Phylogeographical evidence for historical long-distance dispersal in the flightless stick insect Ramulus mikado.

Exploring how organisms overcome geographical barriers to dispersal is a fundamental question in biology. Passive long-distance dispersal events, although infrequent and unpredictable, have a considerable impact on species range expansions. Despite limited active dispersal capabilities, many stick insect species have vast geographical ranges, indicating that passive long-distance dispersal is vital for their distribution. A potential mode of passive dispersal in stick insects is via the egg stage within avian digestive tracts, as suggested by experimental evidence. However, detecting such events under natural conditions is challenging due to their rarity. Therefore, to indirectly assess the potential of historical avian-mediated dispersal, we examined the population genetic structure of the flightless stick insect Ramulus mikado across Japan, based on a multifaceted molecular approach [cytochrome oxidase subunit I (COI) haplotypes, nuclear simple sequence repeat markers and genome-wide single nucleotide polymorphisms]. Subsequently, we identified unique phylogeographic patterns, including the discovery of identical COI genotypes spanning considerable distances, which substantiates the notion of passive long-distance genotypic dispersal. Overall, all the molecular data revealed the low and mostly non-significant genetic differentiation among populations, with identical or very similar genotypes across distant populations. We propose that long-distance dispersal facilitated by birds is the plausible explanation for the unique phylogeographic pattern observed in this flightless stick insect.

Introgression dynamics from invasive pigs into wild boar following the March 2011 natural and anthropogenic disasters at Fukushima.

Natural and anthropogenic disasters have the capability to cause sudden extrinsic environmental changes and long-lasting perturbations including invasive species, species expansion and influence evolution as selective pressures force adaption. Such disasters occurred on 11 March 2011, in Fukushima, Japan, when an earthquake, tsunami and meltdown of a nuclear power plant all drastically reformed anthropogenic land use. Using genetic data, we demonstrate how wild boar (Sus scrofa leucomystax) have persevered against these environmental changes, including an invasion of escaped domestic pigs (Sus scrofa domesticus). Concurrently, we show evidence of successful hybridization between pigs and native wild boar in this area; however in future offspring, the pig legacy has been diluted through time. We speculate that the range expansion dynamics inhibit long-term introgression and introgressed alleles will continue to decrease at each generation while only maternally inherited organelles will persist. Using the gene flow data among wild boar, we assume that offspring from hybrid lineages will continue dispersal north at low frequencies as climates warm. We conclude that future risks for wild boar in this area include intraspecies competition, revitalization of human-related disruptions and disease outbreaks.

Non-destructive DNA extraction from herbarium specimens: a method particularly suitable for plants with small and fragile leaves.

Protocols for DNA extraction from plants generally involve physical and chemical destruction of tissues. Use of these conventional methods precludes preservation of morphological information from herbarium specimens, especially for small plants with few leaves, and reduces the voucher value of specimens. Here, we developed a new, non-destructive DNA extraction protocol (Protocol 1) that only needs a small piece of leaf (< 25 mm2) to obtain DNA suitable for DNA sequencing from fragile herbarium specimens. The protocol was very simple and rapid; an extraction buffer was placed on the leaf surface of an intact specimen for 30 min at room temperature (20 °C). The quality of extracted DNA was checked by PCR amplification of two standard plant DNA barcode regions, the maturase K gene (matK, ca. 850 bp) and the ribulose-1,5-bisphosphatecarboxylase/oxygenase gene (rbcL, ca. 550 bp), for 14 vascular plant species encompassing various taxonomic groups. The protocol retrieved sequences from 80.0% of specimens for matK and 46.2% of specimens for rbcL. Placing of the extraction buffer onto specimens did not cause any tears or deformation, but caused discoloration in some plants. To improve DNA yield for specimens incompatible with Protocol 1, we developed an alternative protocol for DNA extraction with minimally invasive destruction of specimens (Protocol 2). In this protocol, a cut leaf was immersed in the extraction buffer for 30 min and stored subsequently in a fragment pocket on the specimen sheet. This alternative method retrieved matK sequences from 80.0% of specimens and rbcL sequences from 92.8% of specimens. The combination of Protocols 1 and 2 enabled us to obtain matK sequences from 90.0% of specimens and rbcL sequences form 92.8% of specimens. The new protocols facilitate the use of museum specimens for use of DNA of museum specimens while still preserving morphological information.

