2.1 Study area and sampling sites
The Pearl River, the second largest river in China, is in the tropical and subtropical areas of Southeast Asia and serves as a critical water source for Guangdong Province and Hong Kong. The water sources of the Pearl River are essential for supplying drinking water, generating power, and performing irrigation in Guangdong Province. Thus, the ecological health of the Pearl River is important for the sustainable development of the Pearl River Delta. The Pearl River consists of multiple tributaries – the North, West, East, Liuxi, and Zeng Rivers – which ultimately merge into the Pearl River Estuary in southern China. The current survey originated from the Liuxi River and Zeng River (Fig. 1), which are regarded as the water sources with high cultural significance for Guangzhou, the capital city of Guangdong Province in southern China. The Liuxi River and Zeng River are the upstream source waters for the Pearl River, including both upstream wadable and downstream nonwadable habitats. Thus, they are important for the comparison between the eDNA metabarcoding protocol and electrofishing method.
Thirty-eight sampling sites were chosen from the headwaters of the Liuxi (L1 – L14) and Zeng Rivers (Z1 – Z12) to the mainstream of the Pearl River (P1 – P12) and then to the Pearl River mouth connected with the South China Sea (Fig. 1). The landforms within the drainage basin are dominated by medium-low mountains and hills. The mean annual temperature and precipitation are 21.6 °C and 2188 mm, respectively. Approximately 83.3% of the runoff is discharged in the flood season (from April to September). The zonal soil and vegetation are Udic Ferralisols and southern subtropical evergreen broad-leaved forest, respectively. Forest coverage is approximately 50%. Arable land accounts for only 8.7% of the drainage area, with paddy fields accounting for most of this. Environmental data, including physicochemical parameters of water quality, habitat characteristics, and bacterial amounts (see Table S1 in the Supplementary Material), were provided by two nationally accredited (China Metrology Accreditation) third-party testing agencies (Hainan Qingxiao Environmental Testing Co., Ltd. and Hainan Qianchao Ecological Technology Co., Ltd.).