Small phytoplankton contribution to the standing stocks and the total primary production in the Amundsen Sea

Small phytoplankton are anticipated to be more important in a recently warming and freshening ocean condition. However, little information on the contribution of small phytoplankton to overall phytoplankton production is currently available in the Amundsen Sea. To determine the contributions of small phytoplankton to total biomass and primary production, carbon and nitrogen uptake rates of total and small phytoplankton were obtained from 12 productivity stations in the Amundsen Sea. The daily carbon uptake rates of total phytoplankton averaged in this study were 0.42 g C m−2 d−1 (SD=± 0.30 g C m−2 d−1) and 0.84 g C m−2 d−1 (SD=± 0.18 g C m−2 d−1) for nonpolynya and polynya regions, respectively, whereas the daily total nitrogen (nitrate and ammonium) uptake rates were 0.12 g N m−2 d−1 (SD=± 0.09 g N m−2 d−1) and 0.21 g N m−2 d−1 (SD=± 0.11 g N m−2 d−1), respectively, for non-polynya and polynya regions, all of which were within the ranges reported previously. Small phytoplankton contributed 26.9 and 27.7 % to the total carbon and nitrogen uptake rates of phytoplankton in this study, respectively, which were relatively higher than the chlorophyll a contribution (19.4 %) of small phytoplankton. For a comparison of different regions, the contributions for chlorophyll a concentration and primary production of small phytoplankton averaged from all the non-polynya stations were 42.4 and 50.8 %, which were significantly higher than those (7.9 and 14.9 %, respectively) in the polynya region. A strong negative correlation (r2 = 0.790, p<0.05) was found between the contributions of small phytoplankton and the total daily primary production of phytoplankton in this study. This finding implies that daily primary production decreases as small phytoplankton contribution increases, which is mainly due to the lower carbon uptake rate of small phytoplankton than large phytoplankton.


Introduction
The Amundsen Sea is located in West Antarctica between the Ross Sea and Bellingshausen Sea (Fig. 1), which is one of the least biologically studied regions in the Southern Ocean.Recently, several international research programs (KOPRI Amundsen project, iSTAR, ASPIRE and DynaLiFe) were launched to improve the understanding of this remote area.Field-measurement data revealed that annual primary production of phytoplankton reaching to 220 g C m −2 yr −1 in the Amundsen Sea polynya is as high as that of Ross Sea polynya (200 g C m −2 yr −1 ) which was previously known for the highest productivity region in the Southern Ocean (Lee et al., 2012).Given the fact that the chlorophyll a concentration averaged from all the chlorophyll a measured stations was twice as high as that of the only productivity-measured stations, Lee et al. (2012) argued that the annual production in the Amundsen Sea polynya could be even 2 times higher than that of Ross Sea polynya.
Over the past several decades, a rapid climate change has been detected and subsequently physical changes have occurred in the marine ecosystem in the western Antarctic Peninsula (WAP), which was mainly based on the results from Palmer Antarctic Long-Term Ecological Research project focusing on the north of ∼ 69 • S (Ducklow et al., 2007;Montes-Hugo et al., 2009).Recent studies revealed that the Thwaites Glacier in Pine Island Bay is retreating fast and the ice volume loss in the nearby Getz Ice shelf is accelerating (Joughin et al., 2014;Paolo et al., 2015).Shoaling warm Circumpolar Deep Water is believed to be a main cause of the ice sheet mass loss through the ice shelf basal melt underside of the ice shelves (Yager et al., 2012;Schmidtko et al., 2014).Climate change from a cold-dry polar type to a warm-humid sub-Antarctic type has driven subsequent changes in ocean biological productivity along the WAP shelf over the recent 3 decades (Montes-Hugo et al., 2009).Phytoplankton, as the base of oceanic food webs, can be an indicator for changes in marine ecosystems responding to environmental changes (Moline et al., 2004;Wassman et al., 2011;Arrigo and van Dijken, 2015).For example, a recurrent shift in phytoplankton community structure from large diatoms to relatively small cryptophytes could be tightly associated with changes in glacial meltwater runoff (Moline et al., 2004).To date, little information on the contribution of small phytoplankton to primary production is available in the Antarctic Ocean (Saggiomo et al., 1998), especially in the Amundsen Sea with a rapid melting of ice shelf (Yager et al., 2012;Schmidtko et al., 2014).Thus, our main objective in this study is to determine contributions of small phytoplankton to the overall total biomass and primary production of phytoplankton in the Amundsen Sea for monitoring marine ecosystem responding to environmental condition change.