Genetic analysis of Japanese and American specimens of Scirpus hattorianus suggests its introduction from North America.

Scirpus hattorianus is a possible alien species in Japan, and a clarification of its unclear taxonomy is required to reveal its origin. It is not known whether the plants initially described from Japan represent the same species distributed in North America. To clarify the origin of the species, we attempted to sequence old specimens collected about 80 years ago using newly designed primer pairs specific for short sequences, including the variable sites. Chloroplast sequences of ndhF were compared among Japanese and North American S. hattorianus, and the closely related species, S. atrovirens, S. flaccidifolius, and S. georgianus. We succeeded in sequencing all samples, and two haplotypes were detected in S. hattorianus: one was unique to the species and the other, detected from specimens potentially collected from the same population as the types, was shared by both North American S. hattorianus and two closely related species, S. atrovirens and S. flaccidifolius. Our results suggest that Japanese S. hattorianus is an alien species that was introduced from North America at least twice.

Comparative morphological analysis of two parallel mycoheterotrophic transitions reveals divergent and convergent traits in the genus Pyrola (Pyroleae, Ericaceae).

The genus Pyrola includes species with different degree of mycoheterotrophy; some species possess individuals that rely on all carbon through their associations with fungi (full mycoheterotrophy, FM), whereas some species obtain carbon through both fungi and photosynthesis by itself (partial mycoheterotrophy, PM). To investigate how plant functional traits of photosynthesis and reproduction are related to the degree of mycoheterotrophy in the initial stage of the transition from PM to FM, we determined morphological traits in FM (or nearly FM) and PM species in two independent lineages, P. picta and P. japonica complexes. We used herbarium specimens and examined leaf number, leaf area, flower number, and scape length in FM or nearly FM species (P. aphylla and P. subaphylla) and PM species (P. picta s.l. and P. japonica). We found a leaf area reduction in FM (or nearly FM) species in both lineages, suggesting that this is a convergent trait. The number of flowers was not significantly different between FM (or nearly FM) and PM species in both lineages. On the other hand, differences in the variation between FM (or nearly FM) and PM species were found in some traits between the two lineages. The FM (or nearly FM) species in one lineage only possessed rudimentary leaves, whereas that in the other linage possessed a few small, ordinary leaves in addition to those with only rudimentary leaves. The scape length of the FM (or nearly FM) species was significantly longer than that of PM species in one lineage, whereas it was shorter in the other lineage. The different and common variations are divergent and convergent traits, respectively, that could be associated with the transition to FM in Pylora. In addition, shoots of both PM species occasionally lacked ordinary leaves, possibly indicating possession of these shoots is preadaptation for the transition to FM in Pyrola.

Genetic diversity and population structure of Nuphar submersa (Nymphaeaceae), a critically endangered aquatic plant endemic to Japan, and implications for its conservation.

Nuphar submersa (Nymphaeaceae) is a critically endangered freshwater macrophyte indigenous to central Japan, with only four small extant populations represented across its entire range. We investigated the genotypic and genetic diversity as well as the genetic structure of all extant individuals of N. submersa based on analysis of 15 microsatellite loci. Among 278 individual ramets, 52 multilocus genotypes were detected: 30 genotypes in Nikko City (NIK), 18 in Nasukarasuyama City (NAS), 3 in Mooka City (MOK), and 1 in Sakura City (SAK). The average number of alleles per locus ranged from 1.20 to 1.93, whereas the observed and expected heterozygosities ranged from 0.11 to 0.33 and from 0.10 to 0.24, respectively. With the exception of SAK, all populations contained multiple clones, but our results indicated low levels of within-population genetic diversity. The populations NIK and NAS comprised few large or middle-sized genets and many small genets. The populations NIK and NAS were suggested to comprise large old, old fragmented, and/or young small genets resulting from seedling establishment. All four populations were differentiated, and gene flow between the populations was restricted (average level of gene flow (Nm) = 0.122, G' ST  = 0.639). Of the total genetic diversity, 67.20 and 9.13% were attributable to inter- and intra-population diversity, respectively. STRUCTURE analysis revealed two or three well-differentiated groups of populations. Cluster I comprised one population (NIK) and cluster II comprised the remaining populations at K = 2. The populations NIK, NAS, and the remaining populations were assigned to clusters I, II, and III, respectively, at K = 3. For conservation practices, we recommend that each cluster be regarded as a different management unit. We further suggest that artificial gene flow among MOK and SAK populations is an appropriate option, whereas NIK should not be reinforced with genotypes from the remaining populations.

