Genetic diversity and structure of the globally invasive tree, <Emphasis Type="Italic">Paraserianthes lophantha</Emphasis> subspecies <Emphasis Type="Italic">lophantha</Emphasis>, suggest an introduction history characterised by varying propagule pressure

An emerging insight in invasion biology is that intra-specific genetic variation, human usage, and introduction histories interact to shape genetic diversity and its distribution in populations of invasive species. We explore these aspects for the tree species Paraserianthes lophantha subsp. lophantha, a close relative of Australian wattles (genus Acacia). This species is native to Western Australia and is invasive in a number of regions globally. Using microsatellite genotype and DNA sequencing data, we show that native Western Australian populations of P. lophantha subsp. lophantha are geographically structured and are more diverse than introduced populations in Australia (New South Wales, South Australia, and Victoria), the Hawaiian Islands, Portugal, and South Africa. Introduced populations varied greatly in the amount of genetic diversity contained within them, from being low (e.g. Portugal) to high (e.g. Maui, Hawaiian Islands). Irrespective of provenance (native or introduced), all populations appeared to be highly inbred (F _IS ranging from 0.55 to 0.8), probably due to selfing. Although introduced populations generally had lower genetic diversity than native populations, Bayesian clustering of microsatellites and phylogenetic diversity indicated that introduced populations comprise a diverse array of genotypes, most of which were also identified in Western Australia. The dissimilarity in the distribution and number of genotypes in introduced regions suggests that non-native populations originated from different native sources and that introduction events differed in propagule pressure.     

Conservation genetics and geographic patterns of genetic variation of endangered shrub <Emphasis Type="Italic">Ammopiptanthus</Emphasis> (Fabaceae) in northwestern China

Phylogeographic patterns of Ammopiptanthus in northwestern China were examined with internal transcribed spacer (ITS) and three chloroplast intergenic spacers (trnH–psbA, trnL–trnF, and trnS–trnG). Two ITS genotypes (a–b) and 8 chloroplast haplotypes (A–H) were detected. Both ITS genotypes and chloroplast lineages were split in two geographic regions: western Xinjiang and the Alxa Desert. This lineage split was also supported by AMOVA analysis and the Mantel test. AMOVA showed that 89.81 % of variance in Ammopiptanthus occurred between the two geographic regions, and correlation between genetic distances and geographical distances was significant (r = 0.757, p < 0.0001). All populations in western Xinjiang shared haplotype A with high frequency, and range expansion was strongly supported by negative Fu’s F_S value, and mismatch distribution analysis, whereas populations in the Alxa Desert had higher genetic diversity and structure. We speculate that the cold and dry climate during the early Quaternary fragmented habitats of the species, limiting gene flow between regions, and interglacial periods most likely led to the range expansion in western Xinjiang. The low genetic diversity of Ammopiptanthus indicate a significant extinction risk, and protective measures should be taken immediately.     

The origins of global invasions of the German wasp (<Emphasis Type="Italic">Vespula germanica</Emphasis>) and its infection with four honey bee viruses

A successful control or eradication programme using biological control or genetically-mediated methods requires knowledge of the origin and the extent of wasp genetic diversity. Mitochondrial DNA variation in the native and invaded range of the social wasp Vespula germanica was used to examine intra-specific genetic variation and invasive source populations. We also examined wasps for the presence of four viruses found in honey bees: Acute bee paralysis virus, Deformed wing virus, Israeli acute paralysis virus and Kashmir bee virus. German wasps showed reduced genetic diversity in the invaded range compared to that of their native range. Populations in the introduced range are likely to have arrived from different source populations. All four viral honey bee pathogens were found in V. germanica, although they varied in their distribution and strain. Multiple introductions of German wasps have occurred for most invaded regions, though some populations are genetically homogenous. The differing locations of origin will guide researchers searching for biocontrol agents and the reduced genetic diversity may make these wasps a potentially viable target for control via gene drives.     

