Ecotype variation in the endemic tree Callicarpa subpubescens on small oceanic islands: genetic, phenotypic, and environmental insights.

Callicarpa subpubescens, endemic to the Ogasawara Islands, is suggested to have multiple ecotypes in the Hahajima Islands, specifically in the central part of the Ogasawara Islands. In this study, associations between genetic groups and spatial distribution, habitat, leaf morphology, size structure, and flowering time of each genetic group were investigated on Hahajima and the satellite Imoutojima Islands. Genetic groups were identified using EST-SSR markers, revealing four ecotypes named based on morphological features: Dwarf (D), Glabrescent (G), Tall (T), and Middle (M), with M being a result of the hybridization of G and T. Ecotype D, adapted to dry environments, is characterized by small tree size, dense thick leaves with abundant hairs, and is distributed in dry scrub. Ecotype G, adapted to understory of mesic forests, lacks leaf hairs. Ecotype T, adapted to the canopy of mesic forests, has hairy leaves and is tall in tree height. Ecotype M, adapted to the canopy of mesic scrub or edges of mesic forests, has hairy leaves but with a shorter tree height than ecotype T. Flowering peaks differed among all ecotype pairs except G and M, but the flowering times more or less overlapped among all ecotypes, suggesting that pre-mating isolation among ecotypes is not perfect. Post-mating isolation is considered absent, as there were no differences in the results, germination, and survival rates of one-year seedlings among inter- and intra-ecotype crossings. The existence of such ecotypes provides valuable insights into the ongoing speciation processes adapting to the oceanic island environments.

Diversity profiling of soil bacterial and fungal communities in the Ogasawara (Bonin) Islands, Japan.

Island biogeography research provides insight into microbial diversity patterns; however, little is known about the diversity and distribution of soil microbial communities on remote and poorly accessible islands. Here, we present amplicon sequencing data from bacterial and fungal communities in the surface soils of the Ogasawara (Bonin) Islands, Japan.

A DNA barcode reference library for the native woody seed plants of Japan.

DNA barcode databases are increasingly available for a range of organisms, facilitating the wide application of DNA barcode-based studies. Here we announce the development of a comprehensive DNA barcode reference library of Japanese native woody seed plants representing 43 orders, 99 families, 303 genera and 834 species, and comprising 77.3% of the genera and 72.2% of the species of native woody seed plants in Japan. A total of 6216 plant specimens were collected from 223 sites across the subtropical, temperate, boreal and alpine biomes in Japan with most species represented by multiple accessions. This reference library utilized three chloroplast DNA regions (rbcL, trnH-psbA and matK) and consists of 14,403 barcode sequences. Individual regions varied in their identification rates, with species-level and genus-level rates for rbcL, trnH-psbA and matK based on blast being 57.4%/96.2%, 78.5%/99.1% and 67.8%/98.1%, respectively. Identification rates were higher using region combinations, with total species-level rates for two region combinations (rbcL & trnH-psbA, rbcL & matK and trnH-psbA & matK) ranging between 90.6% and 95.8%, and for all three regions being equal to 98.6%. Genus-level identification rates were even higher, ranging between 99.7% and 100% for two region combinations and being 100% for the three regions. These results indicate that this DNA barcode reference library is an effective resource for investigations of native woody seed plants in Japan using DNA barcodes and provides a useful template for the development of libraries for other components of the Japanese flora.

Contrasting genetic diversity between Planchonella obovata sensu lato (Sapotaceae) on old continental and young oceanic island populations in Japan.