Chloroplast DNA sequencing and detailed microsatellite genotyping of all remnant populations suggests that only a single genet survives of the critically endangered plant Rehmannia japonica.

Rehmannia japonica (Thunb.) Makino ex T. Yamaz. is an endangered perennial herb species in Japan. Although earlier the Japanese considered it a variety of R. glutinosa, recent Japanese taxonomists have consistently regarded it as an independent species. According to the historical literature, Rehmannia japonica seems to have been known in China and Japan in the past. However, Chinese taxonomists do not recognize R. japonica at present. In Japan, only two populations are known, and although these populations flower every year, seed reproduction has not been observed. In this study, we aimed to reveal the phylogenetic relationships and levels of genetic diversity of R. japonica. A haplotype network based on two chloroplast DNA regions (trnL-trnF and rps16) showed that the sequences of R. japonica were distinguishable by three or four sites of indels from the most closely related species, R. chingii, consistent with the separate species status of R. japonica. An analysis of genetic diversity using twelve microsatellite loci showed that all of the ramets of R. japonica collected from two geographically isolated populations had an identical multilocus genotype, including identical heterozygous genotypes at six loci. This result indicated asexual origin of all sampled ramets. This study also suggests that the absence of sexual reproduction of R. japonica is explained by self-incompatibility combined with only a single genet remaining in the R. japonica populations.

Variation in vegetative morphology tracks the complex genetic diversification of the mycoheterotrophic species Pyrola japonica sensu lato.

PREMISE OF THE STUDY: Although the evolution of full mycoheterotrophy has attracted many plant researchers, molecular phylogenetic studies that focus on the transition from partial to full mycoheterotrophy are limited to a few taxa. Pyrola japonica sensu lato is an ideal model for examining the evolution of mycoheterotrophy, owing to its variable leaf size, which suggests that the species comprises several transitional stages. METHODS: To elucidate the molecular and morphological changes that occur during the evolutionary transition between partial and full mycoheterotrophy in P. japonica s.l. from 18 populations in Japan, we estimated a parsimony network of plastid haplotypes based on three noncoding regions, measured the leaf size and scape color of the shoots, and compared morphology among haplotypes. KEY RESULTS: The seven haplotypes exhibited star-like relationships, and at least three divergent haplotypes were associated with differences in morphology. The first was mainly observed in large-leaved and green-scaped populations, whereas the second was observed in extremely small-leaved and reddish-scaped populations, which indicated a high degree of mycoheterotrophy, and the last was detected among mixed populations with both green- and reddish-scaped shoots with intermediate leaf sizes. In addition, the inconsistent association between the haplotypes and morphology suggests a complex relationship. CONCLUSIONS: Pyrola japonica s.l. has at least three separate genetic lineages that have different leaf morphologies. The genetic lineages and their coexistence could have led to the variable leaf size and suggest the possibility that gene flow from partial to full mycoheterotrophs could reverse the evolutionary transition to full mycoheterotrophy.

Predominance of a single clone of the most widely distributed bamboo species Phyllostachys edulis in East Asia.

Phyllostachys edulis, one of the most dominant bamboo species with the leptomorph rhizome system, has been asexually expanding its range into adjacent natural forest sites by shooting new culms. The resulting ecological problems include simplification of stand structure and decline in the species diversity of local flora. In this study, the genetic diversity of P. edulis for the entire distribution range from Japan to China was analyzed using 16 microsatellite markers. Among these, 12 loci were fixed by a single allele, whereas only two alleles were detected for each of the remaining 4 loci; all adult samples shared the same genotype at all loci including the four heterozygous loci. These observations indicate that all current samples from Japan and China comprise an identical clone. The clone is distributed over more than 2,800 km with an estimated biomass of approximately 6.6 × 10(11) kg, which is exceptionally large. Among seedlings from flowering events in 2005 and 2006, 20 different genets were generated by recombination through selfing of a single flowering genet. Predominance of a single clone in the wild and a diverse composition of genets among seedlings suggest that the intermittent flowering of P. edulis in the wild has produced a variety of clones through recombination. However, the resulting seedlings cannot compete with other tree species or adult P. edulis, and almost all adult P. edulis growing in Japan and China likely propagated through vegetative reproduction of a single clone by human transplantation, and subsequently expanded into adjacent forest sites by shooting young sprouts. The relatively small size of the flowering area and rapid culm reproduction has led to the stability of P. edulis communities. However, the low genetic diversity is an important consideration for the long-term management of this prevailing bamboo species.

