Lukas et al. 2021: Data from "Diurnal Changes in Hypoxia Shape Predator-Prey Interaction in a Bird-Fish System"

Topic: In many aquatic environments, daily changes in oxygen concentrations occur frequently. When oxygen levels drop to hypoxic levels, many fishes respond with aquatic surface respiration, during which they obtain oxygen by skimming the upper, oxygenated surface layer. By increasing time at the surface, fish become more vulnerable to fish-eating birds. We explored these cascading effects in a sulfidic spring system that harbors the endemic sulfur molly (Poecilia sulphuraria) as prey species and several fish-eating bird species. Study design: All observations stem from the sulphuric spring complex ‘Baños del Azufre’ in southern Mexico. To explore whether the observed diurnal differences in fish behavior are driven by physicochemical water conditions and how they link to predator activity, we conducted two field surveys and one laboratory experiment. During one field season (subsequently termed field survey I), we first quantified how variable physicochemical water conditions were throughout the day. In another field season (subsequently termed field survey II), we investigated the link between fish’s behavior and predatory bird activity. To link observations from both surveys, they were carried out at the same location and followed the same regimen by sampling each morning (07:00 – 09:30), midday (12:00 – 14:30) and afternoon (16:00 – 18:30) for six subsequent days. Surveys were matched for season (i.e., end of dry season; I: 12–17 April 2019, II: 05–10 May 2016) and we subsequently verified that no major deviations from the temperature trend occurred between years (see DATA_FieldSurvey_SI_Temp). DATA_FieldSurvey_WaterChem: Physico-chemical water parametersWe took multiple measurements of water temperature, dissolved oxygen (DO), pH and electrical conductivity (EC), and – due to logistical constraints – one sulfide sample along a cross section of the sulfidic stream. To capture variation introduced through flow regime and/or stratification within the water column, measurements were taken according to a transect grid (i.e., one sample each meter starting at 0.5 m away from the stream bench and at depths of 0.05, 0.25, and 0.5 m as water levels would allow). With a width of 5.6 m and a depth of less than 0.8 m at the site, this resulted in 10–11 subsamples per sampling. DATA_FieldSurvey_Fish: Sulphur mollies’ diving behavior We assessed fish behavior using a focal animal sampling approach. Twenty diving fish of similar size were chosen randomly from a focal shoal and observed from the moment they initiated diving until they resurfaced to calculate the mean dive duration. DATA_FieldSurvey_Birds: Predatory bird activityWe recorded all sightings of piscivorous birds in the predefined study area within a 30-min period. For each bird, we determined species (to lowest feasible taxonomical level), entry and exit times, as well as the number of attacks launched. We calculated presence time as the mean time piscivorous birds spent in the study area (excluding mere fly-throughs, i.e., flying through the transect without landing or attacking) and attacks as the total number of bird attacks launched in a sampling period. DATA_FieldSurvey_SI_Temp: temperature monitoring Air and water temperatures were continuously monitored for the duration of the 2016 field survey. Two HOBO Pendant data loggers (Onset Computer Corp.) were placed in a shaded area at the upstream edge of the study area: one into the water at a depth of 15 centimeters, another 1.5 meters above the water. Temperatures were recorded at a ten-minute interval. For the purpose of this study, we only analyzed temperatures recorded during observational periods of birds and fish (i.e. ~1h per sampling period). DATA_LabExp_ASR: ASR tendency in response to hypoxiaWe exposed fish to dissolved oxygen concentrations ranging from near-anoxic to normoxic conditions (0.6 – 5.1 mg/L DO) in a laboratory setting. We tested a total of 27 groups (5 adult individuals each; n = 135). A trial lasted 10 minutes, but we only analyzed the last 5 minutes to ensue fish had recovered from handling and resumed swimming. Data shown here is group-pooled. We quantified the cumulative time spent at the surface by all five fish and calculated a percentage surface time. We also assessed the total number of dives and mean dive duration performed by each group. Lukas2021_WaterChem.R (see Lukas2021_WaterChem_Report for PDF version) All analyses were performed in R (R Core Team, 2020, version 4.0.2).

