Development of mitochondrial DNA cytochrome c oxidase subunit I primer sets to construct DNA barcoding library using next-generation sequencing.

Insects are one of the most diverse eukaryotic groups on the planet, with one million or more species present, including those yet undescribed. The DNA barcoding system has been developed, which has aided in the identification of cryptic species and undescribed species. The mitochondrial cytochrome c oxidase I region (mtDNA COI) has been utilised for the barcoding analysis of insect taxa. Thereafter, next-generation sequencing (NGS) technology has been developed, allowing for rapid acquisition of massive amounts of sequence data for genetic analyses. Although NGS-based PCR primers designed to amplify the mtDNA COI region have been developed, their target regions were only a part of COI region and/or there were taxonomic bias for PCR amplification. As the mtDNA COI region is a traditional DNA marker for the DNA barcoding system, modified primers for this region would greatly contribute to taxonomic studies. In this study, we redesigned previously developed PCR primer sets that targetted the mtDNA COI barcoding region to improve amplification efficiency and to enable us to conduct sequencing analysis on NGS. As a result, the redesigned primer sets achieved a high success rate (> 85%) for species examined in this study, covering four insect orders (Coleoptera, Lepidoptera, Orthoptera and Odonata). Thus, by combining the primers with developed primer sets for 12S or 16S rRNA regions, we can conduct more detailed taxonomic, phylogeographic and conservation genetic studies using NGS.

Contrasting population genetic structure of three semi-terrestrial brachyuran crabs on the coast of the Japanese archipelago.

Anthropogenic activities have reduced ecotones between the ocean and land, which is likely to threaten the population of brackish-water brachyuran crabs. To assess the current status of these crabs, we examine the population genetic structures of three semi-terrestrial brachyuran crabs widely distributed along the coast of the Japan and to clarify factors determining their genetic structures. We collected 184 Orisarma dehaani, 252 Chiromantes haematocheir, and 151 Helice tridens crabs from 36 localities of the Japanese archipelago. Genome-wide SNP data from these crabs were analyzed using MIG-seq. Bayesian clustering of STRUCTURE and DAPC analysis were used to identify genetically disturbed populations and to visualize genetic differentiation between local populations. Genetic population structure showed clear differentiation between populations on the Pacific coast of the Tohoku region and on other Japanese coasts in O. dehaani, but not in C. haematocheir or H. tridens. The inbreeding coefficient of O. dehaani was significantly higher on the Pacific coast of the Tohoku region compared to other Japanese coasts. C. haematocheir and H. tridens had homogeneous genetic structures along the Japanese coast, but showed genetic differentiation of a local population at their range limits. Thus, O. dehaani showed little gene flow and clear genetic differentiation between populations in the Tohoku Pacific region and those on other Japanese coasts due to ocean currents. Although such a regional differentiation was not found in C. haematocheir and H. tridens, one population of C. haematocheir was genetically isolated at the edge of its distribution range and likely vulnerable to environmental changes.

Fine-scale clonal structure of the lingonberry Vaccinium vitis-idaea under the nurse plant Pinus pumila vegetation in an alpine region, Mt. Norikura.

The nurse effect is a positive interaction in which a nurse plant improves the abiotic environment for another species (beneficiary plant) and facilitates its establishment. The evergreen shrub Vaccinium vitis-idaea (a beneficiary plant) grows mainly under the dwarf shrub Pinus pumila (a nurse plant) in the alpine regions of central Japan. However, whether V. vitis-idaea shrubs under various P. pumila shrubs spread through clonal growth and/or seeds remains unclear. We investigated the clonal structure of V. vitis-idaea under the nurse plant P. pumila in Japanese alpine regions. MIG-seq analysis was conducted to clarify the clonal diversity of V. vitis-idaea in isolated and patchy P. pumila plots on a ridge (PATs), and in a plot covered by dense P. pumila on a slope adjacent to the ridge (MAT) on Mt. Norikura, Japan. We detected 28 multilocus genotypes in 319 ramets of V. vitis-idaea across 11 PATs and MAT. Three genets expanded to more than 10 m in the MAT. Some genets were shared among neighboring PATs or among PATs and MAT. These findings suggest that the clonal growth of V. vitis-idaea plays an important role in the sustainability of populations. The clonal diversity of V. vitis-idaea was positively related with the spatial size of PATs and was higher in MAT than in PATs at a small scale. Therefore, the spatial spread of the nurse plant P. pumila might facilitate the nurse effect on the genetic diversity of beneficiary plants, leading to the sustainability of beneficiary populations.

