Detection of Echinococcus multilocularis in repurposed environmental DNA samples from river water.

Environmental DNA (eDNA) is an increasingly popular tool in biological and ecological studies. As a biproduct of its increasing use, large number of eDNA samples are being collected and stored, that potentially contain information of many non-target species. One potential use for these eDNA samples is a surveillance and early detection of pathogens and parasites that are otherwise difficult to detect. Echinococcus multilocularis is such a parasite with serious zoonotic concern, and whose range has been expanding. If eDNA samples from various studies can be repurposed in detecting the parasite, it can significantly reduce the costs and efforts in surveillance and early detection of the parasite. We designed and tested a new set of primer-probe for detecting E. multilocularis mitochondrial DNA in environmental medium. Using this primer-probe set, we conducted real-time PCR on repurposed eDNA samples collected from three streams flowing through an area of Japan endemic to the parasite. We detected the DNA of E. multilocularis in one of the 128 samples (0.78%). The discovery suggests that while detecting E. multilocularis using eDNA samples is possible, the rate of detection appear to be very low. However, given the naturally low prevalence of the parasite among wild hosts in endemic areas, the repurposed eDNAs may still be a valid option for surveillance in newly introduced areas with the reduced cost and efforts. Further work is needed to assess and improve the effectiveness of using eDNA for detection of E. multilocularis.

Monotropastrum kirishimense (Ericaceae), a new mycoheterotrophic plant from Japan based on multifaceted evidence.

Due to their reduced morphology, non-photosynthetic plants have been one of the most challenging groups to delimit to species level. The mycoheterotrophic genus Monotropastrum, with the monotypic species M. humile, has been a particularly taxonomically challenging group, owing to its highly reduced vegetative and root morphology. Using integrative species delimitation, we have focused on Japanese Monotropastrum, with a special focus on an unknown taxon with rosy pink petals and sepals. We investigated its flowering phenology, morphology, molecular identity, and associated fungi. Detailed morphological investigation has indicated that it can be distinguished from M. humile by its rosy pink tepals and sepals that are generally more numerous, elliptic, and constantly appressed to the petals throughout its flowering period, and by its obscure root balls that are unified with the surrounding soil, with root tips that hardly protrude. Based on genome-wide single-nucleotide polymorphisms, molecular data has provided clear genetic differentiation between this unknown taxon and M. humile. Monotropastrum humile and this taxon are associated with different Russula lineages, even when they are sympatric. Based on this multifaceted evidence, we describe this unknown taxon as the new species M. kirishimense. Assortative mating resulting from phenological differences has likely contributed to the persistent sympatry between these two species, with distinct mycorrhizal specificity.

Odor of achlorophyllous plants' seeds drives seed-dispersing ants.

Seed dispersal by ants is an important means of migration for plants. Many myrmecochorous plants have specialized appendages in their seeds called elaiosome, which provides nutritional rewards for ants, and enable effective seed dispersal. However, some nonmyrmecochorous seeds without elaiosomes are also dispersed by ant species, suggesting the additional mechanisms other than elaiosomes for seed dispersal by ants. The seeds of the achlorophyllous and myco-heterotrophic herbaceous plant Monotropastrum humile are very small without elaiosomes; we investigated whether odor of the seeds could mediate seed dispersal by ants. We performed a bioassay using seeds of M. humile and the ant Nylanderia flavipes to demonstrate ant-mediated seed dispersal. We also analyzed the volatile odors emitted from M. humile seeds and conducted bioassays using dummy seeds coated with seed volatiles. Although elaiosomes were absent from the M. humile seeds, the ants carried the seeds to their nests. They also carried the dummy seeds coated with the seed volatile mixture to the nest and left some dummy seeds inside the nest and discarded the rest of the dummy seeds outside the nest with a bias toward specific locations, which might be conducive to germination. We concluded that, in M. humile seeds, volatile odor mixtures were sufficient to induce seed-carrying behavior by the ants even without elaiosomes.

Environmental DNA revealed the fish community of Hokkaido Island, Japan, after invasion by rainbow trout.