Genetic variation of <Emphasis Type="Italic">Spartina alterniflora</Emphasis> intentionally introduced to China

Spread of smooth cordgrass (Spartina alterniflora) in China is an exceptional example of unanticipated outcomes arising from intentional introductions. It has been proposed that in China, management strategies used to establish S. alterniflora inadvertently promoted evolutionary outcomes that have contributed to other Spartina invasions. In this study, we assessed whether S. alterniflora in China exhibits genetic signatures of mechanisms known to promote invasion success, including large founding populations, evolved self-fertility, ‘superior source ecotypes’, and post-introduction admixture. This involved comparing microsatellite genotype and chloroplast haplotype variation among Chinese populations to other invasive S. alterniflora populations as well as native range populations, inclusive of samples from all reported source areas. We found distinct signatures of source population contributions to Chinese populations, as well as evidence of post-introduction admixture, and no evidence of limitations from a genetic bottleneck. Measures of inbreeding were well below what has been found in other non-native populations that have evolved self-fertility. Differences in genetic diversity among sites were similar to latitudinal patterns in the native range, but could be attributable to introduction history. Comparisons to other invasive populations indicate that a combination of common and idiosyncratic processes have contributed to the success of S. alterniflora in China and elsewhere, with intentional introductions promoting mechanisms that accelerate rates of spread and widespread invasion.     

Haplotype diversity of mt<Emphasis Type="Italic">COI</Emphasis> in the fall webworm <Emphasis Type="Italic">Hyphantria cunea</Emphasis> (Lepidoptera: Arctiidae) in introduced regions in China,Iran, Japan,Korea, and its homeland,the United States

Hyphantria cunea (Drury) has colonized many countries outside its native range of North America and has become a model species for studies of the colonization and subsequent adaptation of agricultural pests. Molecular genetic analyses can clarify the origin and subsequent adaptations to non-native habitats. Using the mitochondrial COI gene, we examined the genetic relationships between invasive populations (China, Iran, Japan, and Korea) and native populations (i.e., the United States). The Jilin (China) and Guilan (Iran) populations showed nine previously unknown haplotypes that differed from those found in the south–central United States, suggesting multiple colonization events and different regions of invasion. A dominant mtDNA haplotype in populations in the United States was shared by all of the populations investigated, suggesting that H. cunea with that haplotype have successfully colonized China, Iran, Japan, and Korea.     

Current status of genetic disturbances in wild medaka populations (<Emphasis Type="Italic">Oryzias latipes</Emphasis> species complex) in Japan

We revealed the range and current status of genetic disturbances in wild medaka populations (Oryzias latipes species complex) using two DNA markers (cytb gene and b-marker). Genetic disturbances were detected in many wild populations throughout Japan and were primarily caused by artificial introduction of the commercial medaka variety, himedaka. We identified native medaka populations without introgressions, which may be significant conservation targets. To conserve the native genetic diversity of the medaka species complex, further introduction of himedaka should be prevented by educating the public about the current status and risks of introducing non-native medaka varieties into the wild.     

Non-congruent geographic patterns of genetic divergence across European species of <Emphasis Type="Italic">Branchinecta</Emphasis> (Anostraca: Branchinectidae)

Three of the five European species of Branchinecta have a disjunct distribution. In this study, we analyze populations of B. ferox and B. orientalis for mitochondrial (cox1) and nuclear (ITS2) molecular markers. We compare intraspecific genetic divergences between geographically distant populations of B. orientalis, from its only known Spanish population (originally described as B. cervantesi) and from a Hungarian population (assigned to B. orientalis since its discovery), with data from two relatively close Iberian populations of B. ferox. Results indicate that isolation between B. ferox and B. orientalis clades is ancient, and that the clade including the two Iberian populations of B. ferox is geographically structured. Conversely, Iberian and Hungarian populations of B. orientalis do not show geographical structure for the mitochondrial fragment. Lack of geographic structure coupled with very low genetic distances indicates that current Iberian and Hungarian populations of B. orientalis originated from a common population stock, and that the time elapsed since their separation has not been long enough to render the clades reciprocally monophyletic. We hypothesize that colonization of the Iberian Peninsula by B. orientalis is probably the consequence of a single recent dispersal event, and consequently we confirm the synonymy between B. cervantesi and B. orientalis.     