Evolutionary radiation of the Eurasian Pinus species under pervasive gene flow.

Evolutionary radiation, a pivotal aspect of macroevolution, offers valuable insights into evolutionary processes. The genus Pinus is the largest genus in conifers with c . 90% of the extant species emerged in the Miocene, which signifies a case of rapid diversification. Despite this remarkable history, our understanding of the mechanisms driving radiation within this expansive genus has remained limited. Using exome capture sequencing and a fossil-calibrated phylogeny, we investigated the divergence history, niche diversification, and introgression among 13 closely related Eurasian species spanning climate zones from the tropics to the boreal Arctic. We detected complex introgression among lineages in subsection Pinus at all stages of the phylogeny. Despite this widespread gene exchange, each species maintained its genetic identity and showed clear niche differentiation. Demographic analysis unveiled distinct population histories among these species, which further influenced the nucleotide diversity and efficacy of purifying and positive selection in each species. Our findings suggest that radiation in the Eurasian pines was likely fueled by interspecific recombination and further reinforced by their adaptation to distinct environments. Our study highlights the constraints and opportunities for evolutionary change, and the expectations of future adaptation in response to environmental changes in different lineages.

Genetic consequences of Last Glacial-Holocene changes in snowfall regime in Arnica mallotopus populations: A plant confined to heavy-snow areas of Japan.

PREMISE: Snow is an important environmental factor affecting plant distribution. Past changes in snowfall regimes may have controlled the demographies of snow-dependent plants. However, our knowledge of changes in the distribution and demographies of such plants is limited because of the lack of fossil records. METHODS: Population genetic and landscape genetic analyses were used to investigate the response of population dynamics of Arnica mallotopus (Asteraceae)-a plant confined to heavy-snow areas of Japan-to changes in snowfall regimes from the Last Glacial Period to the Holocene. RESULTS: The population genetic analysis suggested that the four geographic lineages diverged during the Last Glacial Period. The interaction between reduced snowfall and lower temperatures during this period likely triggered population isolation in separate refugia. Subpopulation differentiation in the northern group was lower than in the southern group. Our ecological niche model predicted that the current distribution was patchy in the southern region; that is, the populations were isolated by topologically flat and climatically unsuitable lowlands. The landscape genetic analysis suggested that areas with little snowfall acted as barriers to the Holocene expansion of species distribution and continued limiting gene flow between local populations. CONCLUSIONS: These findings indicate that postglacial population responses vary among regions and are controlled by environmental and geographic factors. Thus, changes in snowfall regime played a major role in shaping the distribution and genetic structure of the snow-dependent plant.

Double migration of the endangered Tricyrtis formosana (Liliaceae) in Japan.

The Ryukyu Islands of Japan are a biodiversity hotspot due to geographical and historical factors. Tricyrtis formosana is a perennial herbaceous plant that commonly found in Taiwan. But only a few populations have been identified in a limited habitat on Iriomote Island, while populations of unknown origin occur near human settlements in an area on the main island of Okinawa. To better understand these populations of the phylogenetic uniqueness and intrinsic vulnerability, we conducted comparative analyses including (1) phylogeny and population structure with MIG-seq data, (2) photosynthesis-related traits of plants grown under common conditions and (3) transcriptome analysis to detect deleterious variations. Results revealed that T. formosana was split into two clades by the congeners and that Iriomote and Okinawa populations independently derived from ancestral Taiwanese populations in each clade. Photosynthetic efficiency was lowest in the Iriomote population, followed by Okinawa and Taiwan. Transcriptome analysis showed that the Iriomote population accumulated more deleterious variations, suggesting intrinsic vulnerability. These results indicate that each T. formosana population in Japan is phylogenetically unique and has been independently dispersed from Taiwan, and that the Iriomote population presents a high conservation difficulty with a unique photosynthesis-related characteristic and a larger amount of deleterious variations.