In freshwater ecosystems, invasive salmonid fishes can have a significant impact on native fish species. Detecting the invasion and its negative effects is critical for the conservation of native fish communities. We examined the species composition and seasonal changes in the freshwater fish community, including salmonids, on the Kamikawa Plain, Hokkaido Island, Japan, using environmental DNA (eDNA) metabarcoding. We detected 23 fish species in 176 samples collected from 16 sites over 12 months (October 2018 - August 2019). Between 11 and 20 species were detected at each site, including five native salmonids (Oncorhynchus masou, Oncorhynchus keta, Parahucho perryi, Salvelinus leucomaenis leucomaenis and Salvelinus malma krascheninnikova). The invasive alien rainbow trout Oncorhynchus mykiss was detected at all 16 sites and it was the most commonly detected salmonid. Although we found no obvious competitive exclusion of native salmonids by rainbow trout in the study area, the invasive species occurred more often and at more sites than any of the natives. We also determined the occurrence and seasonal changes in the fish community, classified as native salmonids, invasive rainbow trout, Cypriniformes and other benthic fishes. There were fewer species overall in winter, but the sites with higher species richness in winter were on the lower reaches of the river. In addition, we detected domestic invaders, such as the topmouth gudgeon, Pseudorasbora parva, although they were less prevalent than rainbow trout. These results show the effectiveness of eDNA metabarcoding, which can be used for surveying species richness at an ecosystem scale. In particular, the detection of the early stages of establishment and spread of invasive species can be achieved by eDNA monitoring.

Withering corolla remains on the calyx tube enhance reproductive success in Oxalis stricta L.

Several flower traits can affect plant reproductive fitness via pollinator attraction, herbivory defense, and thermal regulation of the pistil. In this study, we focus on thermal regulation of the pistil after flowering. We experimentally investigated the functional significance of the withering corollas that remain attached to the calyx tubes of Oxalis stricta L. We studied thermal regulation of the pistil by removing corollas and comparing the plants with and without corollas, under regulated dark and light periods, with an ambient temperature during the dark period lower than that during the light period. In plants lacking corollas, the pistil temperature was significantly lower than in control plants (with intact corollas) by approximately 2°C. Although fruit set in the corolla-removed plants was not significantly different from that in control plants, the temperature threshold for 50% fruition in the corolla-removed plants was significantly higher than that in the controls. Furthermore, the seed number, total seed weight, and single-grain weight were significantly lower in the corolla-removed plants than in control plants. The estimated annual number of reproductive cycles (from June to October), total seed number, and total seed weights were also lower in corolla-removed plants. These findings indicate that the withering corolla remains play a role in thermoregulation of the pistil, and thereby enhance reproductive success. Our study is the first to validate one of the assumed ecological roles of the withering remains of plant corollas.

Environmental DNA analysis as a non-invasive quantitative tool for reproductive migration of a threatened endemic fish in rivers.

Quantitative information regarding reproduction is essential for conserving endangered animals; however, some conventional quantitative methods can be damaging to the target population and their habitats. In the present study, the reproductive migration of a threatened endemic fish, three-lips (Opsariichthys uncirostris uncirostris), was non-invasively monitored by quantitative PCR of species-specific environmental DNA (eDNA), the usefulness of which has been not sufficiently explored. Water sampling and from-shore visual inspection were performed weekly along a tributary of Lake Biwa (Japan), where adult fish seasonally migrate upstream to reproduce as well as at lake sites near the river mouth. Species-specific eDNA was collected at all locations at times when the fish were visually observed and at certain sites where the fish were not observed. Log-transformed individual counts from visual inspection were positively correlated with log-transformed eDNA concentration in the river sites, indicating that eDNA analysis can be a reliable quantitative tool for fish abundance in rivers. Furthermore, distance from the lake did not influence eDNA concentration, suggesting that eDNA transport by river flow had a negligible effect on eDNA quantification. Both eDNA concentration and individual counts gradually increased from May-July, and decreased in August. Importantly, eDNA analysis showed that the fish occupied more habitats in the peak reproductive season and stayed for longer time at any given site. An additional underwater survey confirmed unexpected eDNA detections as true positives. eDNA analysis has great potential to quantitatively monitor reproductive fish migrations under certain conditions.