Search

Deer species were repeatedly overexploited and protected for their meat and fur and they had strong impacts on ecosystems and human society by damaging crops and planted trees, altering vegetation, deer vehicle collision, and increasing ticks that vector zoonosis. To accomplish appropriate population management, the historical demography and its main driver need to be clarified. In this study, we estimated the historical demography of effective population size ( Ne) of sika deer ( Cervus nippon Temminck) in Hokkaido and Hyogo Prefectures of the Japanese archipelago. We estimated Ne of >100 generations from present (2020) by folded single nucleotide polymorphisms (SNP) frequency spectra and, within 100 generations from present, by linkage disequilibrium between SNP. In Hokkaido, Ne drastically increased around 3.0 ky BP and decreased around 100–150 years ago with the assumption of their generation length as 4 or 9 years. The Ne decreased by a 10th before the recent bottleneck. In Hyogo, Ne increased around 80 and 1 ky BP and decreased around 100–250 years ago. Ne decreased by a 100th before the recent bottleneck. After these recent bottlenecks, Ne of both regions recovered and the current Ne has nearly reaches the highest level of the last 100 ky BP. Literature survey and paleoclimate indicates that the decrease and increase of Ne of sika deer in Japanese archipelago may be caused by variations in the hunting activity of humans rather than climate change or the top predator extinction.

Biogenic volatile organic compounds emitted from plants are important constituents of atmospheric chemistry and play a major role in the resistance of plants against various environmental stresses. However, little is known about how abiotic and biotic environments on a geographic scale relate to diversifications of the emission. Here, we present variations of terpenes stored in and emitted from leaves of a single species in a common garden, using genetically differentiated local populations of Japanese cedar, the most dominant and widely distributed tree species in Japan. Furthermore, we determined the composition of fungal communities in 50 locations, based on the presence or absence of 158 fungal species inhabiting the cedar. The results showed that terpenoids, especially those that are emitted, were highly diversified and geographically structured among the 12 populations. The total amount of stored terpenes was negatively affected by warm and less-snow climates. On the other hand, variations in some emitted terpenoid species among the populations were correlated to antagonistic fungal species inhabiting the Japanese cedar. We propose that the diversification of composition and amount of stored and emitted terpenoids in the tree species is not only structured by climate, but also antagonistic fungal communities through biological interactions.

Aim: We developed the Training Program on Child Abuse Prevention for Citizens (TCAP-C) and tested its effects and acceptability among citizen leaders (CLs). Methods: Community-based participatory research using a pretest–posttest follow-up design was conducted in Tokyo, Japan from September 2021 to March 2022. Participants completed questionnaires before, upon completion, and one month and three months after TCAP-C. Recognition, knowledge, and behaviors regarding child abuse and community consciousness were collected and compared before and one and three months after TCAP-C, and the degree of satisfaction, understanding, and meaningfulness were collected upon completion. We analyzed data using repeated-measures ANCOVA. Results: A total of 111, 98, 101, and 94 participants completed the questionnaires before, upon completion, and one and three months after TCAP-C, respectively. Overall, the recognition, knowledge, and community consciousness scores significantly improved from before to one month and three months after TCAP-C. Regarding the behaviors, only the behaviors of learning and watching over were significantly improved from before to one month after TCAP-C; however, those behaviors were not different between before and three months after TCAP-C. Furthermore, 95% participants reported being entirely satisfied with TCAP-C, and 85% and 91% reported good understanding and meaningfulness of the program. Conclusions: TCAP-C is acceptable and can improve CL recognition, knowledge, and community consciousness.

Shorea parvifolia (Dipterocarpaceae) is a widely distributed tree species which is important in terms of ecosystem functioning as well as forestry in Southeast Asia. During glacial periods, substantial precipitation decline is believed to have occurred in Southeast Asia, which considerably changed the distribution of the species. Repeated glacial and inter-glacial fluctuations were found to have influenced the genetic structure of the species, which is important to know for conservation and sustainable use. Leaf samples were collected from 18 populations covering most of the natural distribution of this species including the Malay Peninsula, Sumatra, and Borneo Islands. We investigated these samples using sequence data for eight chloroplast DNA (cpDNA) regions and 14 nuclear EST-SSR loci. The nucleotide diversity of cpDNA is higher in Malay Peninsula populations but the genetic diversity of nuclear DNA is higher in Borneo populations. The genetic structure revealed by nuclear DNA clearly separated Borneo populations from the rest, with an FST value of 0.150, while the genetic structure obtained from cpDNA was less pronounced (FST value = 0.136). Tajima’s D and Fu and Li’s D* for cpDNA showed statistical significance only in populations from Borneo. These results suggested that there has been recent population expansion of S. parvifolia in Borneo.