The phylogenetic reconstruction of the Neotropical cycad genus Ceratozamia (Zamiaceae) reveals disparate patterns of niche evolution.

The cycad genus Ceratozamia comprises 40 species from Mexico, Guatemala, Belize, and Honduras, where cycads occur throughout climatically varied montane habitats. Ceratozamia has the potential to reveal the history and processes of species diversification across diverse Neotropical habitats in this region. However, the species relationships within Ceratozamia and the ecological trends during its evolution remain unclear. Here, we aimed to clarify the phylogenetic relationships, the timing of clade and species divergences, and the niche evolution throughout the phylogenetic history of Ceratozamia. Genome-wide DNA sequences were obtained with MIG-seq, and multiple data-filtering steps were used to optimize the dataset used to construct an ultrametric species tree. Divergence times among branches and ancestral niches were estimated. The niche variation among species was evaluated, summarized into two principal components, and their ancestral states were reconstructed to test whether niche shifts among branches can be explained by random processes, under a Brownian Motion model. Ceratozamia comprises three main clades, and most species relationships within the clades were resolved. Ceratozamia has diversified since the Oligocene, with major branching events occurring during the Miocene. This timing is consistent with fossil evidence, the timing estimated for other Neotropical plant groups, and the major geological events that shaped the topographic and climatic variation in Mexico. Patterns of niche evolution in the genus do not accord with the Brownian Motion model. Rather, non-random evolution with shifts towards more seasonal environments at high latitudes, or shifts towards humid or dry environments at low latitudes explain the diversification of Ceratozamia. We present a comprehensive phylogenetic reconstruction for Ceratozamia and identify for the first time the environmental factors involved in clade and species diversification within the genus. This study alleviates the controversies regarding the species relationships in the genus and provides the first evidence that latitude-associated environmental factors may influence processes of niche evolution in cycads.

The absence of bumblebees on an oceanic island blurs the species boundary of two closely related orchids.

Oceanic islands offer valuable natural laboratories for studying evolution. The Izu Islands, with their recent geological origin, provide an exceptional opportunity to explore the initial evolution on oceanic islands. Another noteworthy aspect is the absence of bumblebee species on most Izu Islands. We used ecological, morphological, and molecular data to investigate the impact of bumblebee absence on the evolution of two closely related orchid species, Goodyera henryi and Goodyera similis, focusing on Kozu Island, the Izu Islands. Our investigation revealed that while G. henryi exclusively relies on a bumblebee species for pollination on the mainland, G. similis is pollinated by scoliid wasps on both the mainland and the island. Intriguingly, all specimens initially categorized as G. henryi on Kozu Island are hybrids of G. henryi and G. similis, leading to the absence of pure G. henryi distribution on the island. These hybrids are pollinated by the scoliid wasp species that also pollinates G. similis on the island. The absence of bumblebees might result in sporadic and inefficient pollination of G. henryi by scoliid wasps, consequently promoting hybrid proliferation on the island. Our findings suggest that the absence of bumblebees can blur plant species boundaries.

Host-Shift Speciation Proceeded with Gene Flow in Algae Covering Shells.

