Demography and selection analysis of the incipient adaptive radiation of a Hawaiian woody species.

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.

The genome of Shorea leprosula (Dipterocarpaceae) highlights the ecological relevance of drought in aseasonal tropical rainforests.

Hyperdiverse tropical rainforests, such as the aseasonal forests in Southeast Asia, are supported by high annual rainfall. Its canopy is dominated by the species-rich tree family of Dipterocarpaceae (Asian dipterocarps), which has both ecological (e.g., supports flora and fauna) and economical (e.g., timber production) importance. Recent ecological studies suggested that rare irregular drought events may be an environmental stress and signal for the tropical trees. We assembled the genome of a widespread but near threatened dipterocarp, Shorea leprosula, and analyzed the transcriptome sequences of ten dipterocarp species representing seven genera. Comparative genomic and molecular dating analyses suggested a whole-genome duplication close to the Cretaceous-Paleogene extinction event followed by the diversification of major dipterocarp lineages (i.e. Dipterocarpoideae). Interestingly, the retained duplicated genes were enriched for genes upregulated by no-irrigation treatment. These findings provide molecular support for the relevance of drought for tropical trees despite the lack of an annual dry season.

Genotype and transcriptome effects on somatic embryogenesis in Cryptomeria japonica.

Somatic embryogenesis (SE), which is in vitro regeneration 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 of C. 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 in C. japonica and indeed highly expressed in EMs with high-embryogenic potential; therefore, these proteins represent candidate markers for distinguishing embryogenic potential.

Environment-dependent introgression from Quercus dentata to a coastal ecotype of Quercus mongolica var. crispula in northern Japan.

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.

Leaf trichomes in Metrosideros polymorpha can contribute to avoiding extra water stress by impeding gall formation.

BACKGROUND AND AIMS: Plants 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. METHODS: We 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 RESULTS: Our 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. CONCLUSION: Our 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.

Updated Genome Assembly and Annotation for Metrosideros polymorpha, an Emerging Model Tree Species of Ecological Divergence.

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.

Effects of pex1 disruption on wood lignin biodegradation, fruiting development and the utilization of carbon sources in the white-rot Agaricomycete Pleurotus ostreatus and non-wood decaying Coprinopsis cinerea.

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.

Development of microsatellite markers for partially and putative fully mycoheterotrophic varieties of Pyrola japonica sensu lato (Ericaceae).

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.

The population genomic signature of environmental association and gene flow in an ecologically divergent tree species Metrosideros polymorpha (Myrtaceae).

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.

Identification of two mutations that cause defects in the ligninolytic system through an efficient forward genetics in the white-rot agaricomycete Pleurotus ostreatus.

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.

Vertical Structure of Phyllosphere Fungal Communities in a Tropical Forest in Thailand Uncovered by High-Throughput Sequencing.

Phyllosphere fungi harbor a tremendous species diversity and play important ecological roles. However, little is known about their distribution patterns within forest ecosystems. We examined how species diversity and community composition of phyllosphere fungi change along a vertical structure in a tropical forest in Thailand. Fungal communities in 144 leaf samples from 19 vertical layers (1.28-34.4 m above ground) of 73 plant individuals (27 species) were investigated by metabarcoding analysis using Ion Torrent sequencing. In total, 1,524 fungal operational taxonomic units (OTUs) were detected among 890,710 reads obtained from the 144 leaf samples. Taxonomically diverse fungi belonging to as many as 24 orders of Ascomycota and 21 orders of Basidiomycota were detected, most of which inhabited limited parts of the lowest layers closest to the forest floor. Species diversity of phyllosphere fungi was the highest in the lowest layers closest to the forest floor, decreased with increasing height, and lowest in the canopy; 742 and 55 fungal OTUs were detected at the lowest and highest layer, respectively. On the layers close to the forest floor, phyllosphere fungal communities were mainly composed of low frequency OTUs and largely differentiated among plant individuals. Conversely, in the canopy, fungal communities consisted of similar OTUs across plant individuals, and as many as 86.1%-92.7% of the OTUs found in the canopy (≥22 m above ground) were also distributed in the lower layers. Overall, our study showed the variability of phyllosphere fungal communities along the vertical gradient of plant vegetation and environmental conditions, suggesting the significance of biotic and abiotic variation for the species diversity of phyllosphere fungi.

Twenty novel polymorphic microsatellite primers in the critically endangered Melastoma tetramerum var. tetramerum (Melastomataceae).

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.

Genome sequencing of Metrosideros polymorpha (Myrtaceae), a dominant species in various habitats in the Hawaiian Islands with remarkable phenotypic variations.

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.

Development of microsatellite markers for the semi-natural grassland herb Veronicastrum japonicum (Plantaginaceae).

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.

Isolation and characterization of novel microsatellite loci for the endangered orchid Cypripedium japonicum (Orchidaceae).

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.

A quantitative analysis of phenotypic variations of Metrosideros polymorpha within and across populations along environmental gradients on Mauna Loa, Hawaii.

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.

Genetic variation and structure of the endangered Lady Fern Athyrium viridescentipes based on ubiquitous genotyping.

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.

Microsatellite loci in an endangered fern species, Athyrium viridescentipes (Woodsiaceae), and cross-species amplification.

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.