(1) Background: The genetic basis of local adaptation in conifers remains poorly understood because of limited research evidence and the lack of suitable genetic materials. Sakhalin fir (Abies sachalinensis) is an ideal organism for elucidating the genetic basis of local adaptation because its altitudinal adaptation has been demonstrated, and suitable materials for its linkage mapping are available. (2) Method: We constructed P336 and P236 linkage maps based on 486 and 516 single nucleotide polymorphisms, respectively, that were derived from double digest restriction site-associated DNA sequences. We measured the growth and eco-physiological traits associated with morphology, phenology, and photosynthesis, which are considered important drivers of altitudinal adaptation. (3) Results: The quantitative trait loci (QTLs) for growth traits, phenology, needle morphology, and photosynthetic traits were subsequently detected. Similar to previous studies on conifers, most traits were controlled by multiple QTLs with small or moderate effects. Notably, we detected that one QTL for the crown area might be a type-A response regulator, a nuclear protein responsible for the cytokinin-induced shoot elongation. (4) Conclusion: The QTLs detected in this study include potentially important genomic regions linked to altitudinal adaptation in Sakhalin fir.

Summary Introgression from one species in a specific environment to another may facilitate colonization of the environment by the recipient species. However, such environment‐dependent introgression has been clarified in limited plant taxa.In northern Japan, there are two interfertile oak species: Quercus dentata (Qd) in coastal areas and Q. mongolica var. crispula (Qc) in inland areas. However, at higher latitudes where Qd is rare, a coastal Qc ecotype with Qd‐like traits is distributed in the coastal areas. We distinguished inland Qc, coastal Qc, and coastal Qd populations based on genome‐wide genotypes and multitrait phenotypes and verified introgression from coastal Qd to coastal Qc using reduced library sequencing.Genotypes and phenotypes differed among the populations, and coastal Qc was intermediate between inland Qc and coastal Qd. The ABBA–BABA test showed introgression from coastal Qd to coastal Qc. In coastal Qc, we found various stages of introgression after the first generation of backcross but detected no genomic regions where introgression was enhanced.Overall, we show evidence for introgression from a coastal species to an ecotype of an inland species, which has colonized the coastal environment. It remains unclear whether introgressed alleles are selected in the coastal environment., See also the Editorial on this article by https://doi.org/10.1111/nph.16560.

The main purpose of this study was to investigate how a shift in seed sources owing to the implementation of a tree improvement program affected the genetic diversity of plantation forests. We investigated the genetic diversity and pairwise relatedness between individuals in sugi (Cryptomeria japonica D Don) plantations established before (“Pre-B”) and after (“Post-B”) the start of the Japanese tree improvement program. Based on SSR and SNP markers, most genetic diversity indices and the degree of relatedness within plantations were comparable between “Pre-B” and “Post-B”. However, there were large variations in the proportion of closely related pairs within plantations in both groups. Additionally, in “Post-B” plantations, parental contributions greatly diverged among plus-tree clones. The results of this study indicated that the shift in the seed sources, caused by the implementation of a tree improvement program, did not have a significant effect on the genetic diversity of plantations. However, the results also indicated that the degree of relatedness within plantations varied significantly according to how seeds are collected regardless of the type of seed sources. Furthermore, it was also revealed that genetic diversity in “Post-B” plantations could have decreased because of a deviation from equal parental contributions in seed orchards. Our results indicated that effective management of seed orchards is important to secure the genetic diversity of plantation forests.

The evolution of a species depends on multiple forces, such as demography and natural selection. To understand the trajectory and driving forces of evolution of a target species, it is first necessary to uncover that species’ population history, such as past and present population sizes, subdivision and gene flow, by using appropriate genetic markers. Cryptomeria japonica is a long-lived monoecious conifer species that is distributed in Japan. There are two main lines (omote-sugi and ura-sugi), which are distinguished by apparent differences in morphological traits that may have contributed to their local adaptation. The evolution of these morphological traits seems to be related to past climatic changes in East Asia, but no precise estimate is available for the divergence time of these two lines and the subsequent population dynamics in this species. Here, we analyzed the nucleotide variations at 120 nuclear genes in 94 individuals by using amplicon sequencing in combination with high-throughput sequencing technologies. Our analysis indicated that the population on Yakushima Island, the southern distribution limit of C. japonica in Japan, diverged from the other populations 0.85 million years ago (MYA). The divergence time of the other populations on mainland Japan was estimated to be 0.32 MYA suggesting that the divergence of omote-sugi and ura-sugi might have occurred before the last glacial maximum. Although we found modest levels of gene flow between the present populations, the long-term isolation and environmental heterogeneity caused by climatic changes might have contributed to the differentiation of the lines and their local adaptation.

