Solitary ascidians found on complex settlement plates in the Red Sea and the Mediterranean

We constructed unique settlement plates, each representing six different niches, to assess ascidian niche breadth, and deployed them in similar habitats in the Red Sea and the Mediterranean.
Settlement plate design:
To estimate and compare habitat niche breadth between species, we designed settlement plates each representing different niches, based on six substrate squares measures 10 x 10 cm as our basic units. The base of the plate was built from stainless steel, and the substrate squares were glued to the base. We used three substrate types within two current /light regimes to allocate six different "niches” to each settlement plate, with total measurement of 30 x 20 cm. The three substrate types were constructed from three materials: cement with sea shells, non-glazed ceramic and recycled plastic. Since substrate is a major factor for fouling species, we selected three materials that differ substantially from each other: plastic is smooth, the non-glazed ceramic is rough but homogenous, and the cement with sea shells is complex and heterogeneous. The two current and light regimes were achieved by placing the plates facing the pillars, and leaving the upper part of the plate open from three directions (from above and from either side) while the lower part remained open from the bottom only and was thus dark and with restricted water flow. We were interested in the relative patterns across species to these niche differences, and hence do not attempt to quantify the exact differences in light and flow between the upper and lower sections. Nevertheless, light measurements confirm the lower side was ~10 time darker than the upper side. We used all possible permutations (36 in total) of substrate type ordering within each current and light regime treatment in constructing the settlement plates.
Study sites:
The same experimental designs were used in the eastern Mediterranean Sea and northern Red Sea to facilitate direct comparisons. In the Mediterranean, the settlement plates were deployed on three pillars of the Israel Electric Company pier (32°28′ N 34°53 E), and in the Red Sea, on three pillars of the Israeli oil port (29°31′ N 34°56′ E), with minimal distance of 10 meters between pillars. At both sites, the plates were deployed at about 15 meters depth, with the seabed at 20 meters, in order to avoid any bottom effects such as sedimentation. The depth of 15 meters was chosen in order to minimize disturbance to the experiment by the strong winter storms, while still providing sufficient underwater work time when using scuba. As public entrance to both sites is prohibited, the experiment was subjected to minimal human disturbance. Pillars at both sites are located in the open sea (as opposed to closed harbors) and are >30 years old, therefore the fauna found on them represent a climax community.
Study design:
The experiment lasted one year, from February 2014 to February 2015. For analyses, we combined two types of plates, seasonal and full-year. We deployed 15 full-year plates at each site and these remained undisturbed for the entire year of the experiment. In addition, 10 seasonal plates were replaced every three months, totaling 40 seasonal settlement plates at each site. At the end of each experiment, the settlement plates were removed and taken to the lab for taxonomic identification (using Nikon SMZ18 stereomicroscope and dissection tools). Solitary ascidians were counted and identified to species level where possible (649 out of 658 individuals). In addition, we took monthly underwater photos of all settlement plates. Plates were photographed from the exact same distance and angle using a custom-made tripod. These photos were used in order to identify individuals that were present on the plates during the experiment but did not survive to the point of plate removal, in order to increase sample size for the niche breadth calculations. Unfortunately, only four individuals were added using these photos.
We categorized the species found in the Red Sea as Lessepsian (species that are known to establish populations in the Mediterranean) or non-Lessepsian species (species that have not yet been recorded in the Mediterranean, i.e., non-invaders). In the Mediterranean we categorized the species as non-indigenous or indigenous. We included Styela plicata with the indigenous species of the Mediterranean for analysis although it possibly invaded from the Atlantic Ocean (Pineda et al., 2011; Maltagliati et al., 2015) as it is clearly not of tropical origin, unlike the rest of the non-indigenous species, and has been found in the Mediterranean for at least a century (de Barros et al., 2009).
Data file structure:
plate – id for each settlement plate (reminder: the six different habitats described above are within each plate).
site – Mediterranean or Red Sea
species
type – ‘Lessepsian’ and ‘non Lessepsian’ for the species found on plates in the Red Sea site. ‘indigenous’ and ‘non-indigenous’ for species found on plates in the Mediterranean site.
season – ‘spring’, ‘summer’, ‘autumn’, ‘winter’ for the seasonality plates. ‘long term’ for the long term (full year) plates. And ‘added by pictures’ for three records of P. nigra we added in order to increase its sample size for our niche breadth calculations.
niche – ‘su’ for shells-up, ‘cu’ for ceramics-up, ‘pu’ for plastic-up, ‘sd’ for shells down etc…
substrate – shells, ceramics or plastic
light – up – upper part of the plate or down – the lower part of the plate.
number of individuals – the actual data – number of individuals found

本研究构建了专属的附着基盘（settlement plate），每个基盘包含6种不同生态位，用于评估海鞘（ascidian）的生态位宽度，并将其部署于红海与地中海的相似生境中。

