 ± 0.20) was similar to the reported value for autumn in this region (3.58,Matsuno et al., 2012).Most of the C6F had immature gonads and no ovigerous C6Fs were observed (Fig. 4c).These results corresponded with the year-round observation around the Northwind Abyssal Plain (Ashjian et al., 2003).Calanus glacialis C6F is known to occur at the epipelagic layer in April, just before sea-ice melting (Kosobokova, 1999), and to reproduce with grazing ice algae and ice-edge bloom (Campbell et al., 2009).Thus, because this study period (September) greatly varied with regards to their reproduction period (April), most C6Fs were considered to have immature gonads when residing in the lower layer (diapause).
Nocturnal ascent DVM, which is related to nighttime grazing on phytoplankton, was reported for C. glacialis in the Arctic Ocean during spring and autumn (Runge and Ingram, 1988;Conover and Huntley, 1991).In this study, DVM was observed for C3, C4 and C5 (Fig. 3).At high-latitude seas, the magnitude of Calanus spp.DVM is known to vary with the season and copepodid stage, and their DVM intensity is greater during Introduction

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Full spring and autumn when the diel changes in light penetration are large (Falkenhaug et al., 1997).No DVM of Calanus spp. was reported for lipid accumulated C5 (Falk-Petersen et al., 2008).In the present study, nearly half of the C5 population, which was characterized as having a large lipid accumulation, remained in the lower layer throughout the day (Fig. 3).The deep C5 population may have already completed lipid accumulation and ceased DVM in the study period (September), while the remaining C5 population with active DVM may have grazed on phytoplankton in the upper layer during the night and stored lipid in preparation for diapause.These results suggest that the C. glacialis population in this study was at the seasonal phase just before entering diapause, and this interpretation corresponded well with their life cycle in this region (Ashjian et al., 2003).

Grazing of C. glacialis
The gut pigment of C. glacialis in the Arctic Ocean is known to exhibit diel changes and higher levels at night than in the daytime (Conover and Huntley, 1991).Higher gut pigment levels at night were also observed in this study (U test, p < 0.001) (Fig. 5a).
Their gut pigments were correlated with the standing stock of chl a (Fig. 5b) and increased during the high chl a period after the SWE (Fig. 5a).These facts suggest that C. glacialis feeding responded to the small phytoplankton bloom, which was enhanced by the nutrient supply and vertical mixing caused by the SWE (Nishino et al., 2015;Yokoi et al., 2015).
Concerning gut pigment measurement, underestimation by decomposition of phytoplankton pigment through gut passage has been reported (Conover et al., 1986;Head, 1992).This underestimation is reported to be ca.0.1-10 % of grazing (Conover et al., 1986) and varies with light conditions, grazing behavior and phytoplankton species (Head, 1992).To estimate the grazing impact, data on the gut evacuation rate (k, min −1 ) is needed (Mauchline, 1998).The gut evacuation rate is known to have a positive correlation with temperature (Dam and Petersen, 1988).From the equation between k and temperature (T in • C): k = 0.00941 + 0.002575T (Mauchline, 1998) Introduction

