Fine-scale clonal structure of the lingonberry Vaccinium vitis-idaea under the nurse plant Pinus pumila vegetation in an alpine region, Mt. Norikura.

The nurse effect is a positive interaction in which a nurse plant improves the abiotic environment for another species (beneficiary plant) and facilitates its establishment. The evergreen shrub Vaccinium vitis-idaea (a beneficiary plant) grows mainly under the dwarf shrub Pinus pumila (a nurse plant) in the alpine regions of central Japan. However, whether V. vitis-idaea shrubs under various P. pumila shrubs spread through clonal growth and/or seeds remains unclear. We investigated the clonal structure of V. vitis-idaea under the nurse plant P. pumila in Japanese alpine regions. MIG-seq analysis was conducted to clarify the clonal diversity of V. vitis-idaea in isolated and patchy P. pumila plots on a ridge (PATs), and in a plot covered by dense P. pumila on a slope adjacent to the ridge (MAT) on Mt. Norikura, Japan. We detected 28 multilocus genotypes in 319 ramets of V. vitis-idaea across 11 PATs and MAT. Three genets expanded to more than 10 m in the MAT. Some genets were shared among neighboring PATs or among PATs and MAT. These findings suggest that the clonal growth of V. vitis-idaea plays an important role in the sustainability of populations. The clonal diversity of V. vitis-idaea was positively related with the spatial size of PATs and was higher in MAT than in PATs at a small scale. Therefore, the spatial spread of the nurse plant P. pumila might facilitate the nurse effect on the genetic diversity of beneficiary plants, leading to the sustainability of beneficiary populations.

Double migration of the endangered Tricyrtis formosana (Liliaceae) in Japan.

The Ryukyu Islands of Japan are a biodiversity hotspot due to geographical and historical factors. Tricyrtis formosana is a perennial herbaceous plant that commonly found in Taiwan. But only a few populations have been identified in a limited habitat on Iriomote Island, while populations of unknown origin occur near human settlements in an area on the main island of Okinawa. To better understand these populations of the phylogenetic uniqueness and intrinsic vulnerability, we conducted comparative analyses including (1) phylogeny and population structure with MIG-seq data, (2) photosynthesis-related traits of plants grown under common conditions and (3) transcriptome analysis to detect deleterious variations. Results revealed that T. formosana was split into two clades by the congeners and that Iriomote and Okinawa populations independently derived from ancestral Taiwanese populations in each clade. Photosynthetic efficiency was lowest in the Iriomote population, followed by Okinawa and Taiwan. Transcriptome analysis showed that the Iriomote population accumulated more deleterious variations, suggesting intrinsic vulnerability. These results indicate that each T. formosana population in Japan is phylogenetically unique and has been independently dispersed from Taiwan, and that the Iriomote population presents a high conservation difficulty with a unique photosynthesis-related characteristic and a larger amount of deleterious variations.

The phylogenetic reconstruction of the Neotropical cycad genus Ceratozamia (Zamiaceae) reveals disparate patterns of niche evolution.

The cycad genus Ceratozamia comprises 40 species from Mexico, Guatemala, Belize, and Honduras, where cycads occur throughout climatically varied montane habitats. Ceratozamia has the potential to reveal the history and processes of species diversification across diverse Neotropical habitats in this region. However, the species relationships within Ceratozamia and the ecological trends during its evolution remain unclear. Here, we aimed to clarify the phylogenetic relationships, the timing of clade and species divergences, and the niche evolution throughout the phylogenetic history of Ceratozamia. Genome-wide DNA sequences were obtained with MIG-seq, and multiple data-filtering steps were used to optimize the dataset used to construct an ultrametric species tree. Divergence times among branches and ancestral niches were estimated. The niche variation among species was evaluated, summarized into two principal components, and their ancestral states were reconstructed to test whether niche shifts among branches can be explained by random processes, under a Brownian Motion model. Ceratozamia comprises three main clades, and most species relationships within the clades were resolved. Ceratozamia has diversified since the Oligocene, with major branching events occurring during the Miocene. This timing is consistent with fossil evidence, the timing estimated for other Neotropical plant groups, and the major geological events that shaped the topographic and climatic variation in Mexico. Patterns of niche evolution in the genus do not accord with the Brownian Motion model. Rather, non-random evolution with shifts towards more seasonal environments at high latitudes, or shifts towards humid or dry environments at low latitudes explain the diversification of Ceratozamia. We present a comprehensive phylogenetic reconstruction for Ceratozamia and identify for the first time the environmental factors involved in clade and species diversification within the genus. This study alleviates the controversies regarding the species relationships in the genus and provides the first evidence that latitude-associated environmental factors may influence processes of niche evolution in cycads.

