Recognition and utilization of egg maculation signals by twosympatric host species

<b>Study area</b>. This study was conducted in the Liuzhi area (26°13ʹN, 105°42ʹE), Guizhou, southwest China, from March to August during 2018–2022 and 2024.<b>Field experiments</b>. First, the recognition and rejection abilities of the south rock bunting and yellow-throated bunting to plain white budgerigar eggs with no markings were first examined. The method involved placing one budgerigar egg directly into a nest with a full clutch (Fig. 2a, c). After the experiment began, we checked the nests every 2–3 days and ended the experiment on the sixth day (Moksnes et al. 1991). Secondly, to test whether the two host species can use their own spot versus linear markings to identify parasitic eggs, we swapped one egg between the two species and then recorded the parent birds’ rejection of the foreign eggs (Fig. 2b, d). During the nest-checking process, if the experimental eggs were found to have disappeared from the nest or if the host birds had abandoned the nest by the end of the experiment, this was recorded as a rejection; if the foreign eggs were still in the nest and the nest was active on the sixth day, this was recorded as acceptance. Additionally, some nests were monitored with video recordings (JWD DV-58G, JWD Inc) to obtain the methods of rejection by the host.<b>Egg-colour measure. </b>From April to August 2024, we used an optical fiber spectrometer (AvaSpec-ULS2048×64-UA; http://avantes.cn/SensLine/56.html) to measure the reflectance spectra of the background color (300–700 nm) of 15 budgerigar eggs, 15 south rock bunting eggs, and 15 yellow-throated bunting eggs, as well as the reflectance spectra of the maculation (spots or streaks) of the two hosts’ eggs (Supporting information). Each host egg was collected from a separate nest. For the background color measurement, we took two measurements in three regions: the blunt end, the middle, and the sharp end of the egg (only the blunt and sharp end regions of the budgerigar eggs were measured). For the measurement of the maculation on the host eggs, we selected three areas with larger maculation at the blunt end of the egg and measured each area once (for a total of three measurements).<b>Permits. </b>The experiments comply with the current laws of China.Experimental procedures were in accordance with the Animal Research Ethics Committee of Hainan Provincial Education Centre for Ecology and Environment, Hainan Normal University (no.HNECEE-2012-004) and Experimental Animal Ethics Committee of Guizhou Normal University (no. 2021001).

<b>研究区域</b>。本研究于2018—2022年及2024年的3月至8月，在中国西南部贵州省六枝地区（北纬26°13′，东经105°42′）开展。<b>野外实验</b>。首先，本实验旨在探究南岩鹀（south rock bunting）与黄喉鹀（yellow-throated bunting）对无任何斑纹的纯白色虎皮鹦鹉（budgerigar）卵的识别与拒斥能力。实验方法为：将1枚虎皮鹦鹉卵直接置入已产满窝卵的鸟巢中（图2a、c）。实验启动后，每2—3天对鸟巢进行一次检查，并于第6天结束实验（Moksnes等，1991）。其次，为验证两种宿主鸟类能否通过自身卵的斑点与线性斑纹来识别寄生卵，我们在两个物种的鸟巢间互换1枚卵，随后记录亲鸟对异种卵的拒斥行为（图2b、d）。在鸟巢检查过程中，若实验卵从巢中消失，或宿主在实验结束前弃巢，则记为拒斥；若异种卵仍留存于巢中且第6天时鸟巢仍处于活跃状态，则记为接受。此外，部分鸟巢采用录像机（JWD DV-58G，JWD Inc）进行监测，以获取宿主的拒卵行为方式。<b>卵色测量</b>。2024年4月至8月，我们使用光纤光谱仪（optical fiber spectrometer，AvaSpec-ULS2048×64-UA；http://avantes.cn/SensLine/56.html），对15枚虎皮鹦鹉卵、15枚南岩鹀卵及15枚黄喉鹀卵的底色（300—700 nm）反射光谱，以及两种宿主卵的斑纹（斑点或条纹）反射光谱进行了测量（详见补充材料）。每枚宿主卵均采自独立鸟巢。对于底色测量，我们在卵的钝端、中部、锐端三个区域各进行2次测量（虎皮鹦鹉卵仅测量钝端与锐端区域）。对于宿主卵斑纹的测量，我们在卵钝端选取3块斑纹面积较大的区域，每块区域测量1次（共计3次测量）。<b>实验许可</b>。本实验符合中国现行法律法规。实验操作严格遵循海南师范大学海南省生态环境教育中心动物研究伦理委员会（批准号：HNECEE-2012-004）及贵州师范大学实验动物伦理委员会（批准号：2021001）的相关规定。