Twenty polymorphic microsatellite markers were developed, using Next Generation Sequencing (Illumina), from genomic DNA of Parashorea malaanonan, a species of the Dipterocarpa­ceae which is ecologically and economically important in the Philippines. Thirty adult trees from a natural population were used to assess the success of PCR amplification and the degree of polymorphism. The number of alleles per locus varied from three to 13, and observed and expected heterozygosity varied from 0.200 to 0.808 and from 0.301 to 0.890 respectively. Total exclusion probabilities for the first and second parents over the 20 loci were 0.99932499 and 0.99999723 respectively. The high level of polymorphism at these loci makes it possible to obtain precise estimates of genetic parameters and thus the markers will help in studies on population genetics, conservation gene­tics, and molecular ecology of P. malaanonan.

Cryptomeria japonica is a major forestry tree species in Japan. Male sterility of the species is caused by a recessive gene, which shows dysfunction of pollen development and results in no dispersed pollen. Because the pollen of C. japonica induces pollinosis, breeding of pollen-free C. japonica is desired. In this study, single nucleotide polymorphism (SNP) markers located at 1.78 and 0.58 cM to a male sterility locus (MS1) were identified from an analysis of RNA-Seq and RAD-Seq, respectively. SNPs closely linked to MS1 were first scanned by a method similar to MutMap, where a type of index was calculated to measure the strength of the linkage between a marker sequence and MS1. Linkage analysis of selected SNP markers confirmed a higher efficiency of the current method to construct a partial map around MS1. Allele-specific PCR primer pair for the most closely linked SNP with MS1 was developed as a codominant marker, and visualization of the PCR products on an agarose gel enabled rapid screening of male sterile C. japonica. The allele-specific primers developed in this study would be useful for establishing the selection of male sterile C. japonica.

Pyramiding of male-sterile genes in Cryptomeria japonica is currently being carried out in Niigata prefecture, Japan. This is the first attempt to apply pyramid breeding to forest trees. As the breeding materials for male sterility are limited, special attention must be given to increased genetic relatedness in the process of pyramid breeding to avoid the effects of inbreeding depression as much as possible. In this study, we estimated genetic relatedness based on 246 genome-wide SNP markers for male-sterile individuals in Niigata Prefecture (n = 6) and individuals doubly heterozygous for two male-sterile genes (hereafter referred to as “double-hetero”) produced by marker- assisted selection (n = 124). The pairwise relatedness estimates between male-sterile individuals selected from the same area in Niigata Prefecture were low (−0.01 ± 0.08, mean ± standard deviation), suggesting that there will be almost no negative effects even if the F1 of these male-sterile individuals is used for artificial crossing. On the other hand, the pairwise relatedness between double-hetero individuals in this study was higher than the theoretical relatedness values, as individuals with the slightly higher relatedness were used as parents in artificial crossings. However, there was a large variance in pairwise relatedness for double-hetero individuals. This result suggested that it may be possible to avoid the adverse effects of inbreeding depression by using a pair of double-heteros with lower relatedness for artificial crossing, when we produce a double-homo using the limited breeding materials of male- sterile individuals. It will also be important to continue additional selection of new breeding material for male sterility.

Objective Due to the allergic nature of the pollen of Cryptomeria japonica, the most important Japanese forestry conifer, a pollen-free cultivar is preferred. Mutant trees detected in nature have been used to produce a pollen-free cultivar. In order to reduce the time and cost needed for production and breeding, we aimed to develop simple diagnostic molecular markers for mutant alleles of the causative gene MALE STERILITY 1 (MS1) in C. japonica to rapidly identify pollen-free mutants. Results We developed PCR and LAMP markers to detect mutant alleles and to present experimental options depending on available laboratory equipment. LAMP markers were developed for field stations, where PCR machines are unavailable. The LAMP method only needs heat-blocks or a water bath to perform the isothermal amplification and assay results can be read by the naked eye. Because the causative mutations were deletions, we developed two kinds of PCR markers, amplified length polymorphism (ALP) and allele specific PCR (ASP) markers. These assays can be visualized using capillary or agarose gel electrophoresis.