Discrimination of Camellia cultivars using iD-NA analysis.

Recently, many new cultivars have been taken abroad illegally, which is now considered an international issue. Botanical evidence found at a crime scene provides valuable information about the origin of the sample. However, botanical resources for forensic evidence remain underutilized because molecular markers, such as microsatellites, are not available without a limited set of species. Multiplexed intersimple sequence repeat (ISSR) genotyping by sequencing (MIG-seq) and its analysis method, identification of not applicable (iD-NA), have been used to determine several genome-wide genetic markers, making them applicable to all plant species, including those with limited available genetic information. Camellia cultivars are popular worldwide and are often planted in many gardens and bred to make new cultivars. In this study, we aimed to analyze Camellia cultivars/species through MIG-seq. MIG-seq could discriminate similar samples, such as bud mutants and closely related samples that could not be distinguished based on morphological features. This discrimination was consistent with that of a previous study that classified cultivars based on short tandem repeat (STR) markers, indicating that MIG-seq has the same or higher discrimination ability as STR markers. Furthermore, we observed unknown phylogenetic relationships. Because MIG-seq can be applied to unlimited species and low-quality DNA, it may be useful in various scientific fields.

Potential contribution of floral thermogenesis to cold adaptation, distribution pattern, and population structure of thermogenic and non/slightly thermogenic Symplocarpus species.

The genus Symplocarpus in basal Araceae includes both thermogenic and non/slightly thermogenic species that prefer cold environments. If floral thermogenesis of Symplocarpus contributes to cold adaptation, it would be expected that thermogenic species have a larger habitat than non/slightly thermogenic species during an ice age, leading to increased genetic diversity in the current population. To address this question, potential distribution in past environment predicted by ecological niche modeling (ENM), genetic diversity, and population structure of chloroplast and genome-wide single nucleotide polymorphisms were compared between thermogenic Symplocarpus renifolius and non/slightly thermogenic Symplocarpus nipponicus. ENM revealed that the distribution of S. nipponicus decreased, whereas that of S. renifolius expanded in the Last Glacial Maximum. Phylogeographic analyses have shown that the population structures of the two species were genetically segmented and that the genetic diversity of S. renifolius was higher than that of S. nipponicus. The phylogenetic relationship between chloroplast and nuclear DNA is topologically different in the two species, which may be due to the asymmetric gene flow ubiquitously observed in plants. The results of this study imply that floral thermogenesis of Symplocarpus contributes to expanding the distribution during an ice age, resulting in increased genetic diversity due to cold adaptation.

Phylogeography of the temperate grassland plant Tephroseris kirilowii (Asteraceae) inferred from multiplexed inter-simple sequence repeat genotyping by sequencing (MIG-seq) data.

A group of temperate grassland plant species termed the "Mansen elements" occurs in Japan and is widely distributed in the grasslands of continental East Asia. It has been hypothesized that these species are continental grassland relicts in Japan that stretch back to a colder age, but their migration history has not been elucidated. To assess the migration history of the Mansen elements, we performed phylogeographic analyses of Tephroseris kirilowii, a member of this group, using single-nucleotide polymorphisms (SNPs) obtained from multiplexed inter-simple sequence repeat genotyping by sequencing (MIG-seq). It was estimated that the Japanese populations of T. kirilowii were divided from those of continental East Asia at 25.2 thousand years ago (ka) with 95% highest probability density interval (HPD) of 15.3-40.0 ka and that Japanese clades first diverged at 20.2 ka with 95% HPD of 10.4-30.1 ka. As the climatically suitable range during the last glacial maximum (LGM) estimated using ecological niche modeling (ENM) was limited in Japan and there was a slight genetic differentiation among Japanese populations, a post-glacial expansion of T. kirilowii in the Japanese Archipelago was indicated.