2 Materials and methods

Total and size-fractionated chlorophyll a concentration
Water samples for total and size-fractionated chlorophyll a concentrations of phytoplankton were obtained at the 12 productivity stations in the Amundsen Sea (Fig. 1) during the KOPRI Amundsen cruise from 1 to 15 January 2014 onboard the Korean research icebreaker Araon.Based on the sea ice concentration data from the National Snow and Ice Data Center during the cruise period in 2013 (Fig. 1), our study region was further separated into polynya and non-polynya areas for comparison.Four stations (St. 1,St. 2, among the 12 stations belong to the non-polynya region and the rest of the stations belong to the polynya region.St. 3 and St. 3-1 were on the fringe of the polynya area, experiencing approximately 30 % sea ice cover.Following the definition of polynya as an area of open water within sea ice zone, we grouped them into non-polynya regions.Six different light depths (100, 50, 30, 12, 5 and 1 % penetration of the surface irradiance, PAR) were determined with an LI-COR underwater 4π light sensor.Total chlorophyll a concentrations were measured at the six different light depths (100, 50, 30, 12, 5 and 1 % of PAR).For size-fractionated chlorophyll a concentrations, water samples were collected at three light depths (100, 30 and 1 %).Water samples (0.3-0.5 L) for total chlorophyll a concentrations were filtered using Whatman glass fiber filters (GF/F; 25 mm).For different size-fractionated chlorophyll a concentrations water samples (0.7-1 L) were passed sequentially through 20 and 5 µm Nucleopore filters (47 mm) and 0.7 µm GF/F filters (47 mm).7.9 ± 3.5 36.9 ± 4.6 37.0 ± 6.9 14.9 ± 8.4 18.1 ± 6.8 31.6 ± 10.1 23.5 ± 6.0 After the filters were extracted using the method described by Kim et al. (2015), all chlorophyll a concentrations were subsequently determined onboard using a Trilogy fluorometer (Turner Designs, USA).The methods and calculations for chlorophyll a were based on Parsons et al. (1984).

Carbon and nitrogen uptake experiments
Water samples were collected for and carbon and nitrogen uptake measurements of phytoplankton.Using a dual stable isotope technique (Lee et al., 2012;Kim et al., 2015), the experiments of carbon and nitrogen uptake rates of phytoplankton were conducted at 12 selected productivity stations including two revisited stations (St.3-1 and St. 19-1) when on-deck incubations were available during daytime at oceanographic survey stations.Water samples from the six light depths for the uptake experiments were obtained from a CTD-rosette sampler system equipped with twenty-four 10 L Niskin bottles.Water sample from each light depth was transferred into different screened polycarbonate incubation bottles (1 L) which matched each light depth.The bottles were placed in large polycarbonate incubators cooled with running surface seawater on deck under natural light conditions for 4-5 h, after the water samples in the incubation bottles were inoculated with labeled carbon (NaH 13 CO 3 ) and nitrate (K 15 NO 3 ) or ammonium ( 15 NH 4 Cl) substrates.After 4-5 h incubations, the incubated waters were well mixed and distributed into two filtration sets for the carbon and nitrogen uptake rates of total (> 0.7 µm) and small-sized cells (0.7-5 µm).Small-sized cells are generally defined as small phytoplankton in comparison to large diatoms (> 5 µm) (Robineau et al., 1994;references therein).The incubated waters (0.3 L) for total uptake rates were filtered through pre-combusted GF/F filters (25 mm diameter), whereas waters samples (0.5 L) for the uptake rates of small phytoplankton were passed through 5 µm Nuclepore filters (47 mm) to remove large phytoplankton cells (> 5 µm) and then the filtrate was passed through pre-combusted GF/F (25 mm) for the small phytoplankton (Lee et al., 2013).The values for large phytoplankton in this study were obtained from the difference between small and total fractions (Lee et al., 2013).The filters were immediately preserved at −80 • C until mass spectrometric analysis.After acid fuming overnight to remove carbonate, the concentrations of particulate organic carbon (POC) and nitrogen (PON) and the abundance of 13 C and 15 N were determined by a Finnigan Delta+XL mass spectrometer at the Alaska Stable Isotope Facility, USA.All contribution results of small phytoplankton in this study were estimated from comparison of small phytoplankton to total phytoplankton integral values from 100 to 1 % light depth at each station based on the trapezoidal rule.Daily carbon and nitrogen uptake rates of phytoplankton were based on our hourly uptake rates measured in this study and a 24 h photoperiod per day during the summer period in the Amundsen Sea (Lee et al., 2012).