Geographic independence and phylogenetic diversity of red shiner introductions

Identifying areas at risk of invasion can be difficult when the distribution of a non-native species encompasses geographically disjunct regions. Understanding genealogical relationships among native and non-native populations can clarify the origins of fragmented distributions, which in turn can clarify how fast and far a non-native species may spread. We evaluated genetic variation across the native and invasive ranges of red shiner (Cyprinella lutrensis), a minnow known to displace and hybridize with native species, to reconstruct invasion pathways across the United States (USA). Examination of mitochondrial cytochrome-b variation found that native range populations of red shiner fall into four highly divergent lineages that likely warrant species recognition. Introduced red shiner populations in the eastern and western USA are derived from only two of these lineages. Western USA populations originate from the mid-western and western genetic lineages, whereas eastern introductions derive only from the mid-western lineage. Western USA invasive populations exhibit fewer, but more diverse haplotypes compared to eastern USA invasive populations. We also recovered an undescribed, divergent lineage of Cyprinella that has been cryptically introduced into the western USA, which raises the possibility that hybridization has proceeded following secondary contact between previously allopatric lineages. Approximate Bayesian Computation modeling suggests that the disjunct distribution of red shiner across North America is an agglomeration of independent regional invasions with distinct origins, rather than stepwise advance of an invasion front or secondary introductions across regions. Thus localized control may be effective in managing non-native red shiner, including further spread to areas of conservation concern.     

Intra- and interspecific hybridization in invasive Siberian elm

Hybridization creates unique allele combinations which can facilitate the evolution of invasiveness. Frequent interspecific hybridization between the Siberian elm, Ulmus pumila, and native elm species has been detected in the Midwestern United States, Italy and Spain. However, Ulmus pumila also occurs in the western United States and Argentina, regions where no native elm species capable of hybridizing with it occurs. We examined whether inter- or intraspecific hybridization could be detected in these regions. Nuclear markers and the program STRUCTURE helped detect interspecific hybridization and determine the population genetic structure in both the native and the two non-native ranges. Chloroplast markers identified sources of introduction into these two non-native ranges. No significant interspecific hybridization was detected between U. pumila and U. rubra in the western United States or between U. pumila and U. minor in Argentina and vice versa. However, the genetic findings supported the presence of intraspecific hybridization and high levels of genetic diversity in both non-native ranges. The evidence presented for intraspecific hybridization in the current study, combined with reports of interspecific hybridization from previous studies, identifies elm as a genus where both inter- and intraspecific hybridization may occur and help maintain high levels of genetic diversity potentially associated with invasiveness.     

Morphometric and ISSR-Analysis of Local Populations of <Emphasis Type="Italic">Geranium molle</Emphasis> L. from the Southern Coast of the Caspian Sea

Geranium molle is known as Dovefoot Geranium or Awnless Geranium. Dovefoot Geranium is a low-growing herb with pink flowers and sharply toothed leaves. Dovefoot Geranium is native to Eurasia and has been introduced to many habitats of the world. This species is very similar to G. robertianum but its palmate-like leaves and bilobed petals show differences. This plant is considered to be anodyne, astringent and vulnerary. We have no information on its population genetic structure, genetic diversity, and morphological variability in Iran. Therefore, due to the importance of these plant species, we performed a combination of morphological and molecular data for this species. For this study, we used 132 randomly collected plants from 18 geographical populations in 4 provinces. Genetic diversity parameters were determined in these populations. STRUCTURE analysis and K-Means clustering identified 14 gene pools in the country and revealed isolation by distance among the studied populations. The Mantel test showed correlation between genetic and geographical distance. AMOVA revealed a significant genetic difference among populations and showed that 40% of total genetic variation was due to within-population diversity. The consensus tree of both molecular and morphological data identified divergent populations. These data may be used in future breeding and conservation of this important medicinal plant in the country.     

Trophic consequences of non-native pumpkinseed <Emphasis Type="Italic">Lepomis gibbosus</Emphasis> for native pond fishes

Introduced non-native fishes can cause considerable adverse impacts on freshwater ecosystems. The pumpkinseed Lepomis gibbosus, a North American centrarchid, is one of the most widely distributed non-native fishes in Europe, having established self-sustaining populations in at least 28 countries, including the U.K. where it is predicted to become invasive under warmer climate conditions. To predict the consequences of increased invasiveness, a field experiment was completed over a summer period using a Control comprising of an assemblage of native fishes of known starting abundance and a Treatment using the same assemblage but with elevated L. gibbosus densities. The trophic consequences of L. gibbosus invasion were assessed with stable isotope analysis and associated metrics including the isotopic niche, measured as standard ellipse area. The isotopic niches of native gudgeon Gobio gobio and roach Rutilus rutilus overlapped substantially with that of non-native L. gibbosus, and were also substantially reduced in size compared to ponds where L. gibbosus were absent. This suggests these native fishes shifted to a more specialized diet in L. gibbosus presence. Both of these native fishes also demonstrated a concomitant and significant reduction in their trophic position in L. gibbosus presence, with a significant decrease also evident in the somatic growth rate and body condition of G. gobio. Thus, there were marked changes detected in the isotopic ecology and growth rates of the native fish in the presence of non-native L. gibbosus. The implications of these results for present and future invaded pond communities are discussed.     

