Evaluation of fish biodiversity in estuaries using environmental DNA metabarcoding

to the IUCN red list (https://www.iucnredlist.org). Moreover, C. takatsuensis and Japanese eel are registered as endangered species at the EN level by the Ministry of the Environment of Japan (www.env.go.jp). An additional 11 species, detected by eDNA, including Eutaeniichthys gilli, Gymnogobius castaneus, oriental weather loach Misgurnus anguillicaudatus, cherry salmon, Sarcocheilichthys variegatus, slender bitterling Tanakia lanceolata (NT), fourspine sculpin Cottus kazika, Cottus reinii, dark sleeper Odontobutis hikimius (VU), Japanese fluvial sculpin Cottus pollux, and Gymnogobius scrobiculatus (EN), are considered as endangered in Japan (http://ikilog.biodic.go.jp/Rdb/env). Habitat composition of each river A detailed station-by-station analysis (Fig 2B–2F) revealed that in the Tama River, freshwater species were not detected from all stations at the estuary (Fig 2C; S2 Table). Only a small pro- portion of freshwater species occurred at the river mouth and at the station 500 m left along the coast from the mouth of the Sendai River, while no freshwater species occurred at the other stations (Fig 2F). In the Aka River, freshwater species accounted for 30–40% of total spe- cies at the river mouth, but decreased quickly to fewer than 10% along both the left and right sides of the coast. In contrast, seawater species increased at stations in the coastal area (Fig 2B). Similar results were obtained for the Takatsu River, with the proportion of freshwater species decreasing and that of seawater species highly increasing in the coastal area (Fig 2E). In the

Miya River, freshwater species decreased in the coastal area, except for the station at 500 m on the left side (Fig 2D). A different result was observed regarding the number of species in the Aka and Takatsu Rivers (Fig 2B and 2E). More species were detected at the river mouth (37 species at high tide and 34 species at low tide) of the Aka River than in its surrounding coastal area (19–25 spe- cies). In the Takatsu River, diversity was higher in the coastal area (49–59 species) than at the river mouth (32 at high tide and 33 species at low tide). The number of species in the Sendai River decreased during low tide (27 species) compared to high tide (41 species) at the river mouth (Fig 2F). In the Tama River, species composition changed at the river mouth as the tide switched from high to low and seawater species decreased on the low tide, even though the total number of species (12 species) remained the same (Fig 2C). No distinguishable change was found between high and low tides at the river mouth of the other three rivers. The best models examining the effect of salinity or water temperature on the ratio of fresh- water, brackish, or seawater species could be obtained with relatively high accuracy (AUC = 0.825–0.961; Table 2). The proportion of freshwater species decreased as salinity increased (p < 0.01), whereas that of seawater species increased as salinity increased (p < 0.001) for all five rivers. In contrast, the proportion of brackish water fish was not affected by salinity. On the one hand, the proportion of seawater species increased at higher water temperatures

(p < 0.001), while water temperature had no significant effect on brackish and freshwater spe- cies (p > 0.05). Relationships between environmental factors and the number of species Nutrient concentration (TN and TP) was highest in the Tama River (Fig 3), which flows through a mega city (Fig 1), and relatively low in the Aka and Takatsu Rivers, which flow through rural areas. A similar result was obtained regarding the revetment rate. Among the GLMs for evaluating the effect of human impact on the number of fish species, two models with ΔAIC < 2 were retained (Table 3). Both models included TN, whereby the number of species increased as TN decreased (p < 0.001). In the 2nd model, revetment was included but it had no significant effect (p > 0.05). PLOS ONE Biodiversity of five estuaries PLOS ONE | https://doi.org/10.1371/journal.pone.0231127 October 6, 2020 8 / 15