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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.

Crepidiastrum grandicollum is a critically endangered insular endemic herb found only on two oceanic islands of the Bonin Islands in Japan, namely Chichijima Island and Anijima Island. It is explicitly threatened by herbivory pressure from introduced animals. In 2009, a conservation program for C. grandicollum was begun to ensure its future persistence. To provide further information for conservation planning, we investigated the genetic diversity of C. grandicollum using 13 novel microsatellite markers in 55 individuals from four wild populations and an ex situ living collection. Two closely related woody species were also included for interspecific comparison: seven individuals of Crepidiastrum ameristophyllum and 13 of Crepidiastrum linguifolium. The 13 markers were applicable to all three species and identified 129 alleles in total. We found a clear genetic differentiation between C. grandicollum from Anijima Island and Chichijima Island. Crepidiastrum grandicollum also had low expected heterozygosity and allelic richness in populations compared to the two closely related species. Reconstructed divergence history suggested that differentiation between the islands had occurred several thousand generations ago. We suggest separate conservation units for C. grandicollum on Anijima Island and Chichijima Island given the clear (and putatively historical) genetic differentiation, which may result in speciation in the future.

Background and AimsPlants inhabiting arid environments tend to have leaf trichomes, but their adaptive significance remains unclear. Leaf trichomes are known to play a role in plant defence against herbivores, including gall makers. Because gall formation can increase water loss partly through increased surface area, we tested the novel hypothesis that leaf trichomes could contribute to avoiding extra water stress by impeding gall formation, which would have adaptive advantages in arid environments.MethodsWe focused on Metrosideros polymorpha, an endemic tree species in the Hawaiian Islands, whose leaves often suffer from galls formed by specialist insects, Hawaiian psyllids (Pariaconus spp.). There is large variation in the amount of leaf trichomes (0–40 % of leaf mass) in M. polymorpha. Three gall types are found on the island of Hawaii: the largest is the ‘cone’ type, followed by ‘flat’ and ‘pit’ types. We conducted laboratory experiments to quantify the extent to which gall formation is associated with leaf water relations. We also conducted a field census of 1779 individuals from 48 populations across the entire range of habitats of M. polymorpha on the island of Hawaii to evaluate associations between gall formation (presence and abundance) and the amount of leaf trichomes.Key ResultsOur laboratory experiment showed that leaf minimum conductance was significantly higher in leaves with a greater number of cone- or flat-type galls but not pit-type galls. Our field census suggested that the amount of trichomes was negatively associated with probabilities of the presence of cone- or flat-type galls but not pit-type galls, irrespective of environmental factors.ConclusionOur results suggest that leaf trichomes in M. polymorpha can contribute to the avoidance of extra water stress through interactions with some gall-making species, and potentially increase the fitness of plants under arid conditions.

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.

We developed microsatellite markers to compare the genetic variation and reproductive biology between the partially mycoheterotrophic Pyrola japonica var. japonica and the putative fully mycoheterotrophic P. japonica var. subaphylla. Fifteen primer pairs were developed for P. japonica sensu lato and they were tested on 77 ramets from three populations of the two varieties. Thirteen loci were polymorphic in at least one of the two var. japonica populations, whereas only four loci were polymorphic in the var. subaphylla population. The considerably lower genetic variation of the var. subaphylla population may be attributed to frequent selfing and/or inbreeding. The markers developed in this study will be useful for comparing the genetic diversity of P. japonica s. l. populations and measuring gene flow within and between populations and varieties.

Accurate feature annotation as well as assembly contiguity are important requisites of a modern genome assembly. They allow large-scale comparison of genomes across and within species and identification of polymorphisms, leading evolutionary and functional studies. We report an updated genome resource for Metrosideros polymorpha, the most dominant tree species in the Hawaiian native forests and a unique example of rapid and remarkable ecological diversification of woody species. Ninety-one percent of the bases in the sequence assembly (304 Mb) were organized into 11 pseudo-molecules, which would represent the chromosome structure of the species assuming the synteny to a close relative Eucalyptus. Our complementary approach using manual annotation and automated pipelines identified 11.30% of the assembly to be transposable elements, in contrast to 4.1% in previous automated annotation. By increasing transcript and protein sequence data, we predicted 27,620 gene models with high concordance from the supplied evidence. We believe that this assembly, improved for contiguity, and annotation will be valuable for future evolutionary studies of M. polymorpha and closely related species, facilitating the isolation of specific genes and the investigation of genome-wide polymorphisms associated with ecological divergence., G3: Genes, Genomes, Genetics, 9 (11), ISSN:2160-1836

