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.

Environmental DNA (eDNA) has been used to investigate species distributions in aquatic ecosystems. Most of these studies use real-time polymerase chain reaction (PCR) to detect eDNA in water; however, PCR amplification is often inhibited by the presence of organic and inorganic matter. In droplet digital PCR (ddPCR), the sample is partitioned into thousands of nanoliter droplets, and PCR inhibition may be reduced by the detection of the end-point of PCR amplification in each droplet, independent of the amplification efficiency. In addition, real-time PCR reagents can affect PCR amplification and consequently alter detection rates. We compared the effectiveness of ddPCR and real-time PCR using two different PCR reagents for the detection of the eDNA from invasive bluegill sunfish, Lepomis macrochirus, in ponds. We found that ddPCR had higher detection rates of bluegill eDNA in pond water than real-time PCR with either of the PCR reagents, especially at low DNA concentrations. Limits of DNA detection, which were tested by spiking the bluegill DNA to DNA extracts from the ponds containing natural inhibitors, found that ddPCR had higher detection rate than real-time PCR. Our results suggest that ddPCR is more resistant to the presence of PCR inhibitors in field samples than real-time PCR. Thus, ddPCR outperforms real-time PCR methods for detecting eDNA to document species distributions in natural habitats, especially in habitats with high concentrations of PCR inhibitors.

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 5 th 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.

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.

The environmental DNA (eDNA) method has been used to estimate the distributions of aquatic species, and both ethanol precipitation and filtration‐based methods are commonly employed to capture eDNA from sampled water. Although filtration‐based methods can capture more eDNA than that by ethanol precipitation, by processing larger volumes of water (e.g., 1 L vs. 15 mL), ethanol precipitation can immediately preserve eDNA on site and ease downstream processing, which are especially advantageous for eDNA studies conducted with limited resources, such as electric equipment, labor, and time. However, the ethanol precipitation method is limited by small volume of water that can be processed (i.e., 15 mL in a reaction volume of 50 mL). As an alternative, isopropanol could potentially be used to increase the volume of sample water processed, since lower volumes of isopropanol are required for precipitation. Therefore, in the present study, we compared the copy numbers of carp eDNA captured using isopropanol and ethanol precipitation in both mesocosm and field experiments. In both cases, we found that isopropanol precipitation recovered double the amount of eDNA recovered by ethanol precipitation, when the reaction volumes were equal. Therefore, isopropanol precipitation is a superior option for eDNA capture when surveys are conducted under resource‐limited conditions.

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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.Keywords Alien species Á Biological invasion Á Brassicaceae Á Multiple introductions Á Phylogeography Á SSR analysis Electronic supplementary material The online version of this article (

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 gardentransplanted 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.