Bivalve facilitation mediates seagrass recovery from physical disturbance in a temperate estuary

Biomass cores were taken in May 2019 and July 2019 for Experiment 1 and June 2019, July 2019, and June 2020 for Experiment 2 and analyzed for Z. marina and H. wrightii shoot density, aboveground biomass, and belowground biomass. For Experiment 1, cores were taken near the center of each plot. For Experiment 2, cores from the first year of the experiment were taken from the outside edge of subplots to determine the effects of disturbance and clam addition on neighboring shoots. Cores taken in June 2019 were collected from early-season disturbance subplots that had been disturbed 1.5 months earlier; July 2019 cores were collected from late-season disturbance subplots that had been disturbed 1 month earlier. For all core samples, we used a metal corer with a diameter of 10 centimeters that was pushed into the sediment to a depth of 15 centimeters. The core was extracted and sieved in the field to remove sediment, shells, and faunal biomass. The cores were stored in mesh or Ziploc bags in a freezer until processing; all cores were processed within sixty days. To process, cores were thawed under warm running water and then carefully separated into the following categories: aboveground Z. marina biomass, aboveground H. wrightii biomass, and belowground biomass from both species as it was not possible to distinguish belowground biomass between the two species. Shoot count for each species was recorded for each core. Biomass samples were dried in a Fisher Scientific 180L Gravity Convection Oven at 60°C until fully dry and then weighed. Summer growth rates were sampled in 2018 and 2019 for Experiment 1 and 2019 for Experiment 2. To quantify Z. marina growth rate, we used the leaf marking technique first put forth by Zieman (1974) and modified by Dennison (1990). A random location was selected within the treatment plot for Experiment 1 and along the outside edge of the subplots for Experiment 2 and all Z. marina shoots within a three-inch diameter of that random location were pricked completely through the sheath below the meristem. Marked shoots were collected fourteen days later and brought back to the University of North Carolina’s Institute of Marine Science (UNC IMS) where they were processed within twenty-four hours. Up to five shoots were used for each treatment plot or subplot per sampling point. New growth was defined as any tissue below the scar created from the push pin puncture and separated from the old biomass. Belowground biomass was discarded. Samples were dried in a Fisher Scientific 180L Gravity Convection Oven at 60°C until fully dry and then weighed. The growth rate (GR) was calculated as: GR=New Biomass (g)Number of Shoots*Days Between Pricking and Collection (Eq 1) Due to the small size of H. wrightii, it was not possible to mark leaves and thus we used the clipping method from Virnstein (1982). A location within the plot for Experiment 1 or along the outside edge of the subplot for Experiment 2 was selected randomly during each sampling point and the shoots were trimmed with scissors flush to the sediment in a triangular area roughly ~40 cm2. The trimmed area was marked and we returned after fourteen days to collect trimmed shoots for processing in the lab. Up to five shoots per plot or subplot were processed. Growth was determined from the average height of all processed shoots. For Experiment 1, we estimated the epiphytic load on Z. marina and H. wrightii shoots using epiphytic Chlorophyll A as a proxy (see Parsons et al. 1984) in May 2018, July 2018, May 2019, June 2019, and July 2019. We haphazardly selected four individual seagrass blades of each species from each plot (except for Z. marina during the July 2018 and July 2019 sampling points, where only one shoot was collected due to very few Z. marina shoots remaining in the meadow and the high load of epiphytic biomass on the Z. marina shoots by this point in the summer). Shoots were carefully floated into a Ziploc bag with a small amount of seawater. Samples were stored in a cool, dark container for transport to UNC IMS and processed within twenty-four hours. In the laboratory, each sample was transferred to a sorting pan with a small amount of filtered seawater. Blades were carefully scraped to remove all epiphytes using a glass microscope slide and the total surface area of each blade was recorded. The epiphytes and seawater were vacuum-filtered through a Whatman GF/F 0.7μ filter and frozen for no longer than eight weeks until they could be extracted. The filters were sonicated in 90% Acetone for sixty seconds and extracted for 12-24 hours in a freezer. Chlorophyll A concentrations were measured on a Turner Designs Trilogy Laboratory Fluorometer and chlorophyll concentrations were normalized to seagrass surface area. In Experiment 2, we estimated seagrass percent cover to assess seagrass regrowth into experimentally disturbed areas and compare it to percent cover of non-disturbed areas with and without clam additions. Percent cover of subplots was recorded three times in the fall of 2019 (September 3rd, September 16th, and October 2nd) and once in the spring of 2020 (June 6th). Between the two September sampling points, Hurricane Dorian made landfall along the North Carolina coast as a Category 1 hurricane on September 6th, 2019.