Somatic embryogenesis (SE), which isin vitroregeneration of plant bodies from somatic cells, represents a useful means of clonal propagation and genetic engineering of forest trees. While protocols to obtain calluses and induce regeneration in somatic embryos have been reported for many tree species, the knowledge of molecular mechanisms of SE development is still insufficient to achieve an efficient supply of somatic embryos required for the industrial application.Cryptomeria japonica, a conifer species widely used for plantation forestry in Japan, is one of the tree species waiting for a secure SE protocol; the probability of normal embryo development appears to depend on genotype. To discriminate the embryogenic potential of embryonal masses (EMs) and efficiently obtain normal somatic embryos ofC.japonica, we investigated the effects of genotype and transcriptome on the variation in embryogenic potential. Using an induction experiment with 12 EMs each from six genotypes, we showed that embryogenic potential differs between/within genotypes. Comparisons of gene expression profiles among EMs with different embryogenic potentials revealed that 742 differently expressed genes were mainly associated with pattern forming and metabolism. Thus, we suggest that not only genotype but also gene expression profiles can determine success in SE development. Consistent with previous findings for other conifer species, genes encoding leafy cotyledon, wuschel, germin-like proteins, and glutathione-S-transferases are likely to be involved in SE development inC.japonicaand indeed highly expressed in EMs with high-embryogenic potential; therefore, these proteins represent candidate markers for distinguishing embryogenic potential.

Phyllosphere fungi show high species diversity and fulfill important ecological functions not only in natural forests but also in plantations. We sought to estimate the species diversity and community structure of phyllosphere fungi from tropical tree plantation plots in Central Kalimantan, Indonesia. We conducted a massively parallel amplicon sequencing analysis of fungi collected from the leaves of Shorea leprosula (Dipterocarpaceae), an ecologically and commercially important tree species. Phyllosphere fungal compositions and spatial variability were investigated for 31 S. leprosula trees across four plots within a plantation stand. In total, 488 fungal operational taxonomic units (OTUs) were recognized in 153,194 ribosomal internal transcribed spacer reads at 95% OTU identity level. Rare OTUs accounted for the majority of fungal diversity detected in the study site; 200 OTUs (41%) comprised fewer than 10 reads and 465 OTUs (95%) were found in fewer than half of the samples. Fungal OTU compositions of S. leprosula trees were differentiated within a narrow area of the plantation and even between plots that were separated by 15 m. These findings indicate that highly diverse fungal OTUs form spatially structured communities even within a tropical plantation stand of single tree species.

Whole genome sequences, which can be provided even for non-model organisms owing to high-throughput sequencers, are valuable in enhancing the understanding of adaptive evolution. Metrosideros polymorpha, a tree species endemic to the Hawaiian Islands, occupies a wide range of ecological habitats and shows remarkable polymorphism in phenotypes among/within populations. The biological functions of genetic variations observed within this species could provide significant insights into the adaptive radiation found in a single species. Here de novo assembled genome sequences of M. polymorpha are presented to reveal basic genomic parameters about this species and to develop our knowledge of ecological divergences. The assembly yielded 304-Mbp genome sequences, half of which were covered by 19 scaffolds with >5 Mbp, and contained 30 K protein-coding genes. Demographic history inferred from the genome-wide heterozygosity indicated that this species experienced a dramatic rise and fall in the effective population size, possibly owing to past geographic or climatic changes in the Hawaiian Islands. This M. polymorpha genome assembly represents a high-quality genome resource useful for future functional analyses of both intra- and interspecies genetic variations or comparative genomics.

Peroxisomes are well-known organelles that are present in most eukaryotic organisms. Mutant phenotypes caused by the malfunction of peroxisomes have been shown in many fungi. However, these have never been investigated in Agaricomycetes, which include white-rot fungi that degrade wood lignin in nature almost exclusively and play an important role in the global carbon cycle. Based on the results of a forward genetics study to identify mutations causing defects in the ligninolytic activity of the white-rot Agaricomycete Pleurotus ostreatus, we report phenotypes of pex1 disruptants in P. ostreatus, which are defective in two major features of white-rot Agaricomycetes: lignin biodegradation and mushroom formation. Pex1 disruption was also shown to cause defects in the hyphal growth of P. ostreatus on certain sawdust and minimum media. We also demonstrated that pex1 is essential for fruiting initiation in the non-wood decaying Agaricomycete Coprinopsis cinerea. However, unlike P. ostreatus, significant defects in hyphal growth on the aforementioned agar medium were not observed in C. cinerea. This result, together with previous C. cinerea genetic studies, suggests that the regulation mechanisms for the utilization of carbon sources are altered during the evolution of Agaricomycetes or Agaricales.

