Linkage between spatiotemporal distribution of environmental DNA and phenological activity in an amphidromous fish, ayu Plecoglossus altivelis altivelis, in a river located in its northernmost distributional area.

Environmental DNA (eDNA) is a promising tool for the continuous monitoring of fish ecology and diversity. However, its potential for describing the phenological activity of fish has rarely been examined. This study aimed to elucidate a linkage between the spatiotemporal distribution of eDNA and the phenology of an amphidromous fish, ayu Plecoglossus altivelis altivelis, in a river in Hokkaido, Japan, which is its northernmost distributional area. A significant positive correlation between eDNA concentration and catch per unit effort of P. a. altivelis in the river confirmed the use of eDNA as a surrogate for the abundance of P. a. altivelis. eDNA of P. a. altivelis was first detected in late April on a sandy beach adjacent to the river mouth. Subsequent to its first detection at the lowest site in the river in early May, eDNA spread throughout the river, indicating the upstream migration of P. a. altivelis. Spawning activity was also represented by a rapid increase in eDNA concentration and its surge at night in the lowest reaches of the river during September and October. These results suggest that upstream migration and spawning primarily commenced when the water temperature reached 10°C and decreased below 20°C, respectively. This observation is consistent with the behavioral responses observed in P. a. altivelis populations from other regions of Japan. Consequently, this study demonstrated that eDNA distribution was closely linked to the phenological activity of P. a. altivelis and that eDNA is a powerful tool for studying the phenology of migratory fishes.

Evaluating the sampling effort for the metabarcoding-based detection of fish environmental DNA in the open ocean.

Clarifying the effect of the sampling protocol on the detection of environmental DNA (eDNA) is essential for appropriately designing biodiversity research. However, technical issues influencing eDNA detection in the open ocean, which consists of water masses with varying environmental conditions, have not been thoroughly investigated. This study evaluated the sampling effort for the metabarcoding-based detection of fish eDNA using replicate sampling with filters of different pore sizes (0.22 and 0.45 µm) in the subtropical and subarctic northwestern Pacific Ocean and Arctic Chukchi Sea. The asymptotic analysis predicted that the accumulation curves for detected taxa did not saturate in most cases, indicating that our sampling effort (7 or 8 replicates, corresponding to 10.5-40 L of filtration in total) was insufficient to fully assess the species diversity in the open ocean and that tens of replicates or a substantial filtration volume were required. The Jaccard dissimilarities between filtration replicates were comparable with those between the filter types at any site. In subtropical and subarctic sites, turnover dominated the dissimilarity, suggesting that the filter pore size had a negligible effect. In contrast, nestedness dominated the dissimilarity in the Chukchi Sea, implying that the 0.22 µm filter could collect a broader range of eDNA than the 0.45 µm filter. Therefore, the effect of filter selection on the collection of fish eDNA likely varies depending on the region. These findings highlight the highly stochastic nature of fish eDNA collection in the open ocean and the difficulty of standardizing the sampling protocol across various water masses.

Effects of forest cover on richness of threatened fish species in Japan.

Estuaries--one of the most vulnerable ecosystems globally--face anthropogenic threats, including biodiversity loss and the collapse of sustainable fisheries. Determining the factors contributing to the maintenance of estuarine biodiversity, especially that of fish, is vital for promoting estuarine conservation and sustainability. We used environmental DNA metabarcoding analysis to determine fish species composition in 22 estuaries around Japan and measured watershed-scale land-use factors (e.g., population size, urban area percentage, and forest area percentage). We sought to test the hypothesis that the richness of the most vulnerable estuarine fish species (i.e., registered by the Japanese Ministry of the Environment in the national species red-list) is determined by watershed-scale land-use factors. The richness of such species was greater, where forest cover was highest; thus, forest cover contributes to their conservation. The proportion of agriculture cover was associated with low species richness of red-listed fishes (redundancy analysis, adjusted R2 = 43.9% of total variance, df = 5, F = 5.3843, p = 0.0001). The number of red-listed species increased from 3 to 11 along a watershed land-use gradient ranging from a high proportion of agriculture cover to a large proportion of forest cover. Furthermore, the results showed that throughout Japan all the examined watersheds that were covered by >74.8% forest had more than the average (6.7 species per site) richness of red-listed fish species. This result can be attributed to the already high average forest cover in Japan of 67.2%. Our results demonstrate how the land use of watersheds can affect the coastal sea environment and its biodiversity and suggest that proper forest management in conjunction with land-use management may be of prime importance for threatened fish species and coastal ecosystems in general.