本数据集的生物量柱样（biomass cores）于2019年5月、2019年7月（实验1），以及2019年6月、2019年7月、2020年6月（实验2）采集，用于分析大叶藻（Zostera marina，缩写Z. marina）和海韭（Halodule wrightii，缩写H. wrightii）的枝条密度、地上生物量与地下生物量。实验1的柱样采集于每个样方的中心区域；实验2第一年的柱样则取自亚样方的外缘，以探究干扰与添加蛤类对邻近枝条的影响。2019年6月采集的柱样来自于1.5个月前刚完成早期季候干扰的亚样方，2019年7月的柱样则取自1个月前实施后期季候干扰的亚样方。所有柱样均采用直径10厘米的金属采样器，将其推入沉积物至15厘米深度后取出。采样现场即对柱样进行过筛，以去除沉积物、贝壳与动物生物量。柱样被装入网袋或Ziploc保鲜袋后置于冰柜储存，所有样品均在60天内完成处理。处理时，先将柱样置于温流水下解冻，随后仔细分为以下类别：大叶藻地上生物量、海韭地上生物量，以及两种植物的地下生物量（因无法区分二者的地下组织）。记录每个柱样中各物种的枝条数量。生物量样品置于Fisher Scientific 180L重力对流烘箱中，以60℃烘干至恒重后称重。实验1与实验2的夏季生长速率分别于2018、2019年，以及2019年进行采样。对于大叶藻生长速率的量化，采用Zieman（1974）提出、Dennison（1990）改良的叶片标记法。实验1随机选取处理样方内的点位，实验2则选取亚样方外缘的点位，将该点位周围直径3英寸范围内的所有大叶藻枝条，在分生组织下方的叶鞘处完全刺穿。标记后的枝条于14天后采集，送至北卡罗来纳大学海洋科学研究所（University of North Carolina’s Institute of Marine Science, UNC IMS），并在24小时内完成处理。每个处理样方或亚样方的每个采样点最多选取5根枝条。新生长组织定义为穿刺造成的疤痕下方的所有组织，并与旧生物量分离，舍弃地下生物量。样品同样置于Fisher Scientific 180L重力对流烘箱中以60℃烘干至恒重后称重。生长速率（GR）计算公式如下：$GR = frac{ ext{新生物量(g)}}{ ext{枝条数} imes ext{穿刺与采集间隔天数}}$（公式1）。由于海韭个体尺寸较小，无法进行叶片标记，因此采用Virnstein（1982）提出的修剪法。每个采样点随机选取实验1的样方内或实验2的亚样方外缘点位，用剪刀将约40cm²三角形区域内的枝条齐沉积物表面修剪。标记修剪区域后，14天后返回采集修剪后的枝条用于实验室处理。每个样方或亚样方最多处理5根枝条。生长速率通过所有处理枝条的平均高度计算。实验1于2018年5月、2018年7月、2019年5月、2019年6月、2019年7月，采用附生叶绿素a（Chlorophyll A）作为替代指标，估算大叶藻与海韭枝条上的附生生物负载（参考Parsons等1984年的方法）。从每个样方中随机选取4片各物种的海草叶片（但2018年7月与2019年7月的采样中，由于该季节海草床中大叶藻枝条数量极少，且其表面附生生物负载极高，因此仅采集1根枝条）。将枝条轻轻放入装有少量海水的Ziploc保鲜袋中，样品置于阴凉避光容器中运输至UNC IMS，并在24小时内完成处理。实验室中，将每个样品转移至装有少量过滤海水的分选盘中，用玻璃显微镜刮片小心刮除叶片上的所有附生物，并记录每片叶片的总表面积。附生物与海水通过Whatman GF/F 0.7μ滤膜真空过滤，滤膜置于冰柜储存不超过8周后再进行提取。将滤膜置于90%丙酮中超声处理60秒，随后在冰柜中提取12-24小时。采用Turner Designs Trilogy实验室荧光计测定叶绿素a浓度，并将叶绿素浓度标准化至海草表面积。实验2中，通过估算海草盖度，评估海草向实验干扰区域的恢复情况，并对比有无添加蛤类的干扰与非干扰区域的盖度。亚样方的盖度分别于2019年秋季（9月3日、9月16日、10月2日）采样3次，2020年春季（6月6日）采样1次。在两次9月采样之间，飓风多里安（Hurricane Dorian）于2019年9月6日以1级飓风强度登陆北卡罗来纳州海岸。