The chlorophyll a contribution of small phytoplankton was larger in the non-polynya region than in the polynya region although they were not significantly different (t test, p>0.05).
in the non-polynya region, whereas it was 654.8-1213.4mg C m −2 d −1 (837.3 ± 184.1 mg C m −2 d −1 ) in the polynya region.The total daily carbon uptake rates of phytoplankton were significantly higher (t test, p<0.05) in the polynya region than the non-polynya region.The rate of small phytoplankton was 58.6-193.6 mg C m −2 d −1 (126.5 ± 55.2 mg C m −2 d −1 ) in the non-polynya region, whereas it was 62.2-266.4mg C m −2 d −1 (124.1 ± 69.3 mg C m −2 d −1 ) in the polynya region.The daily carbon uptake rates of small phytoplankton were not significantly different (t test, p>0.05) between the polynya and non-polynya stations.The average contributions of small phytoplankton to total daily carbon uptake rates were 50.8 % (±42.8 %) and 14.9 % (±8.4 %), respectively, for the non-polynya and polynya regions (Table 1).The average contributions were largely different between the polynya and non-polynya regions but they were not statistically significant (t test, p>0.05).

Discussion and conclusion
The total daily carbon uptake rates of phytoplankton averaged for the non-polynya and polynya regions were 0.42 g C m −2 d −1 (±0.30g C m −2 d −1 ) and 0.84 g C m −2 d −1 (±0.18 g C m −2 d −1 ), respectively, in this study.According to the previous reports in the Amundsen Sea (Lee et al., 2012;Kim et al., 2015), the total daily carbon uptake rates ranged from 0.2 to 0.12 g C m −2 d −1 in the non-polynya region.Our rate (0.42 g C m −2 d −1 ) in the non-polynya region is somewhat higher than those reported previously but they are not significantly different (t test, p>0.05).In comparison, our total daily carbon uptake rate in the polynya region (0.84 g C m −2 d −1 ) is lower than that (2.2 g C m −2 d −1 ) of Lee et al. (2012) and higher than that (0.2 g C m −2 d −1 ) of Kim et al. (2015).The carbon uptake rates of phytoplankton in Lee et al. (2012) and Kim et al. (2015) were measured during the periods 21 December 2010 to 23 January 2011 and 11 February to 14 March 2012, respectively.Our measurements in this study were executed mainly during the period 1-15 January 2014.For the Amundsen polynya region, a large seasonal variation in the total daily carbon uptake rate of phytoplankton was already reported by Kim et al. (2015) and Arrigo et al. (2012) based on field-measured data and satellitederived approach, respectively.Generally, late December is the time of peak uptake rate in this region (Arrigo et al., 2012).Previous studies reported that the total daily nitrogen uptake rates in non-polynya region were 0.24 g N m −2 d −1 during the period 21 December 2010 to 23 January 2011 and 0.04 g N m −2 d −1 during the period 11 February to 14 March 2012, whereas the uptake rates in polynya region were 0.93 g N m −2 d −1 in 2010/2011 and 0.06 g N m −2 d −1 in 2012 in the Amundsen Sea (Lee et al., 2012;Kim et al., 2015).Our total daily nitrogen uptake rates of phytoplankton in non-polynya (0.12 ± 0.09 g N m −2 d −1 ) and polynya regions (0.21 ± 0.11 g N m −2 d −1 ) were between the ranges of two previous studies (Lee et al., 2012;Kim et al., 2015).
