Tadpole growth performance data

We measured tadpoles’ growth in response to temperature conditions in a complementary set of manipulations conducted in three different experiments. First (Field Enclosures tab), within one catchment we translocated embryos from one source and reared them to metamorphosis in multiple streams with disparate thermal regimes (same experiment as Tsel Trial 1). Second (Common Garden Growth Chambers tab), we reared embryos from different catchments under controlled thermal conditions in the laboratory, and third (Common Garden tab), we used outdoor stream mesocosms to create a common-garden environment (corresponding to Tsel Trial 3). For the Field Enclosures experiment, in 2008 and repeated in 2009, we raised full-sibling tadpoles from shortly after fertilisation until front limb emergence in flowthrough stream enclosures placed in six locations in the catchment of the South Fork Eel River hereafter SF Eel, Appendix S2 in article). Details of animal husbandry methods are in Appendix S3 of article. For each replicate we measured body length and mass of tadpoles weekly and calculated the mean growth rate of all tadpoles in the enclosure (mg/day and mm/day). We used least squares linear regression to relate growth and size at metamorphosis to M30DAT. For the Common Garden Growth Chambers, we collected embryos from three of the coastal study rivers (two with dams) and four of the inland rivers (two with dams) from April to June 2010 (Table S1 of article). We transported embryos in chilled aerated river water to the laboratory and within 8 hr of collection placed them into temperature-controlled diurnal growth chambers (cycle set to 14.5 hr light and warm, 9.5 hr dark and cool). After hatching, we randomly chose five tadpoles from each clutch and placed them in 3.8-L aquaria with an aerated mixture of river water and de-chlorinated tap water (changed 29/week). To mimic warm summer conditions at locations with robust populations
of frogs, one chamber was set at 18°C during the dark cycle, and 22°C during the light cycle. Daily mean temperature, calculated from 3 i-Button data loggers placed in randomly selected aquaria, was 19.5°C. To mimic conditions of the coolest occupied sites, the other incubator was set at 13°C dark cycle, 19°C light cycle, with a daily mean = 16.6°C. Each chamber held 35 aquaria (4–6 replicate clutches 9 7 rivers). We measured body and total length of tadpoles with calipers to the nearest 0.1 mm at weekly intervals after hatching. The response variable was body growth, mm/day. For the Common Garden experiment, we collected embryos in late May and early June 2010 from four rivers, and placed them in outdoor mesocosms at the University of California’s Richmond Field Station (37.913536°N, -122.333303°W). We captured tadpoles in four rivers: a coastal unregulated (South Fork Eel), a coastal regulated (Eel below Scott Dam), an inland unregulated (Middle Fork Feather) and an inland regulated (North Fork Feather). At each river, we obtained embryos from three different egg masses; furthermore, at the North
Fork Feather we collected from two reaches (below the Cresta and Poe dams). After transferring embryos to Richmond and rearing them to tadpole stage, we placed 10 tadpoles in separate mesocosms consisting of re-circulating troughs (total of 15 troughs, 3 for each river except for the North Fork Feather which had 6 troughs).
We mounted PVC pipe cut in half (2.2 m 9 15.3 cm diameter) on saw horses at a slight incline so water drained into a 946 L reservoir. Pumps and hoses continuously re-circulated water to the top of each trough lined with periphyton covered river rocks and loose algae. Tadpoles were reared indoors at 18°C until stage Gosner stage 25, and placed in the troughs on 21 July 2010. We measured and staged tadpoles weekly until front limb emergence (Gosner stage 42). We calculated a mean growth rate for each trough (mm of body length increase/day).

我们通过三组互补操控实验，测量了蝌蚪生长对温度条件的响应。第一组实验对应「野外围栏（Field Enclosures）」标签页：在一个流域内，我们将来自单一来源的胚胎易地移植，于多个热环境差异显著的溪流中饲养至变态发育（该实验与Tsel Trial 1为同一组）。第二组实验对应「普通花园生长箱（Common Garden Growth Chambers）」标签页：我们将来自不同流域的胚胎置于实验室可控温度条件下饲养；第三组实验对应「普通花园（Common Garden）」标签页：我们利用室外溪流中型实验生态系统（mesocosms）构建了普通花园环境（对应Tsel Trial 3）。
针对野外围栏实验，我们于2008年开展并在2009年重复实验：将受精后不久的全同胞蝌蚪饲养至前肢萌出阶段，饲养容器为通流式溪流围栏，放置在南福克伊尔河（South Fork Eel River，下文简称SF Eel，详见文章附录S2）流域内的6个点位。动物饲养方法的细节见文章附录S3。我们每周测量每个重复组中蝌蚪的体长与体重，并计算围栏内所有蝌蚪的平均生长速率（单位为mg/天与mm/天）。我们采用最小二乘线性回归，将变态阶段的生长与体型与M30DAT进行关联分析。
针对普通花园生长箱实验，我们于2010年4月至6月间，从3条沿海研究河流（其中2条建有大坝）与4条内陆研究河流（其中2条建有大坝）采集胚胎（详见文章表S1）。我们将胚胎置于充氧冷却的河水中运输至实验室，并在采集后8小时内将其放入控温昼夜生长箱（光照/升温周期设为14.5小时，黑暗/降温周期设为9.5小时）。孵化后，我们从每个卵簇中随机选取5只蝌蚪，放置于3.8升的水族箱中，水族箱内为充氧的河水与脱氯自来水混合液（每周换水29次）。为模拟青蛙种群繁盛区域的夏季温暖环境，其中一台生长箱的黑暗周期温度设为18℃，光照周期设为22℃，基于随机放置在水族箱中的3个i-Button数据记录仪的测量数据，其日平均温度为19.5℃。为模拟温度最低的适宜生境条件，另一台培养箱的黑暗周期温度设为13℃，光照周期设为19℃，日平均温度为16.6℃。每个生长箱可容纳35个水族箱（对应4~6个重复卵簇×7条河流）。我们在孵化后每周使用游标卡尺测量蝌蚪的体长与总长度，精度达0.1mm，响应变量为体长生长速率（mm/天）。
针对普通花园实验，我们于2010年5月末至6月初从4条河流采集胚胎，并将其放置在加州大学里士满野外站（University of California’s Richmond Field Station，坐标37.913536°N, -122.333303°W）的室外中型实验生态系统中。我们从4条河流采集胚胎：1条沿海未受调控河流（南福克伊尔河）、1条沿海受调控河流（斯科特大坝下游的伊尔河）、1条内陆未受调控河流（中岔费瑟河）与1条内陆受调控河流（北岔费瑟河）。在每条河流中，我们从3个不同的卵块获取胚胎；此外，在北岔费瑟河，我们从两个河段（克雷斯塔大坝下游与坡大坝下游）采集胚胎。将胚胎转移至里士满并饲养至蝌蚪阶段后，我们将10只蝌蚪放置于独立的中型实验生态系统中，该系统为循环水槽（共15个水槽，每条河流对应3个水槽，北岔费瑟河对应6个水槽）。
我们将对半切开的PVC管（长2.2米，直径15.3厘米）架在锯木架上，保持轻微倾斜，使水排入946升的储水箱中。水泵与软管持续将水循环至每个水槽顶部，水槽内壁铺有周丛藻类（periphyton）覆盖的河石与松散藻类。蝌蚪在18℃的室内环境中饲养至戈斯内尔分期（Gosner stage）25期，随后于2010年7月21日放入水槽。我们每周测量并分期蝌蚪，直至前肢萌出（戈斯内尔分期42期）。我们计算每个水槽的平均生长速率（体长增加量mm/天）。