We developed microsatellite markers to compare the genetic variation and reproductive biology between the partially mycoheterotrophic Pyrola japonica var. japonica and the putative fully mycoheterotrophic P. japonica var. subaphylla. Fifteen primer pairs were developed for P. japonica sensu lato and they were tested on 77 ramets from three populations of the two varieties. Thirteen loci were polymorphic in at least one of the two var. japonica populations, whereas only four loci were polymorphic in the var. subaphylla population. The considerably lower genetic variation of the var. subaphylla population may be attributed to frequent selfing and/or inbreeding. The markers developed in this study will be useful for comparing the genetic diversity of P. japonica s. l. populations and measuring gene flow within and between populations and varieties.

Dipterocarp trees are ecologically and commercially important in Southeast Asian tropical rainforests. For sustainable management of forest ecosystems and conservation of biodiversity, it is essential to establish plantation methods ensuring that genetic variation of the planted trees is equivalent to that in natural forests. The genetic diversity and differentiation of Shorea leprosula and Shorea parvifolia on plantations managed by a private-sector forestry company in Indonesia and those in natural populations were compared using microsatellite markers. Genetic diversity in the planted populations was as high as that in the natural populations. No clear genetic differences between each planted population and the natural forest populations were found. The genetic variation present in planted S. leprosula and S. parvifolia populations did not appear to deteriorate in the planting system implemented in Indonesia, known as Tebang Pilih Tanam Jalur (TPTJ). These results indicate that the current plantation method practiced in the region is suitable for maintaining the original genetic composition and achieving sustainable use of tropical rainforests.

White-rot fungi play an important role in the global carbon cycle because they are the species that almost exclusively biodegrade wood lignin in nature. Lignin peroxidases (LiPs), manganese peroxidases (MnPs) and versatile peroxidases (VPs) are considered key players in the ligninolytic system. Apart from LiPs, MnPs and VPs, however, only few other factors involved in the ligninolytic system have been investigated using molecular genetics, implying the existence of unidentified elements. By combining classical genetic techniques with next-generation sequencing technology, they successfully showed an efficient forward genetics approach to identify mutations causing defects in the ligninolytic system of the white-rot fungus Pleurotus ostreatus. In this study, they identified two genes - chd1 and wtr1 - mutations in which cause an almost complete loss of Mn2+ -dependent peroxidase activity. The chd1 gene encodes a putative chromatin modifier, and wtr1 encodes an agaricomycete-specific protein with a putative DNA-binding domain. The chd1-1 mutation and targeted disruption of wtr1 hamper the ability of P. ostreatus to biodegrade wood lignin. Examination of the effects of the aforementioned mutation and disruption on the expression of certain MnP/VP genes suggests that a complex mechanism underlies the ligninolytic system in P. ostreatus.

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

Genomewide markers enable us to study genetic differentiation within a species and the factors underlying it at a much higher resolution than before, which advances our understanding of adaptation in organisms. We investigated genomic divergence in Metrosideros polymorpha, a woody species that occupies a wide range of ecological habitats across the Hawaiian Islands and shows remarkable phenotypic variation. Using 1659 single nucleotide polymorphism (SNP) markers annotated with the genome assembly, we examined the population genetic structure and demographic history of nine populations across five elevations and two ages of substrates on Mauna Loa, the island of Hawaii. The nine populations were differentiated into two genetic clusters distributed on the lower and higher elevations and were largely admixed on the middle elevation. Demographic modelling revealed that the two genetic clusters have been maintained in the face of gene flow, and the effective population size of the high-altitude cluster was much smaller. A FST -based outlier search among the 1659 SNPs revealed that 34 SNPs (2.05%) were likely to be under divergent selection and the allele frequencies of 21 of them were associated with environmental changes along elevations, such as temperature and precipitation. This study shows a genomic mosaic of M. polymorpha, in which contrasting divergence patterns were found. While most genomic polymorphisms were shared among populations, a small fraction of the genome was significantly differentiated between populations in diverse environments and could be responsible for the dramatic adaptation to a wide range of environments.