Norway spruce postglacial recolonization of Fennoscandia.

Contrasting theories exist regarding how Norway spruce (Picea abies) recolonized Fennoscandia after the last glaciation and both early Holocene establishments from western microrefugia and late Holocene colonization from the east have been postulated. Here, we show that Norway spruce was present in southern Fennoscandia as early as 14.7 ± 0.1 cal. kyr BP and that the millennia-old clonal spruce trees present today in central Sweden likely arrived with an early Holocene migration from the east. Our findings are based on ancient sedimentary DNA from multiple European sites (N = 15) combined with nuclear and mitochondrial DNA analysis of ancient clonal (N = 135) and contemporary spruce forest trees (N = 129) from central Sweden. Our other findings imply that Norway spruce was present shortly after deglaciation at the margins of the Scandinavian Ice Sheet, and support previously disputed finds of pollen in southern Sweden claiming spruce establishment during the Lateglacial.

Development of a rational framework for the therapeutic efficacy of fecal microbiota transplantation for calf diarrhea treatment.

BACKGROUND: Establishing fecal microbiota transplantation (FMT) to prevent multifactorial diarrhea in calves is challenging because of the differences in farm management practices, the lack of optimal donors, and recipient selection. In this study, the underlying factors of successful and unsuccessful FMT treatment cases are elucidated, and the potential markers for predicting successful FMT are identified using fecal metagenomics via 16S rRNA gene sequencing, fecal metabolomics via capillary electrophoresis time-of-flight mass spectrometry, and machine learning approaches. RESULTS: Specifically, 20 FMT treatment cases, in which feces from healthy donors were intrarectally transferred into recipient diarrheal calves, were conducted with a success rate of 70%. Selenomonas was identified as a microorganism genus that showed significant donor-recipient compatibility in successful FMT treatments. A strong positive correlation between the microbiome and metabolome data, which is a prerequisite factor for FMT success, was confirmed by Procrustes analysis in successful FMT (r = 0.7439, P = 0.0001). Additionally, weighted gene correlation network analysis confirmed the positively or negatively correlated pairs of bacterial taxa (family Veillonellaceae) and metabolomic features (i.e., amino acids and short-chain fatty acids) responsible for FMT success. Further analysis aimed at establishing criteria for donor selection identified the genus Sporobacter as a potential biomarker in successful donor selection. Low levels of metabolites, such as glycerol 3-phosphate, dihydroxyacetone phosphate, and isoamylamine, in the donor or recipients prior to FMT, are predicted to facilitate FMT. CONCLUSIONS: Overall, we provide the first substantial evidence of the factors related to FMT success or failure; these findings could improve the design of future microbial therapeutics for treating diarrhea in calves. Video abstract.

The gut microbiota induces Peyer's-patch-dependent secretion of maternal IgA into milk.

The evolutionary strategy of transferring maternal antibodies via milk profoundly impacts the survival, lifelong health, and wellbeing of all neonates, including a pronounced impact on human breastfeeding success and infant development. While there has been increased recognition that interorgan connectivity influences the quality of a mother's milk, potentially to personalize it for her offspring, the underlying bases for these processes are incompletely resolved. Here, we define an essential role of Peyer's patches (PPs) for the generation of plasma cells that secrete maternal immunoglobulin A (IgA) into milk. Our metagenomic analysis reveals that the presence of certain residential microorganisms in the gastrointestinal (GI) tract, such as Bacteroides acidifaciens and Prevotella buccalis, is indispensable for the programming of maternal IgA synthesis prior to lactational transfer. Our data provide important insights into how the microbiome of the maternal GI environment, specifically through PPs, can be communicated to the next generation via milk.

A strategic sampling design revealed the local genetic structure of cold-water fluvial sculpin: a focus on groundwater-dependent water temperature heterogeneity.