Instantaneous extracellular solution exchange for concurrent evaluation of membrane permeability of single cells.

The osmotic stress imposed on microorganisms by hypotonic conditions is perceived to regulate water and solute flux via cell membranes, which are crucial for survival. Some cells that fail to perceive osmotic stress die because this results in the rupture of the cell membrane. The flux through the membrane is characterized by the membrane permeability, which is measured using a stopped-flow apparatus in response to a millisecond-order osmolarity change. However, the obtained data are an ensemble average of each cell response. Additionally, the measurement of permeability, considering cellular viability, contributes to a more accurate evaluation of osmoadaptation. Here, we present a novel on-chip instantaneous extracellular solution exchange method using an air-liquid interface. The presented method provides a concurrent evaluation at the single-cell level in response to a millisecond-order osmotic shock, considering cellular viability by solution exchange. This method utilizes a liquid bridge with a locally formed droplet on the surface of a micropillar fabricated inside a microchannel. We evaluated a solution exchange time of 3.6 ms and applied this method to Synechocystis PCC 6803 under two different osmolarity conditions. The live/dead ratio of 1 M to 0.5 M osmotic down shock condition was 78.8/21.2% while that of 1 M to 0.25 M osmotic down shock condition was 40.0/60.0%. We evaluated the water permeability of two groups: cells that were still live before and after osmotic shock (hereafter named cell type 1), and cells that were live before but were dead 10 minutes after osmotic shock (hereafter named cell type 2). The results indicated that the water permeability of cell type 2 was higher than that of cell type 1. The results obtained using the presented methods confirmed that the effect of osmotic stress can be accurately evaluated using single-cell analysis.

Dynamic substrate based on photocleavable poly(ethylene glycol): zeta potential determines the capability of geometrical cell confinement.

Dynamic substrates whose cell adhesiveness changes in response to an external stimulus are useful not only for patterning cells in various geometries but also for inducing cell migration or arraying heterotypic cells. The requirements for such applications are high switching efficiency in cell adhesiveness and long-term persistence of the created cellular patterns. In this study, we prepared a dynamic substrate bearing photocleavable poly(ethylene glycol) (PEG) and examined the effect of the surface PEG density and the charge of cationic base materials on the above-mentioned key requirements. An amino-terminated substrate with a certain amino group density and charge was functionalized with photocleavable PEG5K, with and without subsequent backfilling of photocleavable PEG2K. The PEG chains made the surface non-cell-adhesive, but subsequent near-UV irradiation of the substrate induced photocleavage of the PEG, eventually making the surface cell-adhesive. The substrates were analyzed by atomic force microscopy, contact angle measurements, ellipsometry, and zeta potential measurements, complemented with protein adsorption observations. Although the density of amino group in the base material affected both the grafting efficiency of the backfilling PEG and the electrokinetic potential mainly in the positive range, the latter mainly determined the protein- and cell-repelling abilities of the substrates. Furthermore, varying the surface compositions had almost no effect on the switching efficiency in the early stage of the culture, but it became more significant after culturing cells for a longer time; the cells fouled the nonirradiated PEGylated regions earlier on the surfaces with higher positive zeta potentials. These results indicate that the zeta potential is an essential factor in the long-term persistence of cellular patterns on photoactivatable substrates. This study not only provides a recipe for the development of a dynamic substrate with an adequate time frame but also clarifies how the interfacial nanoarchitectures, composed of the nanometer-scale PEG brushes and charged base materials, affect the biocompatibility.

Complete genotyping in conservation genetics, a case study of a critically endangered shrub, Stachyurus macrocarpus var. prunifolius (Stachyuraceae) in the Ogasawara Islands, Japan.

