Ayako Izuno

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 FST 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.

Development of microsatellite markers for two endangered grassland butterflies, Melitaea ambigua and M. protomedia (Nymphalidae), using Ion Torrent next-generation sequencing

Melitaea ambigua and M. protomedia (Nymphalidae) are widely distributed in the grasslands of Northeast Asia. In Japan, these species have been threatened by habitat loss because of the rapid expansion of land development over recent decades. We isolated and characterised 18 polymorphic microsatellite loci (9 loci for each species) using a next-generation sequencer. The number of alleles and the expected heterozygosity per locus was 2–12 and 2–7, 0.10–0.86 and 0.13–0.76 in M.ambigua and M. protomedia, respectively. The markers can be useful for genetic studies for conservation such as evaluation of genetic diversity and genetic structure.

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.

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.

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.

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 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.

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.