Identifying causative genes for a target trait in conifer reproduction is challenging for species lacking whole-genome sequences. In this study, we searched for the male-sterility gene (MS1) in Cryptomeria japonica, aiming to promote marker-assisted selection (MAS) of male-sterile C. japonica to reduce the pollinosis caused by pollen dispersal from artificial C. japonica forests in Japan. We searched for mRNA sequences expressed in male strobili and found the gene CJt020762, coding for a lipid transfer protein containing a 4-bp deletion specific to male-sterile individuals. We also found a 30-bp deletion by sequencing the entire gene of another individual with the ms1. All nine breeding materials with the allele ms1 had either a 4-bp or 30-bp deletion in gene CJt020762, both of which are expected to result in faulty gene transcription and function. Furthermore, the 30-bp deletion was detected from three of five individuals in the Ishinomaki natural forest. From our findings, CJt020762 was considered to be the causative gene of MS1. Thus, by performing MAS using two deletion mutations as a DNA marker, it will be possible to find novel breeding materials of C. japonica with the allele ms1 adapted to the unique environment of each region of the Japanese archipelago.

Research Highlights: We detected the longitudinal gradients of genetic diversity parameters, such as the number of alleles, effective number of alleles, heterozygosity, and inbreeding coefficient, and found that these might be attributable to climatic conditions, such as temperature and snow depth. Background and Objectives: Genetic diversity among local populations of a plant species at its distributional margin has long been of interest in ecological genetics. Populations at the distribution center grow well in favorable conditions, but those at the range margins are exposed to unfavorable environments, and the environmental conditions at establishment sites might reflect the genetic diversity of local populations. This is known as the central-marginal hypothesis in which marginal populations show lower genetic variation and higher differentiation than in central populations. In addition, genetic variation in a local population is influenced by phylogenetic constraints and the population history of selection under environmental constraints. In this study, we investigated this hypothesis in relation to Abies sachalinensis, a major conifer species in Hokkaido. Materials and Methods: A total of 1189 trees from 25 natural populations were analyzed using 19 EST-SSR loci. Results: The eastern populations, namely, those in the species distribution center, showed greater genetic diversity than did the western peripheral populations. Another important finding is that the southwestern marginal populations were genetically differentiated from the other populations. Conclusions: These differences might be due to genetic drift in the small and isolated populations at the range margin. Therefore, our results indicated that the central-marginal hypothesis held true for the southernmost A. sachalinensis populations in Hokkaido.

To understand how historical and environmental factors have shaped genetic diversity and spatial genetic patterns of tree species, we characterized the genetic diversity and the genetic structure of a coniferous species, Cryptomeria japonica, which is distributed in Japan (two taxonomic groups) and some parts of southeastern China (C. japonica var. sinensis). We investigated population samples for three groups using high-throughput SNP genotyping, sequencing of multiple genes, and then estimated level of genetic diversity, the genetic differentiation, and the divergence time between the three groups. We also investigated the natural distribution during LGM using species distribution modeling. The genetic variation in the Chinese population, var. sinensis, was lower compared to Japanese populations but it harbored some unique genotypes. The species distribution modeling showed that the Chinese population has decreased since Last Glacial Maximum (LGM) because of lower precipitation than present time, but in Japan the favored environment have remained available for this species even in the LGM. Estimated divergence times of the three groups mostly correspond with the time since the Last Glacial. Our data supports the hypothesis that the glacial period, particularly limitations associated with precipitation, strongly influenced the distribution of C. japonica populations. Our findings suggest that climate change plays an important role in speciation processes of C. japonica.

Expressed sequence tagged (EST) microsatellite (SSR) markers were newly developed for Callicarpa subpubescens, a species endemic to the Bonin (Ogasawara) Islands of Japan, to investigate gene flow,...

Maire yew (Taxus chinensis var. mairei (Lemee et Levl) Cheng et L.K. Fu) is an endangered coniferous species in China. We examined the genetic pattern of Maire yew populations and explored any gene...

Todo-matsu (Abies sachalinensis) is one of the most important forestry species in Hokkaido, Japan and is distributed from near sea level to the alpine zone. Due to its wide spatial distribution, the species adapts to its environment, displaying phenotypes of ecological relevance. In order to identify candidate genes under natural selection, we collected the transcriptome from the female and male flower, leaf and inner bark. De novo assembly with 34.7 Gb of sequencing reads produced 158,542 transcripts from 69,618 loci, whose estimated coverage reached 95.6% of conserved eukaryotic genes. Homology searches against publicly available databases identified 134,190 (84.6%) transcripts with at least one hit. In total, 28,944 simple sequence repeats (SSRs) and 80,758 single nucleotide variants (SNVs) were detected from 23,570 (14.9%) and 25,366 (16.0%) transcripts, which were valuable for use in genetic analysis of the species. All the annotations were included in a relational database, TodoFirGene, which provides an interface for various queries and homology search, and can be accessed at http://plantomics.mind.meiji.ac.jp/todomatsu/. This database hosts not only the A. sachalinensis transcriptome but also links to the proteomes of 13 other species, allowing a comparative genomic study of plant species.