Premise of the study: Veronicastrum japonicum (Plantaginaceae) grows in grasslands on Honshu Island, Japan, and is threatened by habitat loss because of rapid land development over recent decades. For the genetic characterization of the remaining populations, microsatellite markers were developed. Methods and Results: Twelve polymorphic microsatellite loci were developed using next-generation sequencing. The number of alleles per locus ranged from two to 24 (mean 7.7), and the expected heterozygosity per locus ranged from 0.35 to 0.94 (mean 0.68). Conclusions: These markers can be used for genetic studies in conservation, such as the evaluation of genetic diversity and genetic structure.

Metrosideros polymorpha, a dominant tree species in the Hawaiian Islands, shows an extreme phenotypic polymorphism both across gradients of climatic/edaphic conditions and within populations, making it a potentially useful model species for evolutionary study. In order to understand how the phenotypic diversity is maintained within populations as well as across populations, we examined the diversities of several leaf and stem functional traits across five elevations and two soil substrates on the volcanic mountain of Mauna Loa, on the island of Hawaii. Leaf dry mass per area (LMA), a key leaf functional trait, was particularly focused on and analyzed in relation to its underlying components-namely, tissue LMA and trichome LMA (LMA = tissue LMA + trichome LMA). Across populations, tissue LMA increased linearly with elevation while trichome LMA showed unimodal patterns with elevation, which were better correlated with temperature and rainfall, respectively. Substantial phenotypic variations were also found within populations. Interestingly, the variations of tissue LMA were often negatively correlated to trichome LMA within populations, which contrasts with the cross-populations pattern, where a strong positive correlation between tissue LMA and trichome LMA was found. This suggests that phenotypic variations within populations were substantially influenced by local ecological processes. Soil depth (an indicator of local water availability) and tree size (an indicator of colonized timing) modestly explained the within-population variations, implying other local environmental factors and/or random processes are also important in local phenotypic diversity. This study provides an insight about how phenotypic diversity of plant species is maintained from local to landscape levels.

Premise of the study: Microsatellite markers were identified for Melastoma tetramerum var. tetramerum (Melastomataceae), a critically endangered shrub endemic to the Bonin Islands, to reveal genetic characteristics in wild and restored populations. Methods and Results: Using next-generation sequencing, 27 microsatellite markers were identified. Twenty of these markers were polymorphic in M. tetramerum var. tetramerum, with two to nine alleles per locus and expected heterozygosity ranging from 0.10 to 0.71. Among the 20 polymorphic markers, 15 were applicable to other closely related taxa, namely M. tetramerum var. pentapetalum, M. candidum var. candidum, and M. candidum var. alessandrense. Conclusions: These markers can be potentially useful to investigate the genetic diversity, population genetic structure, and reproductive ecology of M. tetramerum var. tetramerum as well as of the three related taxa to provide appropriate genetic information for conservation.

Premise of the study: Twenty-six microsatellite markers were developed for the endangered orchid Cypripedium japonicum (Orchidaceae) to estimate the clonal diversity and genetic structure of the remaining populations in Japan. Methods and Results: Microsatellite loci of C. japonicum were isolated using Ion Personal Genome Machine (PGM) sequencing. The primer sets were tested on 55 ramets sampled from two populations in Japan. Sixteen loci showed polymorphism in at least one population, with two to five alleles per locus. Observed and expected heterozygosities for the two populations ranged from 0.00 to 0.92 and 0.00 to 0.71, respectively. Conclusions: The microsatellite markers developed here provide a useful tool to analyze clonal structure and sexual regeneration status and will help to manage the remaining genetic variation within C. japonicum.

• Premise of the study: Microsatellite markers were characterized in Athyrium viridescentipes, a critically endangered fern species in Japan, to investigate its genetic diversity and population structure. • METHODS: and Results: Fifteen microsatellite markers were developed. The 15 loci were successfully amplified in three additional Athyrium species except for one locus in A. vidalii. In A. viridescentipes, the number of alleles per locus ranged from one to five, with an average of 1.9, and the expected heterozygosity ranged from 0.00 to 0.53, with an average of 0.24. • CONCLUSIONS: These markers can be used in studies on conservation programs for A. viridescentipes as well as in further studies involving other Athyrium species.