More than 3,000 species are listed as critically endangered worldwide, and various conservation measures such as habitat restoration, assisted reproduction and establishment of ex situ populations would be required to prevent their extinction. We determined the genotype of all 15 known wild clumps using nuclear microsatellite markers for Stachyurus macrocarpus var. prunifolius, a critically endangered shrub endemic to the Ogasawara (Bonin) Islands of Japan. In addition, the seedlings propagated from seeds taken from one wild clumps were genotyped. The results of complete genotyping showed that both wild and nursery populations had population-specific alleles. Two alleles were detected only in the nursery population, indicating the existence of undiscovered mature individuals in the wild. Four alleles were found only in the wild and were detected in two geographically isolated clumps, and this finding may propose that re-introduction and transplantation between different sites requires sensitive handling in terms of the conservation of evolutionary significant units. These results show that complete genotyping can provide essential genetic and ecological information for effective management of endangered species.

Integration of Microfluidic Chip and Probe with a Dual Pump System for Measurement of Single Cells Transient Response.

The integration of liquid exchange and microfluidic chips plays a critical role in the biomedical and biophysical fields as it enables the control of the extracellular environment and allows for the simultaneous stimulation and detection of single cells. In this study, we present a novel approach for measuring the transient response of single cells using a system integrated with a microfluidic chip and a probe with a dual pump. The system was composed of a probe with a dual pump system, a microfluidic chip, optical tweezers, an external manipulator, an external piezo actuator, etc. Particularly, we incorporated the probe with the dual pump to allow for high-speed liquid change, and the localized flow control enabled a low disturbance contact force detection of single cells on the chip. Using this system, we measured the transient response of the cell swelling against the osmotic shock with a very fine time resolution. To demonstrate the concept, we first designed the double-barreled pipette, which was assembled with two piezo pumps to achieve a probe with the dual pump system, allowing for simultaneous liquid injection and suction. The microfluidic chip with on-chip probes was fabricated, and the integrated force sensor was calibrated. Second, we characterized the performance of the probe with the dual pump system, and the effect of the analysis position and area of the liquid exchange time was investigated. In addition, we optimized the applied injection voltage to achieve a complete concentration change, and the average liquid exchange time was achieved at approximately 3.33 ms. Finally, we demonstrated that the force sensor was only subjected to minor disturbances during the liquid exchange. This system was utilized to measure the deformation and the reactive force of Synechocystis sp. strain PCC 6803 in osmotic shock, with an average response time of approximately 16.33 ms. This system reveals the transient response of compressed single cells under millisecond osmotic shock which has the potential to characterize the accurate physiological function of ion channels.

A historic religious sanctuary may have preserved ancestral genetics of Japanese sika deer (Cervus nippon).

Deer have been a major resource for human populations for thousands of years. Anthropogenic activities, such as hunting, have influenced the genetic structure and distribution of deer populations. In Japan, wild Japanese sika deer (Cervus nippon) have been hunted since ancient times but have also been historically protected as sacred animals in several sanctuaries. Sika deer have been protected for over a thousand years in the religious sanctuary around the Kasuga Taisha Shrine on the Kii Peninsula, located in the center of Japan. Here, we used short sequence repeats (SSR) and mitochondrial DNA (mtDNA) to investigate the genetic diversity, population structure, and demography of Japanese sika deer inhabiting the Kii Peninsula, Japan, and discuss possible anthropogenic influences. Using SSR, three distinct genetic groups were distinguished on the Kii Peninsula: an Eastern genetic group, a Western genetic group, and an isolated genetic group with individuals in the religious sanctuary of Kasuga Taisha Shrine in Nara city. The isolated genetic sanctuary group had only the mtDNA haplotype S4. The SSR genotype data suggested a newer divergence time of the genetic groups of the religious sanctuary than would have occurred as a result of Late Quaternary climate change. This time scale coincided with the establishment of the sanctuary with Kasuga Taisha Shrine. Thus, the religious protection conserved genetic variation over a thousand years.

Rapid survey of de novo mutations in naturally growing tree species following the March 2011 disaster in Fukushima: The effect of low-dose-rate radiation.