Historical range contraction,and not taxonomy,explains the contemporary genetic structure of the Australian tree <Emphasis Type="Italic">Acacia dealbata</Emphasis> Link

Irrespective of its causes, strong population genetic structure indicates a lack of gene flow. Understanding the processes that underlie such structure, and the spatial patterns it causes, is valuable for conservation efforts such as restoration. On the other hand, when a species is invasive outside its native range, such information can aid management in the non-native range. Here we explored the genetic characteristics of the Australian tree Acacia dealbata in its native range. Two subspecies of A. dealbata have previously been described based on morphology and environmental requirements, but recent phylogeographic data raised questions regarding the validity of this taxonomic subdivision. The species has been widely planted within and outside its native Australian range and is also a highly successful invasive species in many parts of the world. We employed microsatellite markers to investigate the population genetic diversity and structure among 42 A. dealbata populations from across the species’ native range. We also tested whether environmental variables purportedly relevant for the putative separation of subspecies are linked with population genetic differentiation. We found no relationship between population genetic structure of A. dealbata in Australia and these environmental features. Rather, we identified two geographically distinct genetic clusters that corresponded with populations in the northeastern part of mainland Australia, and the southern mainland and Tasmanian range of the species. Our results do not support the taxonomic subdivision of the species into two distinct subspecies based on environmental features. We therefore assume that the observed morphological differences between the putative subspecies are plastic phenotypic responses. This study provides population genetic information that will be useful for the conservation of the species within Australia as well as to better understand the invasion dynamics of A. dealbata.     

Heritable bacterial endosymbionts in native and invasive populations of <Emphasis Type="Italic">Harmonia axyridis</Emphasis>

Harmonia axyridis Pallas (1773) (Coleoptera: Coccinellidae) is the well-studied system of invasive insect species. Native and invasive parts of the area of H. axyridis are isolated geographically. We studied the species composition and the distribution of bacterial symbionts Spiroplasma and Rickettsia in seven localities of the native area and six localities of the invasive area of H. axyridis. Rickettsia was detected in H. axyridis populations for the first time. We found that the proportion of beetles infected with Rickettsia in native and invasive populations of H. axyridis is about 0.03. Spiroplasma was found only in native populations of H. axyridis. The proportion of infected individuals with Spiroplasma in native populations of H. axyridis is about 0.08. All studied native populations of H. axyridis are infected with Spiroplasma, while all invasive populations are not. We discuss the possible influence of Spiroplasma and Rickettsia in the formation of invasive populations of H. axyridis.     

Reconciling the biogeography of an invader through recent and historic genetic patterns: the case of topmouth gudgeon <Emphasis Type="Italic">Pseudorasbora parva</Emphasis>

The genetic variability and population structure of introduced species in their native range are potentially important determinants of their invasion success, yet data on native populations are often poorly represented in relevant studies. Consequently, to determine the contribution of genetic structuring in the native range of topmouth gudgeon Pseudorasbora parva to their high invasion success in Europe, we used a dataset comprising of 19 native and 11 non-native populations. A total of 666 samples were analysed at 9 polymorphic microsatellite loci and sequenced for 597 bp of mitochondrial DNA. The analysis revealed three distinct lineages in the native range, of which two haplogroups were prevalent in China (100%), with a general split around the Qinling Mountains. Dating of both haplogroups closely matched past geological events. More recently, its distribution has been influenced by fish movements in aquaculture, resulting in gene flow between previously separated populations in Northern and Southern China. Their phylogeography in Europe indicate as few as two introductions events and two dispersal routes. Microsatellite data revealed native populations had higher genetic diversity than those in the invasive range, a contrast to previous studies on P. parva. This study confirms the importance of extensive sampling in both the native and non-native range of invasive species in evaluating the influence of genetic variability on invasion success.     

