Plankton recruitment from coastal sediments under different temperature and light treatments

In highly seasonal systems, the emergence of planktonic resting stages from the sediment is a key driver for bloom timing and plankton community composition. The termination of the resting phase is often linked to environmental cues, but the extent to which recruitment of resting stages is affected by climate change remains largely unknown for coastal environments. Here we investigate phyto- and zooplankton recruitment from oxic sediments in the Baltic Sea in a controlled experiment under proposed temperature and light increase during the spring and summer. We find that emergence of resting stage differs between seasons and the abiotic environment. Phytoplankton recruitment from resting stages were high in spring with significantly higher emergence rates at increased temperature and light levels for dinoflagellate and cyanobacteria than for diatoms, which had highest emergence under cold and dark conditions. In comparison, copepod hatchlings were most abundant in summer and emergence was not affected by increased temperature and light levels. These results show that activation of plankton resting stages are affected to different degrees by increasing temperature and light levels, indicating that climate change affects plankton dynamics through processes related to resting stage termination with potential consequences for bloom timing, community composition and trophic mismatch.

Methods
Twenty-four sediment cores were collected with a Multicorer (K.U.M. Umwelt und Meerestechnik, Kiel GmbH), fitted with 4 plexiglass tubes measuring 10 cm (inner diameter) and subsampled with plexiglass tubes (25 cm high, 7.4 cm inner diameter with a surface area of 0.004 m2) from a depth of 21 m in the northern Baltic Sea proper (58°50´N, 17°33´E). The same tubes were used for the microcosm experiments. The ratio of sediment to overlaying bottom water (451 ml) in each subsample was about 1:1. The smaller tubes (hereafter called microcosms) were kept cold and dark from collection until being placed in a temperature-controlled room set to in situ bottom temperature for 24 h to allow any suspended material to settle to the bottom. Before deployment of the multicorer, bottom water was collected with a Niskin bottle, and temperature, oxygen and light intensity was measured at 20 m depth by use of a CTD (Sea & Sun Technology GmbH). Sampling was conducted once in the late June 2019 (summer) and early March 2020 (spring). Light and oxygen measurements at 20 m were 2.73 µE m-2 and 7.26 ml l-1 in spring, and 1.69 µE and 8.83 ml l-1 in summer, respectively. In comparison, light values below the water surface were 471 µE m-2 in spring and 1042.46 µE m-2 in summer, respectively. 

Before the experimental start, water overlaying the sediment was siphoned off with syringes as close as possible to the sediment, leaving approximately 5 mm of water. Siphoning off the initial water did, however, not completely remove adult Eurytemora individuals in summer (Fig. S1). Thus, the experiment does not allow for separation of Eurytemoranauplii recruitment from the sediment or hatching from subitaneous eggs and are excluded from the analysis. Sterile, filtered (0.2 mm) bottom water from the collection site was drip-fed onto polystyrene foam disc, placed in each microcosm, to avoid resuspension of the sediment. After re-filling the microcosms with water, they were covered with parafilm with small holes in order to minimize evaporation but also to allow for gas exchange and light aeration through a single thin nylon tube per microcosm. Care was taken to allow minimal aeration of the upper few centimeters of the water to avoid any resuspension of the sediment. The 24 microcosms were placed in incubators divided into four abiotic factor treatments, with six replicates each: control CTRL was kept dark and at in situ bottom temperature (3 °C in spring and 9 °C in summer), elevated temperature (T), kept dark and subjected to an increase of +2 °C, light (L), subjected to the same temperature as CTRL and to weak green light emitting 0.5 mmol m-2 s-1 (Nascimento et al., 2008), and a combination of elevated temperature of +2 °C and weak green light (T+L). Treatment T was covered with a thick, dark plastic sheet to block all possible light from entering. A +2 °C temperature increase was based on average projected temperature increase for the Baltic Sea by the end of this century and weak green light representing underwater light change due to loss of sea ice (HELCOM, 2021).