研究主题：在众多水生环境中，溶解氧浓度常呈现每日周期性波动。当氧含量降至低氧（hypoxic）水平时，多数鱼类会启动水生表面呼吸（aquatic surface respiration, ASR）——即通过掠过水面上方富氧表层来获取氧气。但鱼类增加水面停留时间后，其被捕食性鸟类捕食的风险也会随之升高。本研究以栖息于硫化物泉系统中的特有硫化花鳉（Poecilia sulphuraria）作为猎物物种，搭配多种食鱼鸟类类群，探究这一生态级联效应。

研究设计：所有观测均开展于墨西哥南部的‘Baños del Azufre’硫化泉复合体中。为探明观测到的鱼类行为日差异是否由水体理化条件驱动，以及其与捕食者活动的关联，本研究开展了两次野外调查与一项室内控制实验。
在首个野外季（后续记为野外调查I）中，我们首先量化了一日内水体理化条件的波动情况；在另一野外季（后续记为野外调查II）中，我们探究了鱼类行为与捕食性鸟类活动间的关联。为关联两次调查的观测结果，二者在同一地点开展，并遵循统一采样方案：于每日清晨（07:00–09:30）、正午（12:00–14:30）与午后（16:00–18:30）进行采样，连续开展6天。两次调查的季节匹配（均为旱季末期；调查I：2019年4月12–17日，调查II：2016年5月5–10日），且后续验证了两年间水温趋势无显著偏差（详见DATA_FieldSurvey_SI_Temp）。

DATA_FieldSurvey_WaterChem：水体理化参数
我们对水温、溶解氧（dissolved oxygen, DO）、pH值与电导率（electrical conductivity, EC）进行了多次测量；受后勤条件限制，仅沿硫化溪流断面采集了一份硫化物样品。为捕捉水流动态及/或水柱分层带来的变异，我们按照断面网格方案开展采样：从距离溪岸0.5米处开始，每间隔1米采集1份样品，同时根据水位条件分别在0.05米、0.25米与0.5米深度处取样。该采样点溪流宽度为5.6米，水深不足0.8米，因此每次采样可获得10–11份子样品。

DATA_FieldSurvey_Fish：硫化花鳉的潜水行为
我们采用目标个体采样法评估鱼类行为：从目标鱼群中随机选取20条体型相近的潜水鱼类，对其从开始潜水至再次浮出水面的全过程进行观测，以计算平均潜水时长。

DATA_FieldSurvey_Birds：捕食性鸟类活动
我们在预定义研究区域内，于30分钟时段内记录所有目击到的食鱼鸟类。针对每一只鸟类，我们确定其物种分类（至可行的最低分类阶元）、进入与离开研究区域的时间，以及发起的攻击次数。我们以“捕食性鸟类在研究区域内的停留平均时长”计算停留时间（排除仅飞越区域的个体，即未着陆或发起攻击、仅飞过样带的个体），并以采样时段内鸟类发起的总攻击次数作为攻击频次。

DATA_FieldSurvey_SI_Temp：温度监测
在2016年野外调查期间，我们对空气与水温进行了连续监测。在研究区域上游边缘的荫蔽区域放置了2台HOBO Pendant数据记录仪（Onset Computer Corp.）：1台置于水下15厘米深度处，另1台置于水面上方1.5米处。温度记录间隔为10分钟。本研究仅分析鸟类与鱼类观测时段内记录的温度数据（即每个采样时段约1小时内的数据）。

DATA_LabExp_ASR：低氧胁迫下的水生表面呼吸倾向
我们在实验室环境中将鱼类暴露于从近缺氧至常氧的溶解氧浓度梯度下（0.6–5.1 mg/L DO）。共测试了27组鱼群（每组5条成体个体，总样本量n=135）。单次实验时长为10分钟，但我们仅分析最后5分钟的数据，以确保鱼类已从捕获处理中恢复并恢复游泳行为。本研究展示的数据为组内合并后的结果。我们量化了5条鱼群整体在水面停留的累积时长，并计算了水面停留时间百分比；同时评估了每组鱼群的总潜水次数与平均潜水时长。

Lukas2021_WaterChem.R（PDF版本详见Lukas2021_WaterChem_Report）
所有数据分析均在R语言（R Core Team, 2020, 版本4.0.2）中完成。