Evidence for genetic erosion of a California native tree, <Emphasis Type="Italic">Platanus racemosa</Emphasis>, via recent,ongoing introgressive hybridization with an introduced ornamental species

When non-native, genetically diverse species are introduced, hybridization with native congeners may erode the genetic composition of local species, perhaps even resulting in extinction. While such events may lead to adverse consequences at the community and ecosystem level, few studies exist on ecologically important tree species. In the genus Platanus, introgressive hybridization is widespread, and one common ornamental species, introduced to California during the late 19th century, is itself a hybrid. Our microsatellite analysis of more than 400 Platanus trees from north-central California reveals a complex pattern of invasion and hybridization in an age-structured population. By using size as a proxy for age, we have demonstrated that the Platanus population of north-central California has recently gained genetic diversity and effective population size. Principal coordinate analysis (PCoA) and genetic admixture analysis (STRUCTURE) both reveal a strong differentiation of genotypes into two main genetic clusters, with a large number of admixed genotypes. One of the genetic clusters identified is heavily biased towards younger trees, including samples from locations with relatively recently planted ornamental trees likely to be P. × hispanica (formerly known as P. × acerifolia). We conclude that the two genetic clusters correspond to the native P. racemosa and the introduced invasive hybrid species P. × hispanica. Additional hybridization between the invasive ornamental and the native species has occurred in California, and recent hybrid trees are more likely to be younger than trees without admixture. Our findings suggest that the observed increase in genetic diversity among California Platanus is due to rampant ongoing introgression, which may be threatening the continued genetic distinctiveness of the native species. This is cause for concern from a conservation standpoint, due to a direct loss of genetic distinctiveness, and a potential reduction in habitat value of associated species.     

PhragNet: crowdsourcing to investigate ecology and management of invasive <Emphasis Type="Italic">Phragmites australis</Emphasis> (common reed) in North America

Invasion biology research, often performed by scientists at relatively small spatial scales, provides experimental precision but may be limited in generalizability. Conversely, large-scale invasive species management represents a largely untapped wealth of information on invasion ecology and management, but such data are difficult to capture and synthesize. We developed a network (“PhragNet”) of individuals managing wetlands occupied by native and non-native lineages of the invasive wetland grass Phragmites australis (common reed). This network collected environmental and genetic samples, habitat data, and management information to identify environmental and plant community associations of Phragmites invasion and patterns of management responses. Fifty managers overseeing 209 Phragmites stands in 16 US states and ON, Canada participated. Participants represented federal agencies (26%), municipalities (20%), NGOs (20%), academia (14%), state agencies (12%), and private landowners (8%). Relative to the native lineage, non-native Phragmites occurred in areas with higher nitrate/nitrite and ammonium than non-native Phragmites. Stand interiors had higher soil electrical conductivity than nearby uninvaded areas, consistent with use of road salt promoting spread of Phragmites. Non-native Phragmites co-occurred with fewer plant species than native Phragmites and was actively targeted for management. Herbicide was applied to 51% of non-native stands; surprisingly, 11% of native stands were also treated with herbicide. This project demonstrates the utility of crowdsourcing standardized data from resource managers. We conclude by describing how this approach could be expanded into an adaptive management framework, strengthening connections between wetland management and research.     

Genetic and morphological diversity in <Emphasis Type="Italic">Tragopogon graminifolius</Emphasis> DC. (Asteraceae) in Iran

Tragopogon graminifolius DC. is a medicinal plant species of the genus Tragopogon L. (Asteraceae) that grows in different regions of Iran and is extensively used by locals. There is no report on genetic variability and population structure of this important plant species. Therefore, we studied the genetic diversity, population structure and morphological variability of 14 geographical populations of Tragopogon graminifolius in Iran. AMOVA and Gst analyses revealed significant molecular differences among the studied populations. Mantel test showed significant correlation between genetic distance and geographical distance of the studied populations and indicated that the neighboring populations had a higher degree of gene flow. STRUCTURE plot identified three main gene pools for Tragopogon graminifolius in Iran and population assignment test revealed that gene flow occurred mostly among populations of the same gene flow. The studied populations differed significantly in their morphological and genetic features. These results may be of use for future conservation of this important plant species.     