Incubation of the experiment lasted for two weeks and samples were taken at day 7 and day 14 of the experiment. For this, the overlaying water in each microcosm was removed with syringes, treated with acid Lugol’s solution and placed in clear flasks covered with parafilm to prevent evaporation. Samples were allowed to settle for 6 days, reduced to < 100 ml by siphoning off the water surface and the remaining part transferred to clear 100 ml PVC bottles for storage at 4 °C. At the end of the experiment, sediment from all microcosms was sieved through a 500 mm mesh for identification of benthic macrofauna to determine potential impacts through bioturbation or predation, which did not differ between treatments. All macrofauna retrieved was living, suggesting that the sediment remained well oxygenated throughout the experimental duration. 

Identification and quantification of phytoplankton cells followed the Utermöhl method (Edler and Elbrächter, 2010) with a sedimentation chamber of 100 ml. All samples were allowed to settle for 48 h according to standard guidance (HELCOM, 2017). Phytoplankton were identified to either class (dinoflagellates < 20 mm), order (dinoflagellates > 30 mm), genus (diatoms and cyanobacteria) or dinoflagellate cysts, using http://nordicmicroalgae.org/galleries/HELCOM-PEG (2021). Phytoplankton cell density was calculated per L and carbon content per m2 for comparison with plankton monitoring data and flux estimations from benthic to pelagic systems, respectively. Carbon content are based on Olenina et al. (Olenina et al., 2006). Due to the relatively high taxonomic level of dinoflagellates and to avoid overestimation of C content, all C content calculations for cells > 30 mm, was based on a size class of cells with a size < 30 mm. Zooplankton were identified to genus level using Telesh et al. (2015). Nauplii biomass was calculated on naupliar stages I-III based on Gorokhova et al. (2016).

在高度季节性的生态系统中，浮游生物休眠体从沉积物中的萌发是水华发生时间与浮游生物群落组成的关键驱动因素。休眠期的结束通常与环境信号相关，但就沿海生境而言，气候变化对休眠体补充过程的影响程度仍基本不明。本研究于春夏季在预设升温与增光的受控实验中，探究了波罗的海含氧沉积物中浮游植物与浮游动物的休眠体补充过程。研究发现，休眠体的萌发过程存在季节差异，且受非生物环境调控。春季浮游植物从休眠体的补充量较高：甲藻（dinoflagellate）与蓝细菌（cyanobacteria）在升温与增光条件下的萌发率显著高于硅藻（diatoms）；而硅藻在低温黑暗环境下的萌发率最高。相比之下，桡足类幼体在夏季最为丰富，其萌发过程不受升温与增光的影响。上述结果表明，浮游生物休眠体的激活过程受升温与增光的影响程度存在差异，这意味着气候变化可通过调控休眠期结束相关过程影响浮游生物动态，进而可能对水华发生时间、群落组成及营养级错配产生潜在影响。

方法

24根沉积物柱样通过多管采泥器（Multicorer，K.U.M. Umwelt und Meerestechnik基尔有限公司）采集，该采泥器配备4根内径10 cm的有机玻璃管（plexiglass tubes）；随后于波罗的海北部本部海域（58°50′N，17°33′E）水深21 m处，使用尺寸为25 cm高、内径7.4 cm（表面积0.004 m²）的有机玻璃管进行分样。同一批有机玻璃管用于后续的微宇宙实验（microcosm experiments）。每个分样中沉积物与上覆底层水（体积451 ml）的比例约为1:1。小型分样管（以下简称微宇宙体系）在采集后全程置于低温黑暗环境中，直至转移至设定为原位底层水温的控温室静置24小时，使悬浮物质充分沉降。部署采泥器前，使用尼斯金采水器（Niskin bottle）采集底层水，并通过CTD（Sea & Sun Technology GmbH）测定水深20 m处的水温、溶解氧与光照强度。采样分别于2019年6月下旬（夏季）与2020年3月上旬（春季）各开展一次。春季水深20 m处的光照强度为2.73 µE·m⁻²，溶解氧浓度为7.26 ml·L⁻¹；夏季对应数值分别为1.69 µE与8.83 ml·L⁻¹。相比之下，春季与夏季水面下的光照强度分别为471 µE·m⁻²与1042.46 µE·m⁻²。

