<b>Geographical Variation in Egg Rejection by Azure-winged Magpies (</b><b><i>Cyanopica cyanus</i></b><b>) across China</b>

<b>Study area</b>Fusong County (42°19ʹ N, 127°15ʹ E) is located in Jilin Province, southeastern China (Figure 1) and is characterized by a continental monsoon climate. The dominant vegetation types are coniferous forests and mixed coniferous-broadleaf forests (Liu et al. 2023). Huangpi District (30°42ʹ N, 114°10ʹ E) is an administrative district of Wuhan, situated in Hubei Province, central China (Figure 1). It is characterized by a subtropical monsoon climate, with dry and cold winters and hot, rainy summers. The dominant tree species include camphor (<i>Cinnamomum camphora</i>), the Chinese parasol tree (<i>Firmiana platanifolia</i>), and the golden rain tree (<i>Koelreuteria paniculata</i>) (Liu et al. 2023a). Luqu County (34°34ʹ N, 102°29ʹ E) is located on the eastern edge of the Qinghai-Tibet Plateau in Gansu Province, southwestern China (Figure 1). It is characterized by a humid plateau climate, with cold and damp conditions. The primary vegetation types include meadows, coniferous forests, and shrublands (Gao et al. 2021).<br><b>Egg rejection experiment</b>During the azure-winged magpie breeding season in April to June, 2023 and 2024, we systematically searched for breeding nests in the study area and recorded the nest coordinates and clutch size. Egg recognition experiments were conducted on nests in the early incubation stage. Previous studies have suggested that the color of model eggs can influence hosts’ rejection decisions (Abolins-Abols et al. 2019; Hanley et al. 2021; Liu et al. 2024; Yan and Liang 2024). To investigate the effects of color on the egg rejection behavior of the azure-winged magpie, we tested the egg recognition abilities in all three study sites using both red and blue model eggs. The experiment was divided into three groups: (1) no model egg was added, and the nest was monitored under natural conditions (Figure 2A); (2) one red model egg was added directly to the nest (Figure 2B); and (3) one blue model egg was added directly to the nest (Figure 2C). We observed the host’s response to the experimental egg over six days. If the model egg remained in the nest on the sixth day without peck marks and the magpie continued normal incubation, it was classified as accepted. If the model egg was missing, showed peck marks, or was buried, it was classified as rejected. Nests that were predated during the experiment were excluded from the analysis (Moksnes et al. 1991).While studies have shown that hosts may adjust their defensive behavior according to parasitism risk, whether different geographical populations of the azure-winged magpie (<i>Cyanopica cyanus</i>), which are parasitized by multiple cuckoo species and face varying parasitism risks, exhibit geographical variation in egg rejection behavior is unclear. From April to June in 2023 and 2024, we investigated the egg recognition ability of azure-winged magpie populations in Fusong County, Jilin Province; Huangpi District, Hubei Province; and Luqu County, Gansu Province, China. The results showed that the azure-winged magpie populations in all three regions rejected approximately 100% of the model eggs, with no significant difference in rejection rates between red and blue model eggs. This study revealed that Chinese populations of azure-winged magpies exhibited high egg recognition ability, with no geographical variation in their egg rejection behavior.

<b>研究区域</b>抚松县（42°19′N，127°15′E）位于中国东南部吉林省（图1），属大陆性季风气候，优势植被类型为针叶林与针阔混交林（Liu等，2023）。黄陂区（30°42′N，114°10′E）是湖北省武汉市市辖区，地处中国华中地区（图1），属亚热带季风气候，冬季干燥寒冷、夏季炎热多雨，优势树种包括樟树（<i>Cinnamomum camphora</i>）、梧桐（<i>Firmiana platanifolia</i>）与栾树（<i>Koelreuteria paniculata</i>）（Liu等，2023a）。碌曲县（34°34′N，102°29′E）位于中国西南地区甘肃省青藏高原东缘（图1），属湿润高原气候，环境寒冷湿润，主要植被类型包括草甸、针叶林与灌丛（Gao等，2021）。<br><b>拒卵实验</b>2023至2024年4-6月灰喜鹊（<i>Cyanopica cyanus</i>）繁殖期，我们在研究区域内系统搜寻繁殖巢，记录巢位坐标与窝卵数。针对孵化早期的巢开展卵识别实验。已有研究表明，模拟卵的颜色会影响宿主的拒卵决策（Abolins-Abols等，2019；Hanley等，2021；Liu等，2024；Yan与Liang，2024）。为探究颜色对灰喜鹊拒卵行为的影响，我们在三个研究区域分别使用红色与蓝色模拟卵测试其卵识别能力。实验分为三组：(1) 不添加模拟卵，仅在自然状态下监测巢箱（图2A）；(2) 直接向巢内放入1枚红色模拟卵（图2B）；(3) 直接向巢内放入1枚蓝色模拟卵（图2C）。连续6天观察宿主对实验卵的反应：若第6天时模拟卵仍留存于巢中且无啄痕，灰喜鹊仍保持正常孵化，则判定为接受；若模拟卵缺失、出现啄痕或被掩埋，则判定为拒卵。实验期间被捕食的巢不纳入后续分析（Moksnes等，1991）。<br>已有研究显示宿主可根据寄生风险调整防御行为，但受多种杜鹃寄生、面临不同寄生压力的灰喜鹊不同地理种群，其拒卵行为是否存在地理变异尚不明确。2023至2024年4-6月，我们对中国吉林省抚松县、湖北省武汉市黄陂区及甘肃省碌曲县的灰喜鹊种群开展卵识别能力调查。结果表明，三个区域的灰喜鹊种群对两类模拟卵的拒卵率均接近100%，红色与蓝色模拟卵的拒卵率无显著差异。本研究揭示，中国灰喜鹊种群具备较高的卵识别能力，其拒卵行为未表现出地理变异。