Recently, there has been a continuous increase in the concentration of tropospheric ozone in urban forests in Japan. Since monoterpenes are precursors to ozone, we need to evaluate the effects of ozone exposure on all tree species that are considered to be sources of monoterpenes. Cryptomeria japonica, which is the most widely planted afforestation tree, is classified into three different gene pools. However, the interclone variation for seasonal changes in the ozone exposure effect has not been evaluated. Thus, free-air ozone enhancement experiments were conducted using three representative clones of C. japonica in the summer and winter. After exposure to twice the ozone concentration in the ambient atmosphere, the effect on the monoterpene emission rate was found to be considerably different among the clones and for the different seasons. The monoterpene emission rate after ozone exposure increased in winter and summer in the native clones (Donden and Yakushima) in the snow area and heavy rain area, respectively. Since monoterpenes are antibacterial substances, each clone adapted sensitivity to stress during each season upon considerable damage. These results suggest that not only differences between tree species but also differences between clones are important for evaluating seasonal variation characteristics after ozone exposure.

Genomic regions which affected tree growth and wood property traits were investigated in the major plantation tree of Japan, Cryptomeria japonica, in three replicated common garden experiments planted in contrasting environments in Kyushu and Honshu, Japan. Phenotypic traits measured were stem diameter at breast height, tree height, wood strength (Young’s modulus), heartwood density, sapwood density, heartwood moisture content, and sapwood moisture content. Quantitative trait locus (QTL) analysis identified an average of 53 QTLs across the three environments. There were two QTLs which affected the same traits across all three environments. These stable QTLs were identified as being associated with sapwood density and Young’s modulus and explained 3.5–11.3% and 2.1–18.7% of the total genotypic variation, respectively. In contrast, the majority of QTLs detected were unique to only one environment, a finding which is consistent with QTL mapping studies of other forest trees, indicating a substantial contribution of environmental effects on the mapping progenies. Nonetheless, the two stable QTLs identified in this study could be important genomic regions to target for further research aimed at maximizing breeding efficiency and wood quality of C. japonica across wide environmental gradients.

Clonal reproduction of lianas is a common but important life history strategy. It is necessary to evaluate the clonal structure of liana species because clonal ability is potentially a major determinants distribution pattern of lianas. Therefore, we developed 10 microsatellite markers for Euonymus fortunei and Schizophragma hydrangeoides respectively from genomic sequences obtained from double-digest restriction site associated DNA (ddRAD). The sequence data of the developed markers were deposited on the public database. The expected heterozygosity (HE) of E. fortunei and S. hydrangeoides ranged from 0.727 to 0.847 with an average of 0.766, and from 0.734 to 0.924 with an average of 0.812, respectively. All loci were under HWE except for a locus of S. hydrangeoides (sh07). These markers should contribute to the understanding of the life history of temperate liana species.

Research highlights: Our study is the first to explore the genetic composition of ancient Cryptomeria trees across a distribution range in China. Background and objectives: Cryptomeria japonica var. sinensis is a native forest species of China, it is widely planted in the south of the country to create forests and for wood production. Unlike Cryptomeria in Japan, genetic Chinese Cryptomeria has seldom been studied, although there is ample evidence of its great ecological and economic value. Materials and methods: Because of overcutting, natural populations are rare in the wild. In this study, we investigated seven ancient tree populations to explore the genetic composition of Chinese Cryptomeria through ddRAD-seq technology. Results: The results reveal a lower genetic variation but higher genetic differentiation (Ho = 0.143, FST = 0.1204) than Japanese Cryptomeria (Ho = 0.245, FST = 0.0455). The 86% within-population variation is based on an analysis of molecular variance (AMOVA). Significant excess heterozygosity was detected in three populations and some outlier loci were found, these were considered to be the consequence of selection or chance. Structure analysis and dendrogram construction divided the seven ancient tree populations into four groups corresponding to the geographical provinces in which the populations are located, but there was no obvious correlation between genetic distance and geographic distance. A demographic history analysis conducted by a Stairway Plot showed that the effective population size of Chinese Cryptomeria had experienced a continuing decline from the mid-Pleistocene to the present. Our findings suggest that the strong genetic drift caused by climate fluctuation and intense anthropogenic disturbance together contributed to the current low diversity and structure. Considering the species&rsquo, unfavorable conservation status, strategies are urgently required to preserve the remaining genetic resources.

