Evaluation of fish biodiversity in estuaries using environmental DNA metabarcoding

ecosystems (e.g., coral reefs, mangroves, tropical islands) [2, 4], and other services [9]. In con- trast, biodiversity evaluations that include various regions at the same time have not been car- ried out, because traditional monitoring methods (observations and/or capture) require considerable financial and labor resources to cover a wide range of habitats [10, 11]. Also, par- ticularly for rare and endangered species, monitoring using traditional methods can negatively affect the organisms and their habitat during the survey. Here, we tested environmental DNA (eDNA) metabarcoding as a non-invasive and cost- effective method for monitoring the biodiversity of fishes [12] in multiple estuaries at a nation-wide scale. Environmental DNA, defined as genetic material released from organisms into the environment, has become a convenient tool for molecular biology and ecology over the past decade [13, 14]. By sampling soil, sediment, water, and ice, species can be detected even when they cannot be observed visually. This technique was first reported with regard to amphibians [15], followed by fish [16, 17], crustaceans [18], mammals [19], and plants [20]. In addition, combined with next-generation sequencing technology, eDNA enables the process- ing of massive DNA sequencing data for the identification of various taxa in multiple samples simultaneously, which is termed eDNA metabarcoding [21]. This method is not only practical for assessment of biodiversity, but is also useful to for detection of non-invasive alien, rare,

and endangered species while performing a diversity survey [14, 22, 23]. We used universal primers (MiFish-U and MiFish-E) for the metabarcoding process [24]. Five rivers, indicative of different geographical features and human impact on biodiversity, were selected for this study. As Japan stretches extensively from north to south, the latitude of the target rivers varied from 31.85˚N to 38.85˚N (Fig 1A). The catchment area of the rivers showed considerable variation from natural forest to a megacity. We hypothesized that fish diversity detected from the eDNA survey would reflect those environmental characteristics. Therefore, we confirmed two main aspects. 1) The accordance of fish composition and envi- ronment. 2) The relation of biodiversity and human activity. Materials and methods Water sampling Five rivers, Aka River (Yamagata prefecture, Tohoku area in northern part; 38.8477 N 139.7863 E at river mouth), Tama River (Tokyo, the capitol city in the middle east; 35.5205 N 139.7978 E at river mouth), Miya River (Mie prefecture, Kansai area in the middle west; 34.5396 N 136.7227 E at river mouth), Takatsu River (Shimane prefecture, Chugoku area in western part; 34.6857 N 131.8291 E at river mouth), and Sendai River (Kagoshima prefecture, Kyushu area in southern part; 31.8417 N 130.2087 E at river mouth) with different geographi- cal features and degrees of urbanization were selected (Fig 1). The water at five stations (at the river mouth, and approximately 500 m and 1 km along the coast on both the left and right

sides of the river mouth) was sampled in summer (June or July) 2018. At the river mouth, the water was sampled twice (at high and low tides), and therefore, there was a total of six samples collected from each estuary. For the Tama River, water samples were taken from a boat because the estuary is located between Haneda Airport and the Kawasaki industrial complex, and we could not reach the area from the shore. Moreover, because the airport restricts access to any type of boat near the runway, we could only collect samples from one station of each side of the Tama River estuary and collected four samples instead of six samples (Table 1). All sampling and filtering equipment was cleaned with 10% commercial bleach solution. The surface water at each station was sampled by a bucket and immediately filtered using a 0.45-μm polyethersulfone membrane Sterivex filter unit (Merck Millipore, Billerica, MA, USA) and immersed in 1.6 mL RNAlater Stabilization Solution (Thermo Fisher Scientific, PLOS ONE Biodiversity of five estuaries PLOS ONE | https://doi.org/10.1371/journal.pone.0231127 October 6, 2020 2 / 15 Competing interests: The authors have declared that no competing interest exist.