Genetic diversity of plant populations on islands is likely to be influenced by characteristics such as island origin (oceanic or continental) and their age, size, and distance to continental landmasses. In Japan, Planchonella obovata sensu lato which is found on both continental and oceanic islands of varying age, size, and distance to East Asian continental areas-is an ideal system in which to investigate the factors influencing genetic diversity of island plant species. In this study, we examined the genetic diversity of P. obovata s.l. populations, in the context of the species population genetic structure, demography, and between island migration, from 668 individuals, 28 populations and 14 islands including both continental (the Yaeyama Islands) and oceanic islands (the Daito, Bonin, and Volcano Islands) using 11 microsatellite markers. The Yaeyama and Volcano Islands respectively had the highest and lowest genetic diversity, and island origin and age significantly affected genetic diversity. Clustering analysis revealed that populations were grouped into Bonin, Volcano, and Yaeyama + Daito groups. However, Bonin and Volcano groups were distinct despite the relatively short geographical distance between them. Approximate Bayesian Computation analysis suggested that the population size was stable in Bonin and Yaeyama + Daito groups, whereas population reduction occurred in Volcano group, and migration between groups were very limited. Younger oceanic islands showed lower genetic diversity, probably due to limited gene flow and a lack of time to accumulate unique alleles. Genetic structure was generally consistent with the geographic pattern of the islands, but in Volcano, a limited number of founders and limited gene flow among islands are likely to have caused the large genetic divergence observed.

Predictive Model of Risk Factors of High Flow Nasal Cannula Using Machine Learning in COVID-19

BackgroundWith the rapid increase in the number of COVID-19 patients in Japan, the number of patients receiving oxygen at home has also increased rapidly, and some of these patients have died. An efficient approach to identify high-risk patients with slowly progressing and rapidly worsening COVID-19, and to avoid missing the timing of therapeutic intervention will improve patient prognosis and prevent medical complications. MethodsPatients admitted to medical institutions in Japan from November 14, 2020 to April 11, 2021 and registered in the COVID-19 Registry Japan were included. Risk factors for patients with High Flow Nasal Cannula invasive respiratory management or higher were comprehensively explored using machine learning. Age-specific cohorts were created, and severity prediction was performed for the patient surge period and normal times, respectively. ResultsWe were able to obtain a model that was able to predict severe disease with a sensitivity of 57% when the specificity was set at 90% for those aged 40-59 years, and with a specificity of 50% and 43% when the sensitivity was set at 90% for those aged 60-79 years and 80 years and older, respectively. We were able to identify lactate dehydrogenase level (LDH) as an important factor in predicting the severity of illness in all age groups. DiscussionUsing machine learning, we were able to identify risk factors with high accuracy, and predict the severity of the disease. Using machine learning, we were able to identify risk factors with high accuracy, and predict the severity of the disease. We plan to develop a tool that will be useful in determining the indications for hospitalisation for patients undergoing home care and early hospitalisation.

Efficacy of remdesivir in Japanese patients hospitalised with COVID-19: A large observational study using the COVID-19 Registry Japan

ObjectivesAlthough several randomised controlled trials have compared the efficacy of remdesivir with that of placebo, there is limited evidence regarding its effect in the early stage of nonsevere COVID-19 cases. MethodsWe evaluated the efficacy of remdesivir on the early stage of nonsevere COVID-19 using the COVID-19 Registry Japan, a nationwide registry of hospitalised COVID-19 patients in Japan. Two regimens (start remdesivir therapy within 4 days from admission vs. no remdesivir during hospitalisation) among patients without the need for supplementary oxygen therapy were compared by a three-step processing (cloning, censoring, and weighting) method. The primary outcome was supplementary oxygen requirement during hospitalisation. Secondary outcomes were 30-day fatality risk and risk of invasive mechanical ventilation or extracorporeal membrane oxygenation (IMV/ECMO). ResultsThe data of 12,657 cases met our inclusion criteria. The start remdesivir regimen showed a lower risk of supplementary oxygen requirement (hazard ratio: 0.861, p < 0.001). Both 30-day fatality risk and risk of IMV/ECMO introduction were not significantly different between the two regimens (hazard ratios: 1.05 and 0.886, p values: 0.070 and 0.440, respectively). ConclusionsRemdesivir might reduce the risk of oxygen requirement during hospitalisation in the early stage of COVID-19; however, it had no positive effect on the clinical outcome and reduction of IMV/ECMO requirement.