AbstractHost shifts represent the advancement of a novel niche and often lead to speciation in symbionts. However, its mechanisms are not well understood. Here, we focused on the alga Pseudocladophora conchopheria growing on the shells of intertidal snails. Previous surveys have shown that the alga has host specificity-only attaching to the shell of Lunella correensis-but we discovered that the alga attaches to the shells of multiple sympatric snails. A genome-wide single-nucleotide polymorphism analysis (MIG-seq) was performed to determine whether host-associated speciation occurred in the algae. As a result, there was no gene flow or limited gene flow among the algae from different hosts, and some algae were genetically differentiated among hosts. In addition, the demographic estimate revealed that speciation with gene flow occurred between the algae from different hosts. Therefore, these results support the idea that host-shift speciation gradually proceeded with gene flow in the algae, providing insight into the early evolution of host shifts.

Phylogenetic, population structure, and population demographic analyses reveal that Vicia sepium in Japan is native and not introduced.

Vicia sepium (bush vetch) is a perennial legume widely distributed throughout the Eurasian continent. However, its distribution in Japan is limited to Mt. Ibuki and small parts of central and southern Hokkaido. Therefore, each Japanese V. sepium lineage has been considered to have been introduced separately from Europe. Here, we examined whether the species was introduced or not on the basis of cpDNA sequences and genome-wide SNPs from Japanese and overseas samples. Both the cpDNA haplotype network and the nuclear DNA phylogenetic tree showed that Japanese V. sepium is monophyletic. Furthermore, although the nuclear DNA phylogenetic tree also showed that each lineage is clearly monophyletic, genetic admixture of the genetic cluster dominated in the Hokkaido lineage was also detected in the Mt. Ibuki lineage. Population divergence analysis showed that the two lineages diverged during the last glacial period. The Mt. Ibuki lineage showed a sudden population decline 300-400 years ago, indicating that some anthropogenic activity might be involved, while the Hokkaido lineage showed a gradual population decline from 5000 years ago. Consequently, these two lineages show low current genetic diversity compared with overseas lineages. These results show that the Japanese V. sepium is not introduced but is native.

Molecular phylogeny and taxonomy of Hosta (Asparagaceae) on Shikoku Island, Japan, including five new species, one new subspecies, and two new status assignments.

Japan has 16 native species of the genus Hosta Tratt. (Asparagaceae). A recent study on Hosta based on field surveys and molecular phylogenetic analyses resulted in the discovery of six unknown taxa in Kochi Prefecture, Shikoku Island, southwestern Japan. We aimed to identify these unknown taxa. Therefore, we constructed a finely resolved phylogeny for 320 Hosta samples collected from the Honshu, Shikoku, and Kyushu Islands using multiplex inter-simple sequence repeat genotyping by sequencing (MIG-seq). Based on this phylogenetic analysis and related morphological observations, we describe five new species, H.longipedicellatasp. nov., H.minazukiflorasp. nov., H.polyneuronoidessp. nov., H.samukazemontanasp. nov., and H.takiminazukiflorasp. nov. and one new subspecies, H.takiminazukiflorasubsp.grandissubsp. nov. In addition, we propose two new status assignments, H.tardivasubsp.densinerviacomb. and stat. nov. and H.scabrinerviastat. nov. We also propose classifying H.kikutiivar.tosana as a species, H.tosana. Further studies that combine MIG-seq with careful morphological observations are needed for Hosta plants on all Japanese islands, which may result in the discovery of even more undescribed species.

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.

Discrimination of Camellia cultivars using iD-NA analysis.

Recently, many new cultivars have been taken abroad illegally, which is now considered an international issue. Botanical evidence found at a crime scene provides valuable information about the origin of the sample. However, botanical resources for forensic evidence remain underutilized because molecular markers, such as microsatellites, are not available without a limited set of species. Multiplexed intersimple sequence repeat (ISSR) genotyping by sequencing (MIG-seq) and its analysis method, identification of not applicable (iD-NA), have been used to determine several genome-wide genetic markers, making them applicable to all plant species, including those with limited available genetic information. Camellia cultivars are popular worldwide and are often planted in many gardens and bred to make new cultivars. In this study, we aimed to analyze Camellia cultivars/species through MIG-seq. MIG-seq could discriminate similar samples, such as bud mutants and closely related samples that could not be distinguished based on morphological features. This discrimination was consistent with that of a previous study that classified cultivars based on short tandem repeat (STR) markers, indicating that MIG-seq has the same or higher discrimination ability as STR markers. Furthermore, we observed unknown phylogenetic relationships. Because MIG-seq can be applied to unlimited species and low-quality DNA, it may be useful in various scientific fields.