Shorea platyclados (Dark Red Meranti) is a commercially important timber tree species in Southeast Asia. However, its stocks have dramatically declined due, inter alia, to excessive logging, insufficient natural regeneration and a slow recovery rate. Thus, there is a need to promote enrichment planting and develop effective technique to support its rehabilitation and improve timber production through implementation of Genome-Wide Association Studies (GWAS) and Genomic Selection (GS). To assist such efforts, plant materials were collected from a half-sib progeny population in Sari Bumi Kusuma forest concession, Kalimantan, Indonesia. Using 5900 markers in sequences obtained from 356 individuals, we detected high linkage disequilibrium (LD) extending up to &gt, 145 kb, suggesting that associations between phenotypic traits and markers in LD can be more easily and feasibly detected with GWAS than with analysis of quantitative trait loci (QTLs). However, the detection power of GWAS seems low, since few single nucleotide polymorphisms linked to any focal traits were detected with a stringent false discovery rate, indicating that the species&rsquo, phenotypic traits are mostly under polygenic quantitative control. Furthermore, Machine Learning provided higher prediction accuracies than Bayesian methods. We also found that stem diameter, branch diameter ratio and wood density were more predictable than height, clear bole, branch angle and wood stiffness traits. Our study suggests that GS has potential for improving the productivity and quality of S. platyclados, and our genomic heritability estimates may improve the selection of traits to target in future breeding of this species

A mobile laser-induced fluorescence spectrum (LIFS) lidar was developed for monitoring pollens floating in the atmosphere. The fluorescence spectrum of pollens excited at 355 nm was measured with a fluorescence spectrometer and the results suggested that in general they had peaks at around 460 nm and the ranges were 400–600 nm. A fluorescence spectrum database of 25 different pollens was made with the 355 nm excitation. Based on these results, we developed a LIFS lidar that had features in pollen species identification and daytime operation. The former was achieved by the database and the latter was possible by introducing a synchronous-delay detection to a gated CCD spectrometer in an operation time of 200 ns. Fluorescence detection of pollens floating in the atmosphere was performed using the LIFS lidar in a field where cedars grow in the spring and ragweed in the autumn. The LIFS lidar system successfully detected fluorescence spectrums of the pollens at a distance of approximately 20 m away. We discussed the performance of the LIFS lidar by estimating the number of cedar pollens using a lidar equation, introducing a fluorescence cross section of cedar pollens and a sensitivity of the CCD spectrometer that was measured by ourselves.

The island of Borneo is the diversity center of the Dipterocarpaceae, the most important family of tropical rainforest trees in Southeast Asia. However, changes in land use and climate have affected dipterocarp distributions on the island, raising concerns about the vulnerability (inter alia) of the endemic riparian species Shorea macrophylla. Thus, to aid conservation efforts, we have investigated the genetic diversity, structure, and demographic history of S. macrophylla. The species’ genetic diversity and structure in Kalimantan (part of Indonesia, covering 75% of the island) were explored by examining genotypes of 377 individuals representing 13 populations in three regions (Northeast, Central, and West Kalimantan) using 14 newly developed microsatellite loci. Higher genetic diversity was found, at all loci, in samples from Northeast Kalimantan than in samples from the other regions. Moderate genetic differentiation between populations was detected (FST 0.093). Bayesian clustering, principal coordinate, and neighbor joining tree analyses of the population structure consistently identified two genetically distinct groups, one in the Northeast and the other in the Central and West regions. The higher diversity of the diverged populations in Northeast Kalimantan indicates that the region may have hosted rainforest refugia during the ice age. Accordingly, analysis using DIY ABC software indicated that the Northeast and Central-West Kalimantan groups diverged 194,000 years ago. We conclude that global climate change during the Pleistocene strongly influenced the genetic diversity and structure of S. macrophylla populations in Kalimantan.