The impact of low-dose-rate radiation on genetics is largely unknown, particularly in natural environments. The Fukushima Dai-ich Nuclear Power Plant disaster resulted in the creation of contaminated natural lands. In this study, de novo mutations (DNMs) in germ line cells were surveyed from double-digest RADseq fragments in Japanese cedar and flowering cherry trees exposed to ambient dose rates ranging from 0.08 to 6.86 µGy h-1. These two species are among the most widely cultivated Japanese gymnosperm and angiosperm trees for forestry and horticultural purpose, respectively. For Japanese flowering cherry, open crossings were performed to produce seedlings, and only two candidate DNMs were detected from uncontaminated area. For Japanese cedar, the haploid megagametophytes were used as next generation samples. The use of megagametophytes from open crossing for next generation mutation screening had many advantages such as reducing exposure to radiation in contaminated areas because artificial crossings are not needed and the ease of data analysis owing to the haploid nature of megagametophytes. A direct comparison of the nucleotide sequences of parents and megagametophytes revealed an average of 1.4 candidate DNMs per megagametophyte sample (range: 0-40) after filtering procedures were optimized based on the validation of DNMs via Sanger sequencing. There was no relationship between the observed mutations and the ambient dose rate in the growing area or the concentration of 137Cs in cedar branches. The present results also suggest that mutation rates differ among lineages and that the growing environment has a relatively large influence on these mutation rates. These results suggested there was no significant increase in the mutation rate of the germplasm of Japanese cedar and flowering cherry trees growing in the contaminated areas.

Not an ancient relic: the endemic Livistona palms of arid central Australia could have been introduced by humans.

Livistona mariae is an endemic palm localized in arid central Australia. This species is separated by about 1000 km from its congener L. rigida, which grows distantly in the Roper River and Nicholson-Gregory River catchments in northern Australia. Such an isolated distribution of L. mariae has been assumed to have resulted from contraction of ancestral populations as Australia aridified from the Mid-Miocene (ca 15 Ma). To test this hypothesis at the population level, we examined the genetic relationships among 14 populations of L. mariae and L. rigida using eight nuclear microsatellite loci. Our population tree and Bayesian clustering revealed that these populations comprised two genetically distinct groups that did not correspond to the current classification at species rank, and L. mariae showed closest affinity with L. rigida from Roper River. Furthermore, coalescent divergence-time estimations suggested that the disjunction between the northern populations (within L. rigida) could have originated by intermittent colonization along an ancient river that has been drowned repeatedly by marine transgression. During that time, L. mariae populations could have been established by opportunistic immigrants from Roper River about 15 000 years ago, concurrently with the settlement of indigenous Australians in central Australia, who are thus plausible vectors. Thus, our results rule out the ancient relic hypothesis for the origin of L. mariae.

Genetic variation and structure of the endangered Lady Fern Athyrium viridescentipes based on ubiquitous genotyping.

To clarify the genetic status and provide effective information for the conservation of Athyrium viridescentipes, a critically endangered fern species with only 103 individuals remaining in the wild, we conducted ubiquitous genotyping to determine the genotypes of all remnant individuals of the target species. We analyzed the genetic variation of the 103 known individuals in four populations by using 13 microsatellite loci. The genotypes of single spores from a sporophytic individual were also determined in order to reveal the breeding system of this species. The level of allelic variation in A. viridescentipes was significantly lower than that of closely related Athyrium species. The genetic composition of the four populations was rather similar. Sixty-nine individuals (67%) possessed an identical pattern in the allele combinations at 13 microsatellite loci. The mean pairwise F (ST) among four populations was 0.018. The segregated pattern of alleles, determined by single-spore genotyping, revealed that allelic recombination occurs through meiosis. The results indicate that this species contains a low level of genetic variation, has low population differentiation, and maintains populations by sexual reproduction. These findings could lead to more effective conservation programs, the selection of the most appropriate individuals for ex situ conservation efforts, and separate management of extant populations.

Mutational effects of chronic gamma radiation throughout the life cycle of Arabidopsis thaliana: Insight into radiosensitivity in the reproductive stage.