Potential contribution of floral thermogenesis to cold adaptation, distribution pattern, and population structure of thermogenic and non/slightly thermogenic Symplocarpus species.

The genus Symplocarpus in basal Araceae includes both thermogenic and non/slightly thermogenic species that prefer cold environments. If floral thermogenesis of Symplocarpus contributes to cold adaptation, it would be expected that thermogenic species have a larger habitat than non/slightly thermogenic species during an ice age, leading to increased genetic diversity in the current population. To address this question, potential distribution in past environment predicted by ecological niche modeling (ENM), genetic diversity, and population structure of chloroplast and genome-wide single nucleotide polymorphisms were compared between thermogenic Symplocarpus renifolius and non/slightly thermogenic Symplocarpus nipponicus. ENM revealed that the distribution of S. nipponicus decreased, whereas that of S. renifolius expanded in the Last Glacial Maximum. Phylogeographic analyses have shown that the population structures of the two species were genetically segmented and that the genetic diversity of S. renifolius was higher than that of S. nipponicus. The phylogenetic relationship between chloroplast and nuclear DNA is topologically different in the two species, which may be due to the asymmetric gene flow ubiquitously observed in plants. The results of this study imply that floral thermogenesis of Symplocarpus contributes to expanding the distribution during an ice age, resulting in increased genetic diversity due to cold adaptation.

Phylogeography of the temperate grassland plant Tephroseris kirilowii (Asteraceae) inferred from multiplexed inter-simple sequence repeat genotyping by sequencing (MIG-seq) data.

A group of temperate grassland plant species termed the "Mansen elements" occurs in Japan and is widely distributed in the grasslands of continental East Asia. It has been hypothesized that these species are continental grassland relicts in Japan that stretch back to a colder age, but their migration history has not been elucidated. To assess the migration history of the Mansen elements, we performed phylogeographic analyses of Tephroseris kirilowii, a member of this group, using single-nucleotide polymorphisms (SNPs) obtained from multiplexed inter-simple sequence repeat genotyping by sequencing (MIG-seq). It was estimated that the Japanese populations of T. kirilowii were divided from those of continental East Asia at 25.2 thousand years ago (ka) with 95% highest probability density interval (HPD) of 15.3-40.0 ka and that Japanese clades first diverged at 20.2 ka with 95% HPD of 10.4-30.1 ka. As the climatically suitable range during the last glacial maximum (LGM) estimated using ecological niche modeling (ENM) was limited in Japan and there was a slight genetic differentiation among Japanese populations, a post-glacial expansion of T. kirilowii in the Japanese Archipelago was indicated.

Spiranthes hachijoensis (Orchidaceae), a new species within the S. sinensis species complex in Japan, based on morphological, phylogenetic, and ecological evidence.