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Full and ambient temperature in this study (T = −1.5 to 3.3 • C), k would be estimated to be 0.0055-0.0179min −1 .This range covers the value applied in the present study (k = 0.017, Tande and Båmstedt, 1985).The value was also at the range observed by our independent laboratory experiments in September 2010 (0.006-0.041,Matsuno et al. unpublished data).These facts suggest that the value applied in this study (k = 0.017) was reasonable for C. glacialis in this region.
Assuming that half of the C5 population performed nocturnal ascent and grazing on phytoplankton at night, using C : Chl a ratio (29.9, Sherr et al., 2003), the grazing impact (mg C m −2 day −1 ) of C. glacialis C5 was calculated (Table 2).The grazing impact of this study was estimated to be 4.14 mg C m −2 day −1 , which was lower than the reported value (Tande and Båmstedt, 1985).In the Arctic Ocean, the grazing impact of copepods is greatly affected by the extent of sea-ice, environmental conditions and food concentration (Hirche et al., 1991;Campbell et al., 2009).The low grazing impact of this study may be caused by the low feeding activity of C5 just before their diapause and may also be related to the low ambient chl a (mean: 14.3 mg m −2 ) during the study period.
We also estimated the food requirement of C. glacialis C5 to support their metabolism under ambient temperature (Ikeda and Motoda, 1978;Ikeda et al., 2001).The proportion of phytoplankton food to food requirements was 12.6 % for C. glacialis C5 (Table 2).This result indicates that C. glacialis C5 could not maintain its population solely on phytoplankton food; other food sources are important.Regarding food for C. glacialis, Campbell et al. (2009) reported that this species prefers microzooplankton rather than phytoplankton in the Chukchi Sea, and Levinsen et al. (2000) noted that the micro-size (> 10 µm) ciliates and dinoflagellates are important food sources during post-bloom.For the microplankton community during the study period, Yokoi et al. (2015) noted that not only diatoms (1.64-14.11cells mL −1 ) but also dinoflagellates (0.54-2.42 cells mL −1 ) and ciliates (0.14-2.76 cells mL −1 ) were abundant.Thus, as food sources for C. glacialis, microzooplankton (dinoflagellates and ciliates) are considered to be important in the Chukchi Sea during autumn.

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Conclusions
Throughout this study, short-term changes in the mesozooplankton community and the grazing impact of dominant copepods were evaluated in the Chukchi Sea during autumn.During the 16 day sampling/observation period, the zooplankton community showed no clear changes related to the SWE and dominant copepods prepared for diapause (i.e., stored lipids in the pre-adult stage or adults with immature gonads).On the other hand, the feeding intensity of dominant copepods increased with the reflection of the temporal phytoplankton bloom, which was enhanced by the SWE (Nishino et al., 2015).Thus, the temporal phytoplankton bloom caused by the atmospheric turbulence (SWE) during autumn may have had a positive indirect effect on mesozooplankton (SWE → nutrient supply from the deep layer → small phytoplankton bloom → copepod feeding) within a short period.However, because of the relatively long generation length of copepods, a smaller effect was detected for their abundance, population structure, lipid accumulation and gonad maturation within the short-term period.Introduction

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Full  1. List of mesozooplankton taxa and calanoid copepod species and their mean abundances (ind.m −2 ) at a fixed station in the Chukchi Sea from 10 to 18 September, before the strong wind event (SWE), and 19-25 September (after the SWE), 2013.Differences between the two periods (before vs. after the SWE) were tested with the U test.Full  Table 2. Calanus glacialis: comparison of food requirements (ingestion) and grazing rate on phytoplankton and the proportion of phytoplankton food in the Chukchi Sea from 10 to 25 September 2013.Food requirements were calculated from the metabolism, which was estimated by the body mass, temperature (Ikeda et al., 2001), respiratory quotient (Gnaiger, 1983) and individual carbon budget (Ikeda and Motoda, 1978).Details on the values used in this calculation: dry mass (DM: mg ind.−1 ) of C. glacialis C5 was 0.654 (Matsuno et al., unpublished data).Oxygen consumption (µL O 2 ind.−1 h −1 ) = exp(−0.399+0.801 × ln(DM)+0.069× T ) (Ikeda et al., 2001).Respiratory quotient ([CO 2 /O 2 ]) was assumed to be 0.97 (Gnaiger, 1983).
Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | −1 ), k is the gut evacuation rate (0.017 min −1 , Tande and Båmstedt, 1985), and T is the length of the nighttime (mean 13 h = 780 min during study period).The grazing impact of C. glacialis C5 on micro-size (> 10 µm) chl a (GI, % on chl a standing stock day −1 ) was calculated by the Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Screen / Esc Printer-friendly Version Interactive Discussion Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | significantly after the SWE (0.11 vs. 0.18 mg pigment m −2 day −1 , U test, p < 0.05).
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