Efectos de la Cobertura Forestal sobre la Riqueza de Especies Amenazadas de Peces en Japón Resumen Los estuarios-uno de los ecosistemas más vulnerables a nivel mundial-enfrentan amenazas causadas por el hombre, incluyendo la pérdida de biodiversidad y el colapso de las pesquerías sustentables. La determinación de los factores que contribuyen al mantenimiento de la biodiversidad estuarina, especialmente la de los peces, es vital para fomentar la conservación y sustentabilidad estuarinas. Usamos un análisis de metasecuenciación de ADN ambiental para determinar la composición de especies de peces en 22 estuarios de Japón y medimos los factores del uso de suelo a nivel de cuenca (p. ej.: tamaño poblacional, porcentaje de área urbana y porcentaje de área forestal). Buscamos probar la hipótesis de que la riqueza de las especies de peces más vulnerables (es decir, aquellas registradas por el Ministerio Japonés del Ambiente en la lista roja de especies a nivel nacional) está determinada por los factores de uso de suelo a nivel de cuenca. La riqueza de dichas especies fue mayor en donde la cobertura forestal era la más alta; por lo tanto, la cobertura forestal contribuye a la conservación de estas especies. La proporción de la cobertura agrícola estuvo asociada con una baja riqueza de especies de peces en la lista roja (análisis de redundancia, R2 ajustada = 43.9% de la varianza total, gl = 5, F = 5.3843, p = 0.0001). El número de especies en la lista roja incrementó de 3 a 11 a lo largo del gradiente de uso de suelo de la cuenca, yendo desde una proporción alta de cobertura agrícola a una proporción alta de cobertura forestal. Además, los resultados mostraron que, en Japón, todas las cuencas analizadas que contaban con una cobertura forestal >74.8% tenían mayor riqueza de especies de peces en la lista roja que el promedio (6.7 especies por sitio). Este resultado puede atribuirse a la ya de por sí elevada cobertura forestal de Japón (67.2%). Nuestros resultados demuestran cómo el uso de las cuencas puede afectar al ambiente costero marino y a su biodiversidad y sugiere que la gestión adecuada de los bosques en conjunto con el manejo del uso de suelo puede ser de suma importancia para las especies amenazadas de peces y los ecosistemas costeros en general.

Intraspecific genetic variability and diurnal activity affect environmental DNA detection in Japanese eel.

Environmental DNA (eDNA) analysis with species-specific primer/probe sets is promising as a tool to quantify fish abundance and distribution. Nevertheless, several factors could reduce the accuracy of this method. Here, we aimed to analyze whether intraspecific variability and diel activity rhythm affect eDNA detection in Japanese eels (Anguilla japonica). For this purpose, we performed tank experiments focusing on two points. First, we assessed the effects of base pair sequences with probe region polymorphism on eDNA detection. Next, we evaluated the influences of diel rhythm, activity, and individual differences in eDNA release rate on eDNA concentration. We examined the base pair sequences of the probe regions of 20 individuals and found genetic mismatches in two of them. The eDNA concentration was estimated to be much lower in these variants than it was in the other individuals. We conducted a rearing experiment on four non-variant individuals to explore the influences of diel activity and inter-individual differences in eDNA detection. Nocturnal eel activity was reflected in the eDNA detection but the inter-individual differences remained large. The observed weak positive correlations between eDNA concentration and activity suggest that eDNA emission is highly dependent on basal metabolism. The present study suggests that consideration of polymorphic sites at the probe region and diel activity rhythms should improve the accuracy and precision of abundance estimation through eDNA. Such fine-tuning is applicable not only for eels but also for other fishes to be targeted by eDNA technology.

Estimating fish population abundance by integrating quantitative data on environmental DNA and hydrodynamic modelling.

Molecular analysis of DNA left in the environment, known as environmental DNA (eDNA), has proven to be a powerful and cost-effective approach to infer occurrence of species. Nonetheless, relating measurements of eDNA concentration to population abundance remains difficult because detailed knowledge on the processes that govern spatial and temporal distribution of eDNA should be integrated to reconstruct the underlying distribution and abundance of a target species. In this study, we propose a general framework of abundance estimation for aquatic systems on the basis of spatially replicated measurements of eDNA. The proposed method explicitly accounts for production, transport and degradation of eDNA by utilizing numerical hydrodynamic models that can simulate the distribution of eDNA concentrations within an aquatic area. It turns out that, under certain assumptions, population abundance can be estimated via a Bayesian inference of a generalized linear model. Application to a Japanese jack mackerel (Trachurus japonicus) population in Maizuru Bay revealed that the proposed method gives an estimate of population abundance comparable to that of a quantitative echo sounder method. Furthermore, the method successfully identified a source of exogenous input of eDNA (a fish market), which may render a quantitative application of eDNA difficult to interpret unless its effect is taken into account. These findings indicate the ability of eDNA to reliably reflect population abundance of aquatic macroorganisms; when the "ecology of eDNA" is adequately accounted for, population abundance can be quantified on the basis of measurements of eDNA concentration.

Evaluation of fish biodiversity in estuaries using environmental DNA metabarcoding.

Biodiversity is an important parameter for the evaluation of the extant environmental conditions. Here, we used environmental DNA (eDNA) metabarcoding to investigate fish biodiversity in five different estuaries in Japan. Water samples for eDNA were collected from river mouths and adjacent coastal areas of two estuaries with high degrees of development (the Tama and Miya Rivers) and three estuaries with relatively low degrees of development (the Aka, Takatsu, and Sendai Rivers). A total of 182 fish species across 67 families were detected. Among them, 11 species occurred in all the rivers studied. Rare fishes including endangered species were successfully detected in rich natural rivers. Biodiversity was the highest in the Sendai River and lowest in the Tama River, reflecting the degree of human development along each river. Even though nutrient concentration was low in both the Aka and Sendai Rivers, the latter exhibited greater diversity, including many tropical or subtropical species, owing to its more southern location. Species composition detected by eDNA varied among rivers, reflecting the distribution and migration of fishes. Our results are in accordance with the ecology of each fish species and environmental conditions of each river.