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An environmental DNA (eDNA) analysis method has been recently developed to estimate the distribution of aquatic animals by quantifying the number of target DNA copies with quantitative real-time PCR (qPCR). A new quantitative PCR technology, droplet digital PCR (ddPCR), partitions PCR reactions into thousands of droplets and detects the amplification in each droplet, thereby allowing direct quantification of target DNA. We evaluated the quantification accuracy of qPCR and ddPCR to estimate species abundance and biomass by using eDNA in mesocosm experiments involving different numbers of common carp. We found that ddPCR quantified the concentration of carp eDNA along with carp abundance and biomass more accurately than qPCR, especially at low eDNA concentrations. In addition, errors in the analysis were smaller in ddPCR than in qPCR. Thus, ddPCR is better suited to measure eDNA concentration in water, and it provides more accurate results for the abundance and biomass of the target species than qPCR. We also found that the relationship between carp abundance and eDNA concentration was stronger than that between biomass and eDNA by using both ddPCR and qPCR; this suggests that abundance can be better estimated by the analysis of eDNA for species with fewer variations in body mass.

Integrated weed management (IWM) is currently the most appropriate and effective method of agricultural weed control. To determine the most effective strategy, it is necessary to compare the effects of different control options and their rotation. Avena fatua (common wild oat) is one of the most common and economically threatening grass weed species of cereal crops worldwide. To examine the effects of non-chemical weed management options (farmland use, delayed sowing, and summer irrigation) on control of A . fatua , we recorded coverage levels and field conditions in 41 sites during the spring growing season of winter wheat for about 10 years. A transition matrix model was then constructed to project coverage levels of A . fatua under each management option using ordinal logistic regression. The results showed that farmland use had a remarkable effect on coverage; notably, planting of paddy rice and vegetables, which respectively eliminated the effect of coverage in the previous year and facilitated rapid convergence of coverage to 0%. Thus, although 90% of fields under continuous wheat cultivation were found to be at risk of A . fatua colonization, the risk was reduced to almost 0% with rotation of effective farmland use. As summer irrigation was also effective, more than 50% of wheat fields with the option continuously converged to no risk for A . fatua colonization. When the different management cycles were repeated, the effects were observed within 3 years, with a steady state reached in less than 10 years. Overall, these results suggest that simplified monitoring data could help decision-making on IWM, thereby helping to improve the efficiency of agricultural production.

The environmental DNA (eDNA) method has increasingly been recognized as a powerful tool for monitoring aquatic animal species; however, its application for monitoring aquatic plants is limited. To evaluate eDNA analysis for estimating the distribution of aquatic plants, we compared its estimated distributions with eDNA analysis, visual observation, and past distribution records for the submerged species Hydrilla verticillata. Moreover, we conducted aquarium experiments using H. verticillata and Egeria densa and analyzed the relationships between eDNA concentrations and plant biomass to investigate the potential for biomass estimation. The occurrences estimated by eDNA analysis closely corresponded to past distribution records, and eDNA detections were more frequent than visual observations, indicating that the method is potentially more sensitive. The results of the aquarium experiments showed a positive relationship between plant biomass and eDNA concentration; however, the relationship was not always significant. The eDNA concentration peaked within three days of the start of the experiment in most cases, suggesting that plants do not release constant amounts of DNA. These results showed that eDNA analysis can be used for distribution surveys, and has the potential to estimate the biomass of aquatic plants.

Using convolutional neural networks (CNNs) for image recognition is effective for early weed detection. However, the impact of training data curation, specifically concerning morphological changes during the early growth phases of weeds, on recognition robustness remains unclear. We focused on four weed species (giant ragweed [Ambrosia trifida L.], red morningglory [Ipomoea coccinea L.], pitted morningglory [Ipomoea lacunosa L.], and burcucumber [Sicyos angulatus L.]) with varying cotyledon and true leaf shapes. Creating 16 models in total, we employed four dataset patterns with different growth stage combinations, two image recognition algorithms (object detection: You Look Only Once [YOLO] v5 and image classification: Visual Geometry Group [VGG] 19), and two conditions regarding the number of species treated (four and two species). We evaluated the effects of growth stage training on weed recognition success using two datasets. One evaluation revealed superior results with a single class/species training dataset, achieving >90% average precision for detection and classification accuracy under most conditions. The other dataset revealed that merging different growth stages with different shapes as a class effectively prevented misrecognition among different species when using YOLOv5. Both results suggest that integrating different shapes in a plant species as a single class is effective for maintaining robust recognition success amid temporal morphological changes during the early growth stage. This finding not only enhances early detection of weed seedlings but also bolsters the robustness of general plant species identification.

Floral organ number is often fixed within families, and the basic floral ground plan of Brassicaceae is well conserved.Cardamine hirsutaL. (Brassicaceae) shows variation in lateral stamen number, that is, zero to two lateral stamens. The aim of this study was to examine whether temperature conditions alter stamen number. Temporal changes in the frequency of flowers with zero, one, and two lateral stamens were assessed during the flowering periods of a natural population and a garden-transplanted population in Japan. We conducted an experiment to evaluate how temperature regimes during flowering (15°/15°C and 15°/5°C) alter the number of stamens. The proportion of flowers with zero lateral stamens increased in both the natural and the garden-transplanted population as the flowering season progressed. In the growth experiment, lateral stamen numbers fluctuated even within individual inflorescences, but the frequency of flowers with zero lateral stamens was higher in the high-temperature condition than in the low-temperature condition. Temperature-dependent phenotypic plasticity is likely to be the cause of the field-observed variation in lateral stamen number forC. hirsuta. Developmentally unstable but partly temperature-dependent production of the lateral stamen may be an indication of some epigenetic regulations as an underlying mechanism.