Pollinosis caused by Japanese cedar (Cryptomeria japonica) is a widespread social problem in Japan. To date, 23 male-sterile C. japonica trees have been selected in Japan to address pollinosis, from which four male-sterility loci (MS1, MS2, MS3, and MS4) have been identified from test crossing results. For efficient breeding of male-sterile C. japonica trees, more male-sterile individuals and individuals heterozygous for male-sterile genes are required. Therefore, we aimed to develop DNA markers for marker-assisted selection of four types of male-sterile genes from populations without a family structure. First, for four families exhibiting segregation of each male-sterile locus (MS1, MS2, MS3, and MS4), genome-wide single-nucleotide polymorphism and insertion/deletion (indel) genotyping was performed using the Axiom myDesign Targeted Genotyping Array method. Four high-density linkage maps for mapping the MS1, MS2, MS3, and MS4 families were constructed, which included 4923, 1722, 1896, and 2247 markers, respectively. In these maps, 15, 4, 2, and 2 markers were located 0.0, 3.3, 1.1, and 0.0 cM from the MS1, MS2, MS3, and MS4 loci, respectively. Second, for the markers located 0.0 cM from a male-sterile locus (i.e., MS1 and MS4), to clarify the most tightly linked markers, we calculated the prediction rate of male-sterile gene genotypes from marker genotypes for 78 trees. The markers with the highest prediction rates were AX-174127446 (0.95) for MS1 and AX-174121522 (1.00) for MS4. The AX-174121522 marker was considered to be suitable for selecting trees homozygous or heterozygous for the MS4 gene from plus-trees without a pollination test, which requires a large amount of time and effort. The nearest markers to the male-sterile loci found in this study may facilitate the isolation of male-sterile genes in C. japonica via combination with the draft genomic sequence that is currently being collated.

Cryptomeria japonica is a major forestry tree species in Japan. Male sterility of the species is caused by a recessive gene, which shows dysfunction of pollen development and results in no dispersed pollen. Because the pollen of C. japonica induces pollinosis, breeding of pollen-free C. japonica is desired. In this study, single nucleotide polymorphism (SNP) markers located at 1.78 and 0.58 cM to a male sterility locus (MS1) were identified from an analysis of RNA-Seq and RAD-Seq, respectively. SNPs closely linked to MS1 were first scanned by a method similar to MutMap, where a type of index was calculated to measure the strength of the linkage between a marker sequence and MS1. Linkage analysis of selected SNP markers confirmed a higher efficiency of the current method to construct a partial map around MS1. Allele-specific PCR primer pair for the most closely linked SNP with MS1 was developed as a codominant marker, and visualization of the PCR products on an agarose gel enabled rapid screening of male sterile C. japonica. The allele-specific primers developed in this study would be useful for establishing the selection of male sterile C. japonica.

Deep sequencing of small RNAs (sRNAs) in developing male strobili of second-generation offspring originating from a nuclear genic male sterile tree of Cryptomeria japonica were performed to characterize sRNA populations in the male strobili at early pollen developmental stages. Comparing to sequences of microRNA (miRNA) families of plant species and sRNAs expressed in the reproductive organs of representative vascular plants, 37 conserved miRNA families were detected, of which eight were ubiquitously expressed in the reproductive organs of land plant species. In contrast, miR1083 was common in male reproductive organs of gymnosperm species but absent in angiosperm species. In addition to conserved miRNAs, 199 novel miRNAs candidates were predicted. The expression patterns of the obtained sRNAs were further investigated to detect the differentially expressed (DE) sRNAs between genic male sterile and fertile individuals. A total of 969 DE sRNAs were obtained and only three known miRNA families were included among them. These results suggest that both conserved and species-specific sRNAs contribute to the development of male strobili in C. japonica.

Selective logging is one of several silvicultural practices used in sustainable forest management in the lowland dipterocarp forest in Indonesia. Selecting only trees with diameters >50 cm for logging can reduce the density of reproductive trees, thereby affecting pollen dispersal and influencing the mating system among remaining trees. We evaluated the effect of logging rotations on the mating system, gene flow and genetic diversity in populations of Shorea parvifolia in primary forest, and in first and second rotation forest. Our results revealed that multiple (or at least two) selective logging events with a 30-year logging rotation had a significant impact on the genetic diversity of pollen clouds. However, the average pollen dispersal distance did not differ significantly among the multiple selective logging rotations. The multiple rotations reduced the outcrossing rate and the number effective of pollen donors in the logged forest. Moreover, the number of pollen donors in a plot was affected by the basal area of reproductive trees present. These results suggest that reducing the number of reproductive trees by logging with multiple rotations might increase the bi-parental inbreeding rate due to the reduction in density of reproductive trees in a selectively logged forest. We conclude that multiple rotations with a 30-year cycle of selective logging as currently practiced would reduce the density of reproductive trees, and would not be sustainable in terms of maintaining genetic diversity in tropical forests of Southeast Asia.

