Contents lists available at ScienceDirect Marine Pollution Bulletin journal homepage: www.elsevier.com/locate/marpolbul Fish and crustacean biodiversity in an outer maritime estuary of the Pearl River Delta revealed by environmental DNA Chi-chiu Cheang a, ⁎ , Bo-yee Lee a , Brian Ho-yeung Ip a , Wai-hong Yiu a , Ling-ming Tsang b , Put O. Ang Jr. c a Department of Science and Environmental Studies, The Education University of Hong Kong, Taipo, N.T., Hong Kong, China b School of Life Sciences, The Chinese University of Hong Kong, Shatin, N.T., Hong Kong, China c Institute of Space and Earth Information Science, The Chinese University of Hong Kong, Shatin, N.T., Hong Kong, China ARTICLE INFO Keywords: Cytochrome oxidase I (COI) High-throughput sequencing Marine Metabarcoding Subtropical ABSTRACT Understanding the faunal community structure in the estuary would be crucial in assessing the health of the ecosystem. The poor visibility in the estuarine area due to the outfow from the Pearl River hinders the con- ventional visual census in assessing the megafaunal biodiversity. In this study, the fsh and crustacean biodi- versity of Hong Kong's western waters, i.e. the outer maritime estuary of the PRD, were studied through the metabarcoding of environmental DNA (eDNA). eDNA from the seawater and sediment samples was extracted from fve sites in the region. After testing the performance of two genetic markers, amplicons of the cytochrome oxidase I, amplifed by polymerase chain reaction, were subjected to Illumina high-throughput sequencing (MiSeq) analysis. A total of 22 fsh species from 17 families and 34 crustacean species from 27 families were identifed by blasting the sequences against the NCBI GenBank database, demonstrating segregation between samples from diferent sites. This study provides insight on the detail distribution of fsh assembly in PRD, when compared with a previous eDNA study in the inner brackish PRD. 1. Introduction Water pollution caused in estuarine areas by anthropogenically polluted freshwater outfow from rivers has long been documented (Ram et al., 2014; Lam et al., 2016; O’Mara et al., 2017). The Pearl River Delta (PRD) region in southern China is regarded as the largest urban area in the world in terms of size and population (Deuskar et al., 2015). The rapid economic development in the PRD in recent decades has raised concern over water pollution in the region (Liu et al., 2018). Apart from pollution originating within the PRD region itself (Zhu et al., 2002), pollution from the upstream rivers of the PRD and the surrounding marine area in southern China has also been reported (e.g. Luo et al., 2004; Ni et al., 2008; Lam et al., 2016). A number of pol- lutants, such as trace metals, polychlorinated and polybrominated bi- phenyls (Xia et al., 2011, 2012) and polyfuoroalkyl substances (Kwok et al., 2015), have been identifed to have exerted profound adverse impacts on the marine life in the South China Sea (SCS) (Lam et al., 2009; Leung et al., 2010; Xia et al., 2011). The discharge from rivers afects not just the health of marine life in the downstream estuary (e.g. Xia et al., 2011; Lam et al., 2016) but also the structure of the whole community (Yüksek et al., 2006). To assess the impact of pollution, as well as the holistic functioning of ecosystem services, in the polluted nearshore and estuarine areas, researchers have focused on identifying sentinel species or species of indicative value to conduct index-based assessment of the biota in the region (Dauvin, 2007; Borja et al., 2009). Phytoplankton, macrophytes, benthic organ- isms and fsh (Borja, 2005) are some common biotic indicators used to assess the ecosystem health in estuarine area. In contrast to the intense research into environmental risk assess- ment, limited studies have investigated the community structure, especially of metazoans, in the PRD-associated region or the SCS. One of the earliest studies on community structure in the region dates back to 1981 (Wu and Richards, 1981), in which the benthic community was found to be dominated by various gastropods and crabs, with a decrease in the abundance of the dominant species along the salinity gradient from the PRD towards the SCS. Another study elucidated the dominance of decapods and stomatopods in the benthic environment in the western waters of Hong Kong, i.e. the outer estuary of the PRD (Lui et al., 2007). The community structure of phytoplankton has been well studied by ecological investigations (Zhou et al., 2016), pigment analysis (Chai et al., 2016) and DNA barcoding (Jiang et al., 2015). Through meta- barcoding, a site-specifc variation in the microbial communities among https://doi.org/10.1016/j.marpolbul.2020.111707 Received 3 July 2020; Received in revised form 13 September 2020; Accepted 21 September 2020 ⁎ Corresponding author. E-mail address: cccheang@eduhk.hk (C.-c. Cheang). Marine Pollution Bulletin 161 (2020) 111707 Available online 13 October 2020 0025-326X/ © 2020 Elsevier Ltd. All rights reserved. T