Based on the nitrate and ammonium uptake rates in this study, f ratios (nitrate uptake rate/nitrate + ammonium uptake rates) averaged for non-polynya and polynya regions were 0.62 (±0.08) and 0.54 (±0.20), respectively.These ratios were also between the ranges of two previous studies.Although they were not significantly different because of a large spatial variation, larger f ratios in non-polynya than in polynya region are consistent with the results of the previous studies (Lee et al., 2012;Kim et al., 2015).At this point, we do not have a solid explanation for that and a further future study is needed for the higher f -ratio mechanism in nonpolynya region.
The percentage contributions of small phytoplankton to chlorophyll a, POC/PON, daily carbon and nitrogen uptake rates are shown in Table 1.The result of significantly higher chlorophyll a contribution than the POC contribution of small phytoplankton is consistent with the result in the Chukchi Sea, Arctic Ocean reported by Lee et al. (2013).
They explain that higher POC content per chlorophyll a unit of small phytoplankton could have caused the higher POC contribution in their study (Lee et al., 2013).Given C / N ratio (6.6 ± 0.6) and δ 13 C (−25.9 ± 1.0 ‰) of sample filters attained for POC and PON in this study, our filtered samples are believed to be mainly phytoplankton-originated POC and PON (Kim et al., 2016).Thus, a significant potential overestimated contribution of POC caused by non-phytoplankton materials could be excluded for the higher POC contribution than chlorophyll a contribution of small phytoplankton.Therefore, small phytoplankton contributions based on conventional assessments of chlorophyll a concentration might lead to an underestimated contribution of small phytoplankton (Lee et al., 2013).In fact, several authors argue that chlorophyll a concentration might not be a good index for phytoplankton biomass since it depends largely on environmental factors such as nutrient and light conditions, as well as dominant groups and physiological status of phytoplankton (Desortová, 1981;Behrenfeld et al., 2005;Kruskopf and Flynn, 2006;Behrenfeld and Boss, 2006).However, the effects of non-phytoplankton carbon materials such as extracellular carbon mucilage cannot be completely excluded for the POC contribution as discussed below.
The overall contributions of carbon (26.9 %) and nitrogen (27.7 %) uptake rates of small phytoplankton at all the productivity stations in this study are relatively higher than the chlorophyll a contribution of small phytoplankton but they are not statistically different (t test, p>0.05).In general, the contribution of daily ammonium uptake rate of small phytoplankton is significantly (t test, p<0.05) higher than the contribution of daily nitrate uptake rate of small phytoplankton at all the stations in this study.This is well known for the ammonium preference of small phytoplankton in various regions (Koike et al., 1986;Tremblay et al., 2000;Lee et al., 2008Lee et al., , 2013)).
In terms of the contributions in different regions, all the contributions (chlorophyll a, POC/PON, carbon and nitrogen uptake rates) of small phytoplankton were higher in the non-polynya region than in the polynya region (Table 1).In addition, the chlorophyll a contribution of small phytoplankton (7.9 ± 3.5 %) was significantly (t test, p<0.05) lower than the POC contribution (36.9 ± 4.6 %) in the polynya region, whereas they were not statistically different in the non-polynya region (Table 1).This indicates that small phytoplankton contributed more to the total POC than to the chlorophyll a concentration in the polynya region.We do not have species compositions of phytoplankton in this study, but previous results reported that Phaeocystis spp.are dominant in the Amundsen Sea polynya region (Lee et al., 2012).Generally, Phaeocystis spp. release a large amount (up to 46 %) of extracellular carbon mucilage which constitutes their colonial form (Matrai et al., 1995).This non-phytoplankton carbon material without chlorophyll a might have caused a high POC contribution of small phytoplankton in the polynya region in this study.In fact, the contribution of the daily carbon www.biogeosciences.net/14/3705/2017/Biogeosciences, 14, 3705-3713, 2017

Table 1 .
Percentage contributions (%) of small phytoplankton to depth-integrated total concentrations of chlorophyll a, POC, PON and carbon and nitrogen uptake rates in the Amundsen Sea.