A key piece of information for ecosystem management is the relationship between the environment and population genetic structure. However, it is difficult to clearly quantify the effects of environmental factors on genetic differentiation because of spatial autocorrelation and analytical problems. In this study, we focused on stream ecosystems and the environmental heterogeneity caused by groundwater and constructed a sampling design in which geographic distance and environmental differences are not correlated. Using multiplexed ISSR genotyping by sequencing (MIG-seq) method, a fine-scale population genetics study was conducted in fluvial sculpin Cottus nozawae, for which summer water temperature is the determinant factor in distribution and survival. There was a clear genetic structure in the watershed. Although a significant isolation-by-distance pattern was detected in the watershed, there was no association between genetic differentiation and water temperature. Instead, asymmetric gene flow from relatively low-temperature streams to high-temperature streams was detected, indicating the importance of low-temperature streams and continuous habitats. The groundwater-focused sampling strategy yielded insightful results for conservation.

Morphological and molecular evidence reveals three new species of Lithocarpus (Fagaceae) from Bidoup-Nui Ba National Park, Vietnam.

Three new species, Lithocarpusbidoupensis Ngoc & Tagane, L.congtroiensis Ngoc & Yahara, and L.hongiaoensis Ngoc & Binh are described from Bidoup-Nui Ba National Park, Central Highland of Vietnam. Morphological analyses and Maximum likelihood tree based on genome-wide SNPs support the distinction of those species from the previously known taxa in the region. The three new species are considered to be endemic to the Bidoup-Nui Ba National Park and the preliminary conservation status for each species is evaluated as Critically Endangered.

A Narrow Endemic or a Species Showing Disjunct Distribution? Studies on Meehania montis-koyae Ohwi (Lamiaceae).

Meehania montis-koyae Ohwi (Lamiaceae), which has been considered a narrow endemic and endangered species in Japan, was found in eastern China in 2011. China and Japan belong to the same floristic region and share many plant species, but it is very rare that Japanese narrow endemic species are newly found outside of the country. We examined herbarium specimens of both countries, and conducted analyses of molecular phylogenetics, population genetics, and divergence time estimation using two nuclear (ITS and ETS) gene regions and MIG-seq data. Chinese plants tend to become larger than Japanese, and they are different in leaf shape and floral features. Molecular phylogenetic analysis shows Chinese and Japanese M. montis-koyae are the closest relatives to each other. Population genetic analysis indicates no current gene flow between the Chinese and Japanese populations, and divergence time analysis shows they were separated during the late Miocene. We reach the conclusion that Chinese and Japanese M. montis-koyae have already become distinct biological entities, and a new taxon name Meehania zheminensis A. Takano, Pan Li, G.-H.Xia is proposed for the Chinese plants. A key to Asian Meehania species is provided.

Elucidation of the Effects of a Current X-SCID Therapy on Intestinal Lymphoid Organogenesis Using an In Vivo Animal Model.

BACKGROUND & AIMS: Organ-level research using an animal model lacking Il2rg, the gene responsible for X-linked severe combined immunodeficiency (X-SCID), is clinically unavailable and would be a powerful tool to gain deeper insights into the symptoms of patients with X-SCID. METHODS: We used an X-SCID animal model, which was first established in our group by the deletion of Il2rg gene in pigs, to understand the clinical signs from multiple perspectives based on pathology, immunology, microbiology, and nutrition. We also treated the X-SCID pigs with bone marrow transplantation (BMT) for mimicking a current therapeutic treatment for patients with X-SCID and investigated the effect at the organ-level. Moreover, the results were confirmed using serum and fecal samples collected from patients with X-SCID. RESULTS: We demonstrated that X-SCID pigs completely lacked Peyer's patches (PPs) and IgA production in the small intestine, but possessed some dysfunctional intestinal T and B cells. Another novel discovery was that X-SCID pigs developed a heterogeneous intestinal microflora and possessed abnormal plasma metabolites, indicating that X-SCID could be an immune disorder that affects various in vivo functions. Importantly, the organogenesis of PPs in X-SCID pigs was not promoted by BMT. Although a few isolated lymphoid follicles developed in the small intestine of BMT-treated X-SCID pigs, there was no evidence that they contributed to IgA production and microflora formation. Consistently, most patients with X-SCID who received BMT possessed abnormal intestinal immune and microbial environments regardless of the presence of sufficient serum IgG. CONCLUSIONS: These results indicate that the current BMT therapies for patients with X-SCID may be insufficient to induce the organogenesis of intestinal lymphoid tissues that are associated with numerous functions in vivo.

Genetic consequences of plant edaphic specialization to solfatara fields: Phylogenetic and population genetic analysis of Carex angustisquama (Cyperaceae).