附着基盘设计方案：
为估算并比较不同物种的生境生态位宽度，本研究以6块10×10 cm的基底正方形为基本单元，设计了可代表不同生态位的附着基盘。基盘主体采用不锈钢制成，基底正方形则通过黏合固定于主体之上。
本研究在两种水流/光照条件下设置3种基底类型，为每个附着基盘分配6种不同“生态位”，基盘整体尺寸为30×20 cm。3种基底类型分别由三种材料制成：带贝壳的水泥、无釉陶瓷以及回收塑料。
鉴于基底是附着生物的关键影响因子，本研究选取了三种差异显著的材料：塑料表面光滑，无釉陶瓷粗糙但质地均一，带贝壳的水泥则结构复杂且非均质。
两种水流与光照条件通过以下方式实现：将基盘朝向柱体安装，基盘上部从上方及两侧三个方向保持开放，而下部仅从底部开放，因此光照较弱且水流受限。
本研究聚焦于不同物种对这些生态位差异的相对响应模式，因此未尝试量化上下部之间光照与水流的精确差异。不过光照测量结果显示，基盘下部的光照强度约为上部的1/10（即下部比上部暗约10倍）。
在构建附着基盘时，本研究对每种水流/光照处理下的基底类型排序采用了全部可能的排列组合（共计36种）。

研究站点：
为便于直接对比，本研究在东地中海与北红海采用了相同的实验设计。在地中海区域，附着基盘部署于以色列电力公司码头的3根柱体上（北纬32°28′，东经34°53′）；在红海区域，则部署于以色列石油港的3根柱体上（北纬29°31′，东经34°56′），柱体间最小间距为10米。
两个站点的基盘均部署于约15米水深处，而海底深度为20米，以此避免沉降等底栖效应的干扰。选取15米水深的原因是，既可降低冬季强风暴对实验的干扰，又能保证水肺潜水时有充足的水下作业时间。
由于两个站点均禁止公众进入，实验受到的人为干扰极小。两处站点的柱体均位于开阔海域（而非封闭港湾），且已有超过30年的历史，因此柱体上附着的动物群落属于顶极群落。

实验设计：
实验周期为1年，自2014年2月至2015年2月。为开展分析，本研究结合了两种类型的基盘：季节性基盘与全年基盘。每个站点部署15块全年基盘，在整个实验周期内保持不更换。此外，每3个月更换10块季节性基盘，因此每个站点的季节性基盘总数为40块。
实验结束后，将附着基盘取回实验室，进行分类鉴定（使用尼康SMZ18体式显微镜及解剖工具）。对单个海鞘进行计数，并尽可能鉴定至物种水平（658个个体中成功鉴定649个）。
此外，本研究每月对所有附着基盘进行水下拍摄，使用定制三脚架确保每次拍摄的距离与角度完全一致。通过这些照片，可识别出实验期间存在于基盘上但在基盘取回时已死亡的个体，以此扩大生态位宽度计算的样本量。但遗憾的是，通过该方法仅补充了4个个体。
本研究将红海区域发现的物种分为莱塞普西亚种（Lessepsian），即已知可在地中海建立种群的物种，与非莱塞普西亚种（non-Lessepsian），即尚未在地中海有记录的物种，也就是非入侵物种。在地中海区域，则将物种分为非本土种与本土种。
尽管柄海鞘（Styela plicata）可能源自大西洋入侵（Pineda等，2011；Maltagliati等，2015），但鉴于其并非热带起源，且与其他非本土种存在明显差异，且已在地中海被记录超过一个世纪（de Barros等，2009），本研究将其归入地中海本土种进行分析。

数据文件结构：
plate：每个附着基盘的唯一标识（注：每个基盘内包含前文所述的6种不同生境）
site：站点类型，分为地中海（Mediterranean）与红海（Red Sea）
species：物种名称
type：物种类型，红海站点的物种分为莱塞普西亚种（Lessepsian）与非莱塞普西亚种（non Lessepsian）；地中海站点的物种分为本土种（indigenous）与非本土种（non-indigenous）
season：季节类型，季节性基盘分为"spring"（春季）、"summer"（夏季）、"autumn"（秋季）、"winter"（冬季）；全年基盘为"long term"（长期）；另有3条通过照片补充的黑尾海鞘（P. nigra）记录，归类为"added by pictures"，用于扩大该物种生态位宽度计算的样本量
niche：生态位标识，"su"代表贝壳朝上（shells-up），"cu"代表陶瓷朝上（ceramics-up），"pu"代表塑料朝上（plastic-up），"sd"代表贝壳朝下（shells down）等
substrate：基底材料，分为贝壳（shells）、陶瓷（ceramics）与塑料（plastic）
light：光照区域，分为"up"（基盘上部）与"down"（基盘下部）
number of individuals：个体数量，即实际观测到的个体总数