Correction to: Population genetic structure and demography of Magnolia kobus: variety borealis is not supported genetically.

The original article was updated.

Genetic diversity, structure, and demography of Pandanus boninensis (Pandanaceae) with sea drifted seeds, endemic to the Ogasawara Islands of Japan: Comparison between young and old islands.

Pandanus boninensis, endemic to the Ogasawara Islands, Japan, is distributed on both the older Bonin and younger Volcano Islands. In this study, we conducted population genetic analyses of P. boninensis on these islands to examine the population diversity and structure across old and young islands, to assess potential differences in population demography with island age, and to collect any evidence of migration between old and young islands. We found that the genetic diversity of expressed sequence tag (EST)-based microsatellite (SSR) markers, the nucleotide diversity of nuclear DNA sequences, and the haplotype diversity of chloroplast DNA on young islands were lower than those on old islands. Clustering analyses of EST-SSR indicated that populations on old islands were strongly diverged from those on young islands. Approximate Bayesian computation analysis of EST-SSR suggested that population expansion occurred on old islands while population reduction occurred on young islands. We also found evidence of migration among old islands (mostly from south to north), while it appears that there have been very few migration events between old and young islands. These differences could be due to the fact that young islands tend to be geographically isolated and support smaller populations that began a shorter time ago from limited founders. The P. boninensis populations on the Volcano Islands are interesting from an evolutionary perspective as they constitute a classic example of the early stages of progressive colonization on oceanic islands with small effective population sizes and low genetic diversity.

Population genetic structure and demography of Magnolia kobus: variety borealis is not supported genetically.

Species delimitations by morphological and by genetic markers are not always congruent. Magnolia kobus consists of two morphologically different varieties, kobus and borealis. The latter variety is characterized by larger leaves than the former. For the conservation of M. kobus genetic resources in natural forests, the relationships between morphological and genetic variation should be clarified. We investigated variations in nuclear microsatellites, chloroplast DNA (cpDNA) sequences and leaf morphological traits in 23 populations of M. kobus over the range of species. Two genetically divergent lineages, northern and southern were detected and their geographical boundary was estimated to be at 39°N. The northern lineage consisted of two genetic clusters and a single cpDNA haplotype, while the southern one had multiple genetic clusters and cpDNA haplotypes. The northern lineage showed significantly lower genetic diversity than the southern. Approximate Bayesian computation indicated that the northern and southern lineages had experienced, respectively, population expansion and long-term stable population size. The divergence time between the two lineages was estimated to be 565,000 years ago and no signature of migration between the two lineages after divergence was detected. Ecological niche modeling showed that the potential distribution area in northern Japan at the last glacial maximum was very small. It is thus considered that the two lineages have experienced different population histories over several glacial-inter-glacial cycles. Individuals of populations in the central to northern part of Honshu on the Sea of Japan side and in Hokkaido had large leaf width and area. These leaf characteristics corresponded with those of variety borealis. However, the delimitation of the northern and southern lineages detected by genetic markers (39°N) was not congruent with that detected by leaf morphologies (36°N). It is therefore suggested that variety borealis is not supported genetically and the northern and southern lineages should be considered separately when identifying conservation units based not on morphology but on genetic markers.

Reduced incompatibility in the production of second generation hybrids between two Magnolia species revealed by Bayesian gene dispersal modeling.