实验开始前，使用注射器尽可能贴近沉积物表面抽吸上覆水，仅保留约5 mm厚的水层。但抽吸初始上覆水无法完全去除夏季样本中的成体真哲水蚤属（Eurytemora）个体（补充图S1），因此本实验无法区分真哲水蚤无节幼体是来自沉积物中休眠体的补充，还是从暂存卵孵化而来，故该类数据被排除在后续分析之外。将采集自采样点的无菌过滤（0.2 mm）底层水滴加至置于每个微宇宙体系中的聚苯乙烯泡沫圆盘上，以避免沉积物被扰动悬浮。重新向微宇宙体系注水后，用扎有小孔的封口膜（parafilm）覆盖，以减少水分蒸发，同时通过每个微宇宙体系的单根细尼龙管进行气体交换与曝气，并保证光照透过。操作过程中尽量减少对水体上层几厘米的曝气，以避免沉积物再次悬浮。将24个微宇宙体系置于分隔为4组非生物因子处理的培养箱中，每组设置6个重复：①对照组（CTRL）：保持黑暗，维持原位底层水温（春季3℃，夏季9℃）；②升温组（T）：保持黑暗，水温升高+2℃；③增光组（L）：维持对照组相同水温，施加光照强度为0.5 mmol·m⁻²·s⁻¹的弱绿光（Nascimento等，2008）；④升温增光组合组（T+L）：同时施加+2℃升温与弱绿光处理。升温组（T）用厚黑色塑料布覆盖，以阻挡所有光线进入。本次+2℃的升温幅度基于波罗的海至本世纪末的预测平均升温值，而弱绿光则代表海冰消融导致的水下光环境变化（赫尔辛基海洋环境委员会，HELCOM，2021）。

实验培养周期为两周，分别于实验第7天与第14天采集样本。采集时使用注射器抽取每个微宇宙体系中的上覆水，加入酸性鲁格氏液（Lugol’s solution）固定后，转移至封口膜覆盖的透明烧瓶中以防止蒸发。样本静置沉降6天后，通过抽吸水面上层水体将体积浓缩至<100 ml，随后将剩余样品转移至100 ml透明PVC瓶中，于4℃下保存。实验结束后，将所有微宇宙体系中的沉积物过500 mm筛网，以分离底栖大型动物（benthic macrofauna），用于评估生物扰动或捕食作用的潜在影响，结果显示各组间无显著差异。回收的所有大型底栖动物均为活体，表明实验全程沉积物始终保持良好的含氧状态。

浮游植物细胞的鉴定与定量采用乌特莫尔沉降法（Utermöhl method，Edler与Elbrächter，2010），使用100 ml沉降室。所有样本均按照标准规程静置沉降48小时（赫尔辛基海洋环境委员会，HELCOM，2017）。浮游植物的分类阶元包括：甲藻按体长分为<20 mm的类群（归为纲级）与>30 mm的类群（归为目级），硅藻与蓝细菌鉴定至属级，同时可鉴定甲藻孢囊，分类参考来源为http://nordicmicroalgae.org/galleries/HELCOM-PEG（2021）。浮游植物细胞密度以每升为单位计算，碳储量以每平方米为单位计算，分别用于与浮游生物监测数据及底栖-浮游系统间的碳通量估算结果进行对比。碳储量计算参考Olenina等（2006）的方法。考虑到甲藻的分类阶元相对较粗，为避免碳储量估算高估，所有>30 mm的甲藻细胞的碳含量均基于<30 mm的细胞尺寸类别进行计算。浮游动物鉴定至属级，参考Telesh等（2015）的方法。无节幼体的生物量基于I-III期无节幼体，参考Gorokhova等（2016）的方法进行计算。