Invasion genetics of <Emphasis Type="Italic">Senecio vulgaris</Emphasis>: loss of genetic diversity characterizes the invasion of a selfing annual,despite multiple introductions

Genetic variation in invasive populations is affected by a variety of processes including stochastic forces, multiple introductions, population dynamics and mating system. Here, we compare genetic diversity between native and invasive populations of the selfing, annual plant Senecio vulgaris to infer the relative importance of genetic bottlenecks, multiple introductions, post-introduction genetic drift and gene flow to genetic diversity in invasive populations. We scored multilocus genotypes at eight microsatellite loci from nine native European and 19 Chinese introduced populations and compared heterozygosity and number of alleles between continents. We inferred possible source populations for introduced populations by performing assignment analyses and evaluated the relative contributions of gene flow and genetic drift to genetic diversity based on correlations of pairwise genetic and geographic distance. Genetic diversity within Chinese populations was significantly reduced compared to European populations indicating genetic bottlenecks accompanying invasion. Assignment tests provided support for multiple introductions with populations from Central China and southwestern China descended from genotypes matching those from Switzerland and the UK, respectively. Genetic differentiation among populations in China and Europe was not correlated with geographic distance. However, European populations exhibited less variation in the relation between G _ST and geographical distance than populations in China. These results suggest that gene flow probably plays a more significant role in structuring genetic diversity in native populations, whereas genetic drift appears to predominate in introduced populations. High rates of selfing in Chinese populations may restrict opportunities for pollen-mediated gene flow. Repeated colonization-extinction cycles associated with ongoing invasion is likely to maintain low genetic diversity in Chinese populations.     

Assessment of genetic diversity and differentiation of <Emphasis Type="Italic">Liposcelis bostrychophila</Emphasis> (Psocoptera: Liposcelidae) in China using inter-simple sequence repeat (ISSR) fingerprinting

Liposcelis bostrychophila (Psocoptera: Liposcelidae) is a widely distributed pest that can cause considerable economic losses and pose human health risks. Rapid development of insecticide resistance has made L. bostrychophila increasingly difficult to control. To obtain information potentially useful for pest management, genetic diversity and differentiation of L. bostrychophila from five geographic locations in China was studied using inter-simple sequence repeat (ISSR). A total of 104 loci were found by ISSR markers and amplified using 9 selected primers. The percentage of polymorphic bands (PPB) was 91.4%. Shannon’s information index (I) and Nei’s gene diversity (He) indicated high genetic diversity at the species level. Population differentiation (Gst = 0.484) was average in these populations. Analysis of molecular variation (AMOVA) indicated that genetic variation was mainly distributed within populations. Gene flow (Nm = 0.534) was moderate. Cluster analysis showed that genotypes isolated from the same locations displayed higher genetic similarity and permitted the grouping of isolates of L. bostrychophila into three distinct clusters. The correlation between genetic distance and geographic distance was not significant.     

Genetic structure and diversity in relation to the recently reduced population size of the rare conifer, <Emphasis Type="Italic">Pseudotsuga japonica</Emphasis>, endemic to Japan

Rare species consisting of small populations are subject to random genetic drift, which reduces genetic diversity. Thus, determining the relationship between population size and genetic diversity would provide key information for planning a conservation strategy for rare species. We used six microsatellite markers to investigate seven extant populations of the rare conifer Pseudotsuga japonica, which is endemic to the Kii Peninsula and Shikoku Island regions that are geographically separated by the Kii Channel in southwest Japan. The population differentiation of P. japonica was relatively high (F_ST = 0.101) for a coniferous species, suggesting limited gene flow among populations. As expected, significant regional differentiation (AMOVA; p?<?0.05) indicated genetic divergence across the Kii Channel. A strong positive correlation between census population size and the number of rare alleles (r?=?0.862, p?<?0.05) was found, but correlations with major indices of genetic diversity were not significant (allelic richness: r?=?0.649, p?=?0.104, expected heterozygosity: r?=?0.361, p?=?0.426). The observed order of magnitude of correlation with three genetic diversity indices corresponded with the theoretically expected order of each index’ sensitivity (i.e., the rate of decline per generation) to the bottleneck event. Thus, features that exhibit a faster response, i.e., the number of rare alleles, would have been subject to deleterious effects of the recent decline in population size, which is presumably caused by the development of extensive artificial plantations of other tree species over the last several decades. Finally, we propose a conservation plan for P. japonica based on our findings.