PREMISE OF THE STUDY: Hybrid zones are areas where gene flow between related species is currently occurring, so information on the compatibility between related species and their hybrids is essential for predicting the dynamics of such zones generated by introgressive hybridization. In this study, we quantified the compatibility among Magnolia stellata, M. salicifolia, and their hybrids in a hybrid zone using gene dispersal modeling. METHODS: After determining the genealogical classes of adult trees in the hybrid zone, the paternity of 574 open-pollinated seeds from 37 known maternal trees was analyzed with microsatellite markers. A neighborhood-based Bayesian gene dispersal model developed by us for estimating compatibility was then applied to the paternity data. KEY RESULTS: When M. stellata or M. salicifolia were mothers, interspecific mating to produce F1 hybrids yielded significant incompatibility, but backcrossing with F1 hybrids did not. Furthermore, when F1 hybrids became mothers, no significant incompatibility resulted from backcrossing to parental species or intra-F1 mating to produce F2 hybrids. The estimated proportion of F1 hybrids in the outcrossed seeds (1.7%) in the hybrid zone was much lower than that in the adult trees (14.0%). CONCLUSIONS: While it is difficult to obtain F1 hybrids, their low incompatibility makes it easy to produce advanced generation hybrids, once they have been successfully obtained. Although the production of F1 seeds is rare, heterosis and/or weak selection pressure in an empty niche between the parental species' niches may have contributed to the increased proportion of adult F1 hybrids in the hybrid zone.

Development of microsatellite markers for the clonal shrub Orixa japonica (Rutaceae) using 454 sequencing.

PREMISE OF THE STUDY: Microsatellite markers were developed for a dioecious shrub, Orixa japonica (Rutaceae). Because O. japonica vigorously propagates by vegetative growth, microsatellite markers can be used to identify clonal relationships among its ramets. METHODS AND RESULTS: Sixteen polymorphic microsatellite markers were identified by 454 next-generation sequencing. The number of alleles and expected heterozygosity for each locus among four populations ranged from two to 10 and from 0.140 to 0.875, respectively. Five of the 16 loci showed a low null allele frequency. Because Orixa is a monotypic genus, cross-amplification in a consubfamilial species, Skimmia japonica, was tested, and only one locus showed polymorphism. CONCLUSIONS: These microsatellite markers developed for O. japonica contribute to clone identification for studies examining the clonal structure and true sex ratio in the wild. Moreover, five markers that have a low null allele frequency can also be used for estimating mating systems or performing parentage analysis.

Novel microsatellite markers for Distylium lepidotum (Hamamelidaceae) endemic to the Ogasawara Islands.

BACKGROUND: Distylium lepidotum is a small tree endemic to the Ogasawara Islands located in the northwestern Pacific Ocean. This species is a sole food for an endemic locust, Boninoxya anijimensis. Here, we developed microsatellite markers to investigate genetic diversity and genetic structure and to avoid a genetic disturbance after transplantation to restore the Ogasawara Islands ecosystem. RESULTS: Microsatellite markers with perfect dinucleotide repeats were developed using the next-generation sequencing Illumina MiSeq Desktop Sequencer. Thirty-two primer pairs were characterized in two D. lepidotum populations on Chichijima and Hahajima Islands of the Ogasawara Islands. The number of alleles for the markers ranged from three to 23 per locus in the two populations. Expected heterozygosity per locus in each population ranged from 0.156 to 0.940 and 0.368 to 0.845, respectively. CONCLUSIONS: These microsatellite markers will be useful for future population genetics studies of D. lepidotum and provide a basis for conservation management of the Ogasawara Islands.

Diet analysis by next-generation sequencing indicates the frequent consumption of introduced plants by the critically endangered red-headed wood pigeon (Columba janthina nitens) in oceanic island habitats.

Oceanic island ecosystems are vulnerable to the introduction of alien species, and they provide a habitat for many endangered species. Knowing the diet of an endangered animal is important for appropriate nature restoration efforts on oceanic islands because introduced species may be a major component of the diets of some endangered species. DNA barcoding techniques together with next-generation sequencing may provide more detailed information on animal diets than other traditional methods. We performed a diet analysis using 48 fecal samples from the critically endangered red-headed wood pigeon that is endemic to the Ogasawara Islands based on chloroplast trnL P6 loop sequences. The frequency of each detected plant taxa was compared with a microhistological analysis of the same sample set. The DNA barcoding approach detected a much larger number of plants than the microhistological analysis. Plants that were difficult to identify by microhistological analysis after being digested in the pigeon stomachs were frequently identified only by DNA barcoding. The results of the barcoding analysis indicated the frequent consumption of introduced species, in addition to several native species, by the red-headed wood pigeon. The rapid eradication of specific introduced species may reduce the food resources available to this endangered bird; thus, balancing eradication efforts with the restoration of native food plants should be considered. Although some technical problems still exist, the trnL approach to next-generation sequencing may contribute to a better understanding of oceanic island ecosystems and their conservation.