The systematics of the Old World Spiranthes sinensis (Pers.) Ames species complex (Orchidaceae) has been complicated by its wide distribution and morphological variations. Within the species complex, S. australis Lindl. has been generally accepted as the only Spiranthes Rich. species distributed on the Japanese mainland. The present study provides morphological, phylogenetic, and ecological evidence for the recognition of S. hachijoensis Suetsugu as a new species of the S. sinensis species complex on the Japanese mainland. Spiranthes hachijoensis is morphologically similar to S. hongkongensis S.Y. Hu & Barretto and S. nivea T.P. Lin & W.M. Lin, sharing a degenerated rostellum, pollinia without a viscidium, and distinctly trilobed stigma. However, the taxon can be morphologically distinguished from S. hongkongensis by its glabrous rachis, ovaries, and sepals, and from S. nivea by its papillate labellum disc, larger papillate basal labellum callosities, and glabrous rachis, ovaries, and sepals. The autogamy and flowering phenology (i.e., earlier flowering) of S. hachijoensis are most likely responsible for premating isolation from the sympatric S. australis. A MIG-seq-based high-throughput molecular analysis indicated that the genetic difference between S. hachijoensis and its putative sister species S. sinensis is comparable to, or even greater than, the genetic difference between pairs of other species within the S. sinensis species complex. Our multifaceted approach strongly supports the recognition of S. hachijoensis as a morphologically, phenologically, phylogenetically, and ecologically distinct species.

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.

Plants, pollinators and their interactions under global ecological change: The role of pollen DNA metabarcoding.

Anthropogenic activities are triggering global changes in the environment, causing entire communities of plants, pollinators and their interactions to restructure, and ultimately leading to species declines. To understand the mechanisms behind community shifts and declines, as well as monitoring and managing impacts, a global effort must be made to characterize plant-pollinator communities in detail, across different habitat types, latitudes, elevations, and levels and types of disturbances. Generating data of this scale will only be feasible with rapid, high-throughput methods. Pollen DNA metabarcoding provides advantages in throughput, efficiency and taxonomic resolution over traditional methods, such as microscopic pollen identification and visual observation of plant-pollinator interactions. This makes it ideal for understanding complex ecological networks and their responses to change. Pollen DNA metabarcoding is currently being applied to assess plant-pollinator interactions, survey ecosystem change and model the spatiotemporal distribution of allergenic pollen. Where samples are available from past collections, pollen DNA metabarcoding has been used to compare contemporary and past ecosystems. New avenues of research are possible with the expansion of pollen DNA metabarcoding to intraspecific identification, analysis of DNA in ancient pollen samples, and increased use of museum and herbarium specimens. Ongoing developments in sequencing technologies can accelerate progress towards these goals. Global ecological change is happening rapidly, and we anticipate that high-throughput methods such as pollen DNA metabarcoding are critical for understanding the evolutionary and ecological processes that support biodiversity, and predicting and responding to the impacts of change.

Genetic Structure of the Liriope muscari Polyploid Complex and the Possibility of Its Genetic Disturbance in Japan.

Anthropogenic activities, such as the movement of plants through greening, can result in genetic disturbance that can interfere with local adaptation in wild populations. Although research is underway to prevent genetic disturbance associated with greening, genetic disturbance of intraspecific polyploidy, which is estimated to be present in 24% of vascular plants, has not been well studied. Liriope muscari is a polyploid complex with known diploid (2n = 36), tetraploid (2n = 72), and hexaploid (2n = 108) forms. The plants of this species tolerate dry and hot conditions and are therefore frequently used for greening and gardening. However, the distribution of this polyploid in Japan, its genetic structure, and genetic disturbance are not known. In this study, we investigated the polyploidy distribution and genetic structure in naturally distributed L. muscari in Japan using chloroplast DNA (cpDNA) haplotypes and nuclear DNA (nDNA). Commercially produced individuals were also studied and compared with natural populations to assess any genetic disturbance of the ploidy complex in this species. Chromosome counts, cpDNA, and nDNA results showed three genetically and cytologically distinct groups in Japan: first, a tetraploid group in mainland Japan; second, a hexaploid group in the Ryukyu Islands; and third, a diploid and tetraploid group in the Ryukyu Islands. Significant isolation by distance was also detected within the three groups (p = 0.001). Genetic disturbance due to greening and gardening should be avoided among the three groups. Genetic disturbance can be reduced by using individuals derived from natural populations that are close to the sites used for greening and gardening. For commercially produced individuals, genetic disturbance is unlikely in the Kanto region, an area of high usage, while genetic disturbance is thought possible in the Ryukyu Islands.