Environmental DNA (eDNA) methods are increasingly used to detect aquatic organisms. To optimize survey efficiency in natural environments, it is necessary to understand the seasonal change in eDNA concentrations of target species and identify the season and environmental conditions in which eDNA concentrations are highest. Recently, eDNA methods have been developed to detect aquatic plant species, but the seasonal change in the eDNA concentrations remains uninvestigated. Many aquatic plants undergo considerable changes in abundance, size, and shape throughout the year. Therefore, their eDNA concentration may change along with their phenology. We investigated the seasonal change in the eDNA concentration of an aquatic submerged species, Hydrilla verticillata, in agricultural ponds in Japan by measuring the eDNA concentration of H. verticillata from 5 ponds 5× in a year. This species has a dormant period during winter and a growth period from spring to autumn. The eDNA concentrations were higher during the growth period than during the dormant period. Management and conservation surveys that use eDNA for species detection should be done when eDNA concentrations are highest to maximize detection probabilities and therefore survey efficiency.

The first step toward solving the problems caused by an invasive alien species is to know the distribution of the species. However, species in underwater environments are difficult to investigate. The recent development of environmental DNA (eDNA) analysis has made it possible to investigate the distribution of a target species simply by analyzing the DNA in the water. To date, few investigators have used eDNA detection of aquatic plants. We established an eDNA detection method for Egeria densa, an invasive aquatic plant species in Japan; used eDNA detection to survey the species in aquaria; and applied this method to water samples from 23 outdoor ponds. We also used visual observations of the ponds. The aquarium experiments revealed that the eDNA concentration in the water increased rapidly and peaked 1 or 2 d after starting the experiment, after which it decreased rapidly, reaching its lowest point on the 5th day. In the field surveys, we visually observed E. densa at 5 ponds, and the eDNA of E. densa was detected from the same 5 ponds. Thus, the eDNA results perfectly matched the observational results. Our work confirms that detection of aquatic plants by eDNA analysis is feasible.

Floral organ number is often fixed within families, and the basic floral ground plan of Brassicaceae is well conserved. Cardamine hirsuta L. (Brassicaceae) shows variation in lateral stamen number, that is, zero to two lateral stamens. The aim of this study was to examine whether temperature conditions alter stamen number. Temporal changes in the frequency of flowers with zero, one, and two lateral stamens were assessed during the flowering periods of a natural population and a garden-transplanted population in Japan. We conducted an experiment to evaluate how temperature regimes during flowering (15°/15°C and 15°/5°C) alter the number of stamens. The proportion of flowers with zero lateral stamens increased in both the natural and the garden-transplanted population as the flowering season progressed. In the growth experiment, lateral stamen numbers fluctuated even within individual inflorescences, but the frequency of flowers with zero lateral stamens was higher in the high-temperature condition than in the low-temperature condition. Temperature-dependent phenotypic plasticity is likely to be the cause of the field-observed variation in lateral stamen number for C. hirsuta. Developmentally unstable but partly temperature-dependent production of the lateral stamen may be an indication of some epigenetic regulations as an underlying mechanism.

Multiple introductions of a species are thought to enhance its invasion success by increasing genotypic diversity; this involves frequent crossing among different lineages. However, genetic diversity through crossing is less likely in autogamous species. To understand the impact of multiple introductions on the colonization success of autogamous species, we studied hairy bittercress, Cardamine hirsuta, which invaded Japan several decades ago. We detected temporal changes in its population structure using nine microsatellite markers amplified from leaf samples collected from 87 sites between 2009 and 2010, and herbarium specimens collected between 1988 and 2007. To examine whether the phenotypic variation corresponded with the genetic population structure, we also investigated the geographic variation in the lateral stamen number of this species across 49 sites. The present populations can be divided into three genetic groups, which are distributed in northern, eastern, and western Japan. This finding suggests that there are three invasive lineages (North, East, and West) in Japan. The geographic variation in lateral stamen number corresponded to the distributions of these lineages. The former distributions of the North and West lineages mostly corresponded to those found at present, but they were also historically found in eastern Japan. However, the East lineage has apparently expanded into eastern Japan, resulting in a change in dominant lineages over only a few decades. For the autogamous C. hirsuta, multiple introductions contributed toward colonization success over a wider range, which was associated with a local change in the dominant lineages.

An environmental DNA (eDNA) analysis method has been recently developed to estimate the distribution of aquatic animals by quantifying the number of target DNA copies with quantitative real-time PCR (qPCR). A new quantitative PCR technology, droplet digital PCR (ddPCR), partitions PCR reactions into thousands of droplets and detects the amplification in each droplet, thereby allowing direct quantification of target DNA. We evaluated the quantification accuracy of qPCR and ddPCR to estimate species abundance and biomass by using eDNA in mesocosm experiments involving different numbers of common carp. We found that ddPCR quantified the concentration of carp eDNA along with carp abundance and biomass more accurately than qPCR, especially at low eDNA concentrations. In addition, errors in the analysis were smaller in ddPCR than in qPCR. Thus, ddPCR is better suited to measure eDNA concentration in water, and it provides more accurate results for the abundance and biomass of the target species than qPCR. We also found that the relationship between carp abundance and eDNA concentration was stronger than that between biomass and eDNA by using both ddPCR and qPCR; this suggests that abundance can be better estimated by the analysis of eDNA for species with fewer variations in body mass.