Local adaptation of plant species is a central issue for survival during global climate change, especially for long-lived forest trees, with their lengthy regeneration time and spatially limited gene flow. Identification of loci and/or genomic regions associated with local adaptation is necessary for knowledge of both evolution and molecular breeding for climate change. Cryptomeria japonica is an important species for forestry in Japan; it has a broad natural distribution and can survive in a range of different environments. The genetic structure of 14 natural populations of this species was investigated using 3930 SNP markers. Populations on the Pacific Ocean side of Japan are clearly different from those on the Japan Sea side, as discussed in previous studies. Structure analysis and population network trees show that peripheral populations, including the most northerly and southerly ones, have unique features. We found that the genetic differentiation coefficient is low, FST = 0.05, although it must account for the presence of important genes associated with adaptation to specific environments. In total, 208 outlier loci were detected, of which 43 were associated with environmental variables. Four clumped regions of outlier loci were detected in the genome by linkage analysis. Linkage disequilibrium (LD) was quite high in these clumps of outlier loci, which were found in linkage groups (LGs) 2, 7, 10, and 11, especially between populations of two varieties, and when interchromosomal LD was also detected. The LG7 region is characteristic of the Yakushima population, which is a large, isolated, peripheral population occupying a specific environment resulting from isolation combined with volcanic activity in the region. The detected LD may provide strong evidence for selection between varieties.

Population structure and genome-wide linkage disequilibrium (LD) were investigated in a Cryptomeria japonica core collection consisting of 510 individuals and providing comprehensive coverage of the natural distribution range of the species, using 4,896 SNP markers. Both Bayesian clustering and principal coordinate analyses clearly subdivided the sample into two distinct groups corresponding to the Japan Sea side and the Pacific Ocean side of Japan. The clustering analysis also detected two subgroups located in Northern Tohoku and Yakushima Island, as was also the case in the natural population. This finding is in accord with the ecogeographical origin of the cultivars; however, the spatial distribution of these clusters was slightly different between the natural population and the core collection, especially in the Chugoku district, an effect which might have resulted from historical human activity. The genetic diversity of the core collection is comparable to, or a little higher than, that of natural populations, indicating that the individuals in the core collection have not suffered from diversity losses caused by a domestication bottleneck. LD analysis with mapped markers indicated that the LD decays rapidly, within approximately 5 cM, in both the core collection and natural populations. The results show that the population described here is a valuable resource for both basic and applied research on C. japonica.

Clonal reproduction is a crucial factor shaping the demography and genetic structure of many plant populations. However, we still do not understand the extent to which environmental and genetic factors influence clonal reproduction. Natural forests of Cryptomeria japonica are distributed under a range of environmental conditions in the Japanese Archipelago. In this study, the spatial genetic structures of 13 natural old-growth C. japonica forest plots of about 0.25 ha were investigated using eight nuclear microsatellite markers. Clonal reproduction was detected in 10 plots, the clonal frequency in each plot varied. Patterns of spatial genetic structure at the ramet level showed significant positive autocorrelation over short distances, which may reflect clonal reproduction and the limited extent to which seedlings establish in gaps. The clonality of individual genets was influenced by the Q value of cluster 1 of the results of STRUCTURE analysis as the genetic factor, snow depth as the environmental factor, the average basal area (BA) and the interaction between Q value and snow depth. This result indicated that clonal genets had low Q values for cluster 1 and small BA, especially in regions experiencing heavy snow. On the other hand, the average BA had a negative effect for a number of ramets, however the parameter estimate was small. Our results suggest that clonal reproduction may have been the most important of the strategies exploited in order to establish in harsh environments like frequent disturbance conditions such as heavy snow region in this species.

Long-distance dispersal (LDD) of seeds has a critical impact on species survival in patchy landscapes. However, relative to pollen dispersal, empirical data on how seed LDD affects genetic diversity in fragmented populations have been poorly reported. Thus, we attempted to indirectly evaluate the influence of seed LDD by estimating maternal and paternal inbreeding in the seed rain of fragmented 8 Pinus densiflora populations. In total, the sample size was 458 seeds and 306 adult trees. Inbreeding was estimated by common parentage analysis to evaluate gene flow within populations and by sibship reconstruction analysis to estimate gene flow within and among populations. In the parentage analysis, the observed probability that sampled seeds had the same parents within populations was significantly larger than the expected probability in many populations. This result suggested that gene dispersal was limited to within populations. In the sibship reconstruction, many donors both within and among populations appeared to contribute to sampled seeds. Significant differences in sibling ratios were not detected between paternity and maternity. These results suggested that seed-mediated gene flow and pollen-mediated gene flow from outside population contributed some extent to high genetic diversity of the seed rain (H E > 0.854). We emphasize that pine seeds may have excellent potential for gene exchange within and among populations.