Organisms living on Earth have always been exposed to natural sources of ionizing radiation, but following recent nuclear disasters, these background levels have often increased regionally due to the addition of man-made sources of radiation. To assess the mutational effects of ubiquitously present radiation on plants, we performed a whole-genome resequencing analysis of mutations induced by chronic irradiation throughout the life cycle of Arabidopsis thaliana grown under controlled conditions. We obtained resequencing data from 36 second generation post-mutagenesis (M2) progeny derived from 12 first generation (M1) lines grown under gamma-irradiation conditions, ranging from 0.0 to 2.0 Gray per day (Gy/day), to identify de novo mutations, including single base substitutions (SBSs) and small insertions/deletions (INDELs). The relationship between de novo mutation frequency and radiation dose rate from 0.0 to 2.0 Gy/day was assessed by statistical modeling. The increase in de novo mutations in response to irradiation dose fit the negative binomial model, which accounted for the high variability of mutation frequency observed. Among the different types of mutations, SBSs were more prevalent than INDELs, and deletions were more frequent than insertions. Furthermore, we observed that the mutational effects of chronic radiation were greater during the reproductive stage. These results will provide valuable insights into practical strategies for analyzing mutational effects in wild plants growing in environments with various mutagens.

Japanese "nameko" mushrooms (Pholiota microspora) produced via sawdust-based cultivation exhibit severe genetic bottleneck associated with a single founder.

Pholiota microspora ("nameko" in Japanese) is one of the most common edible mushrooms, especially in Japan, where sawdust-based cultivation is the most dominant method accounting for 99% of the production. The current strains for sawdust cultivation in Japan are considered to have been derived from a single wild strain collected from Fukushima, Japan, implying that commercial nameko mushrooms are derived from a severe genetic bottleneck. We tested this single founder hypothesis by developing 14 microsatellite markers for P. microspora to evaluate the genetic diversity of 50 cultivars and 73 wild strains isolated from across Japan. Microsatellite analysis demonstrated that sawdust-cultivated strains from Japan were significantly less genetically diverse than the wild strains, and the former displayed a significant bottleneck signature. Analyzing the genetic relationships among all genotypes also revealed that the sawdust-cultivated samples clustered into one monophyletic subgroup. Moreover, the sawdust-cultivated samples in Japan were more closely related than full-sibs. These results were consistent with the single founder hypothesis that suggests that all commercial nameko mushrooms produced in Japan are descendants of a single ancestor. Therefore, we conclude that cultivated P. microspora originated from a single domestication event that substantially reduced the diversity of commercial nameko mushrooms in Japan.

Radiocesium accumulation and germline mutations in chronically exposed wild boar from Fukushima, with radiation doses to human consumers of contaminated meat.

Genetic effects and radioactive contamination of large mammals, including wild boar (Sus scrofa), have been studied in Japan because of dispersal of radionuclides from the Fukushima Dai-ichi Nuclear Power Plant in 2011. Such studies have generally demonstrated a declining trend in measured radiocesium body burdens in wildlife. Estimating radiation exposure to wildlife is important to understand possible long-term impacts. Here, radiation exposure was evaluated in 307 wild boar inhabiting radioactively contaminated areas (50-8000 kBq m-2) in Fukushima Prefecture from 2016 to 2019, and genetic markers were examined to assess possible germline mutations caused by chronic radiation exposures to several generations of wild boar. Internal Cs activity concentrations in boar remained high in areas near the power plant with the highest concentration of 54 kBq kg-1 measured in 2019. Total dose rates to wild boar ranged from 0.02 to 36 µGy h-1, which was primarily attributed to external radiation exposure, and dose rates to the maximally exposed animals were above the generic no-effects benchmark of 10 µGy h-1. Using the estimated age of each animal, lifetime radiation doses ranged from <0.1 mGy to 700 mGy. Despite chronic exposures, the genetic analyses showed no significant accumulation of mutation events. Because wild boar is an occasional human dietary item in Japan, effective dose to humans from ingesting contaminated wild boar meat was calculated. Hypothetical consumption of contaminated wild boar meat from radioactively contaminated areas in Fukushima, at the per capita pork consumption rate (12.9 kg y-1), would result in an average effective annual dose of 0.9 mSv y-1, which is below the annual ingestion limit of 1 mSv y-1. Additionally, a consumption rate of about 1.4 kg y-1 of the most contaminated meat in this study would not exceed annual ingestion limits.