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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.

Ecological divergence in a species provides a valuable opportunity to study the early stages of speciation. We focused on Metrosideros polymorpha, a unique example of the incipient radiation of woody species, to examine how an ecological divergence continues in the face of gene flow. We analyzed the whole genomes of 70 plants collected throughout the island of Hawaii, which is the youngest island with the highest altitude in the archipelago and encompasses a wide range of environments. The continuous M. polymorpha forest stands on the island of Hawaii were differentiated into three genetic clusters, each of which grows in a distinctive environment and includes substantial genetic and phenotypic diversity. The three genetic clusters showed signatures of selection in genomic regions encompassing genes relevant to environmental adaptations, including genes associated with light utilization, oxidative stress, and leaf senescence, which are likely associated with the ecological differentiation of the species. Our demographic modeling suggested that the glaberrima cluster in wet environments maintained a relatively large population size and two clusters split: polymorpha in the subalpine zone and incana in dry and hot conditions. This ecological divergence possibly began before the species colonized the island of Hawaii. Interestingly, the three clusters recovered genetic connectivity coincidentally with a recent population bottleneck, in line with the weak reproductive isolation observed in the species. This study highlights that the degree of genetic differentiation between ecologically-diverged populations can vary depending on the strength of natural selection in the very early phases of speciation., Author summary Knowledge about how genetic barriers are formed between populations in distinct environments is valuable to understand the processes of speciation and conserve biodiversity. Metrosideros polymorpha, an endemic woody species in the Hawaiian Islands, is a good system to study developing genetic barriers in a species, because it colonized the diverse environments and diversified the morphology for a relatively short period of time. We analyzed the genomes of 70 M. polymorpha plants from a broad range of environments on the island of Hawaii to infer the current and past genetic barriers among them. Currently, M. polymorpha plants growing in different environments have substantially different genomes, especially at the genomic regions with genes putatively controlling physiology to fit in distinct environment. However, in its history, they had hybridized with one another, possibly because plants formerly growing in different environments came into close contact due to the climate changes. It is suggested that genetic barriers can easily strengthen or weaken depending on environments splitting the ecology of a species before reproductive isolation becomes complete.

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.

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.

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.