Genetic differentiation in Elaeocarpus photiniifolia (Elaeocarpaceae) associated with geographic distribution and habitat variation in the Bonin (Ogasawara) Islands.

Gene flow between populations in different environmental conditions can be limited due to divergent natural selection, thus promoting genetic differentiation. Elaeocarpus photiniifolia, an endemic tree species in the Bonin Islands, is distributed in two types of habitats, dry scrubs and mesic forests. We aim to elucidate the genetic differentiation in E. photiniifolia within and between islands and between the habitat types. We investigated genotypes of 639 individuals from 19 populations of E. photiniifolia and its closely-related E. sylvestris at 24 microsatellite loci derived from expressed sequence tags. The data revealed genetic differentiation (1) between E. photiniifolia and E. sylvestris (0.307 ≤ F ST ≤ 0.470), (2) between the E. photiniifolia populations of the Chichijima and Hahajima Island Groups in the Bonin Islands (0.033 ≤ F ST ≤ 0.121) and (3) between E. photiniifolia populations associated with dry scrubs and mesic forests in the Chichijima Island Group (0.005 ≤ F ST ≤ 0.071). Principal coordinate analysis and Bayesian clustering analysis also showed that genetically distinct groups were associated with the habitat types, and isolation by distance was not responsible for the genetic differentiation. These findings suggest that E. photiniifolia is divided into genetically differentiated groups associated with different environmental conditions in the Bonin Islands.

Pollen flow and effects of population structure on selfing rates and female and male reproductive success in fragmented Magnolia stellata populations.

BACKGROUND: Fragmentation of plant populations may affect mating patterns and female and male reproductive success. To improve understanding of fragmentation effects on plant reproduction, we investigated the pollen flow patterns in six adjacent local populations of Magnolia stellata, an insect-pollinated, threatened tree species in Japan, and assessed effects of maternal plant (genet) size, local genet density, population size and neighboring population size on female reproductive success (seed production rates), and effects of mating distance, paternal genet size, population size and separation of populations on male reproductive success. RESULTS: The seed production rate, i.e. the proportion of ovules that successfully turned into seeds, varied between 1.0 and 6.5%, and increased with increasing population size and neighboring population size, and with decreasing maternal genet size and local genet density. The selfing rate varied between 3.6 and 28.9%, and increased with increasing maternal genet size and with declining local genet density. Male reproductive success increased with increasing paternal genet size, and decreased with increasing mating distance and separation of population. Pollen flow between the populations was low (6.1%) and highly leptocurtic. CONCLUSIONS: Our results indicate that habitat fragmentation, separation and reduced size of populations, affected mating patterns and reproductive success of M. stellata. Local competition for pollinators and plant display size were likely to alter the reproductive success.

Development of EST-SSR markers for Elaeocarpus photiniifolia (Elaeocarpaceae), an endemic taxon of the Bonin Islands.

PREMISE OF THE STUDY: Expressed sequence tag (EST)-derived microsatellite markers were developed for Elaeocarpus photiniifolia, an endemic taxon of the Bonin Islands. METHODS AND RESULTS: Initially, a complementary DNA (cDNA) library was constructed by de novo pyrosequencing of total RNA extracted from a seedling. A total of 267 primer pairs were designed from the library. Of the 48 tested loci, 25 loci were polymorphic among 41 individuals representing the entire geographical range of the species, with the number of alleles per locus and expected heterozygosity ranging from two to 14 and 0.09 to 0.86, respectively. Most loci were transferable to a related species, E. sylvestris. CONCLUSIONS: The developed markers will be useful for evaluating the genetic structure of E. photiniifolia.

Isolation and characterization of EST-SSR markers in Schima mertensiana (Theaceae) using pyrosequencing technology.

PREMISE OF THE STUDY: Microsatellite or simple sequence repeat (SSR) markers from expressed sequence tags (ESTs) in Schima mertensiana, an evergreen tree that is endemic to the Bonin Islands, were developed to facilitate evaluation of the species's population genetic structure. METHODS AND RESULTS: Using 149831 ESTs derived by pyrosequencing S. mertensiana complementary DNA (cDNA), 683 EST-SSRs were developed, 48 of which were tested for amplification. Thirty of the 48 showed clear amplification and detected polymorphism in sampled populations, with mean expected heterozygosity at the amplified loci ranging from 0.045 to 0.874 (average 0.545). CONCLUSIONS: The markers developed in this study can be used for future studies of the genetic structure of S. mertensiana and related species.

Rapid development of microsatellite markers for Pandanus boninensis (Pandanaceae) by pyrosequencing technology.

PREMISE OF THE STUDY: To facilitate rapid development of microsatellite or simple sequence repeat (SSR) markers, an expressed sequence tags (EST) database was constructed for Pandanus boninensis, an evergreen tree endemic to the Bonin Islands, using pyrosequencing technology. METHODS AND RESULTS: We designed primers for 340 EST-SSRs identified from 109620 pyrosequencing reads, 48 of which were tested for PCR amplification. Thirty-four primers provided clear amplification, and 26 of those 34 displayed clear polymorphic patterns in sampled populations, with mean expected heterozygosity at the amplified loci ranging from 0.022 to 0.742 (average 0.262). CONCLUSIONS: The developed markers are promising tools for future genetic studies of P. boninensis and related species.

Microsatellite markers derived from Calophyllum inophyllum (Clusiaceae) expressed sequence tags.

PREMISE OF THE STUDY: Robust markers are required (inter alia) for assessing origins of Calophyllum inophyllum populations on the Bonin Islands, Japan. Therefore, informative expressed sequence tag (EST)-based microsatellite or simple sequence repeat (SSRs) markers in the species were sought. METHODS AND RESULTS: Using 135378 ESTs derived from de novo pyrosequencing, primers for 475 EST-SSRs were developed, 48 of which were tested for PCR amplification. Thirty-six of the 48 primers showed clear amplification, with 23 displaying polymorphism in sampled populations. Expected heterozygosity in the samples from the Bonin Islands and Ryukyu Islands populations ranged from 0.041 to 0.697 and from 0.041 to 0.773, respectively. CONCLUSIONS: As EST-SSRs are potentially tightly linked with functional genes, and reportedly more transferable to related species than anonymous genomic SSRs, the developed primers have utility for future studies of the origins, genetic structure, and conservation of C. inophyllum and related species.

Interpopulation variation in mating system and late-stage inbreeding depression in Magnolia stellata.

Inbreeding has the potential to cause evolutionary changes in populations, although these changes are likely to drive populations to extinction through inbreeding depression and reductions in genetic diversity. We investigated the mating system and late-stage inbreeding depression (delta) in 10 populations of Magnolia stellata using nine microsatellite markers and evaluated the effects of population size and the degree of population isolation through inbreeding and inbreeding depression on the persistence of populations. The outcrossing rates were very similar (approximately 0.7) among populations, but the correlations of paternity, fractions of biparental inbreeding and inbreeding coefficients at the seed stage (F(S)) varied among populations, suggesting that the level of outcrossing was similar among populations, while the quality of it was not. A significant negative correlation was detected between F(S) and population size. The average value of delta was 0.709, and the values in six of the 10 populations were significant. The values of delta differed among populations, although clear relationships with population size and the degree of population isolation were not detected. However, in one population, which was very small and located in the edge of the species' range, we obtained a very low value of delta (-0.096), which may be indicative of purging or the fixation of deleterious alleles. Existing M. stellata populations that are small (and thus might be expected to have higher frequencies of inbreeding) and have large values of delta may be in danger of declining, even if the populations are located within the central region of the species' range.