Edaphic specialization is one of the main drivers of plant diversification and has multifaceted effects on population dynamics. Carex angustisquama is a sedge plant growing only on heavily acidified soil in solfatara fields, where only extremophytes can survive. Because of the lack of closely related species in similar habitats and its disjunct distribution, the species offers ideal settings to investigate the effects of adaptation to solfatara fields and of historical biogeography on the genetic consequences of plant edaphic specialization to solfatara fields. Here, genome-wide single nucleotide polymorphisms were used to reveal the phylogenetic origin of C. angustisquama, and 16 expressed sequence tag-simple sequence repeat markers were employed to infer population demography of C angustisquama. Molecular phylogenetic analysis strongly indicated that C. angustisquama formed a monophyletic clade with Carex doenitzii, a species growing on nonacidified soil in the sympatric subalpine zone. The result of population genetic analysis showed that C. angustisquama has much lower genetic diversity than the sister species, and notably, all 16 loci were completely homozygous in most individuals of C. angustisquama. Approximate Bayesian computation analysis supported the model that assumed hierarchical declines of population size through its evolutionary sequence. We propose that the edaphic specialist in solfatara fields has newly attained the adaptation to solfatara fields in the process of speciation. Furthermore, we found evidence of a drastic reduction in genetic diversity in C. angustisquama, suggesting that the repeated founder effects associated with edaphic specialization and subsequent population demography lead to the loss of genetic diversity of this extremophyte in solfatara fields.

Genet dynamics of a regenerating dwarf bamboo population across heterogeneous light environments in a temperate forest understorey.

Despite the advantage of plant clonality in patchy environments, studies focusing on genet demography in relation to spatially heterogeneous environments remain scarce. Regeneration of bamboos in forest understoreys after synchronous die-off provides an opportunity for assessing how they come to proliferate across heterogeneous light environments. In a Japanese forest, we examined genet demography of a population of Sasa kurilensis over a 7-year period starting 10 years after die-off, shortly after which some genets began spreading horizontally by rhizomes. The aboveground biomass was estimated, and genets were discriminated in 9-m2 plots placed under both canopy gaps and closed canopies. Overall, the results suggest that the survival and spread of more productive genets and the spatial expansion of genets into closed canopies underlie the proliferation of S. kurilensis. Compared to canopy gaps, the recovery rate of biomass was much slower under closed canopies for the first 10 years after the die-off, but became accelerated during the next 7 years. Genet survival was greater for more productive genets (with greater initial number of culms), and the spaces occupied by genets that died were often colonized afterward by clonal growth of surviving genets. The number of genets decreased under canopy gaps due to greater mortality, but increased under closed canopies where greater number of genets colonized clonally from outside the plots than genets died. The colonizing genets were more productive (having larger culms) than those originally germinated within the plots, and the contribution of colonizing genets to the biomass was greater under closed canopies. Our study emphasizes the importance of investigating genet dynamics over relevant spatiotemporal scales to reveal processes underlying the success of clonal plants in heterogeneous habitats.

Female and male fitness consequences of clonal growth in a dwarf bamboo population with a high degree of clonal intermingling.

BACKGROUND AND AIMS: Although many studies have reported that clonal growth interferes with sexual reproduction as a result of geitonogamous self-pollination and inbreeding depression, the mating costs of clonal growth are expected to be reduced when genets are spatially intermingled with others. This study examined how clonal growth affects both female and male reproductive success by studying a population of a mass-flowering plant, Sasa veitchii var. hirsuta, with a high degree of clonal intermingling. METHODS: In a 10 × 10 m plot, genets were discriminated based on the multilocus genotypes of 11 nuclear microsatellite loci. The relationships between genet size and the components of reproductive success were then investigated. Male siring success and female and male selfing rates were assessed using paternity analysis. KEY RESULTS: A total of 111 genets were spatially well intermingled with others. In contrast to previous studies with species forming distinct monoclonal patches, seed production linearly increased with genet size. While male siring success was a decelerating function of genet size, selfing rates were relatively low and not related to genet size. CONCLUSIONS: The results, in conjunction with previous studies, emphasize the role of the spatial arrangement of genets on both the quantity and quality of offpsring, and suggest that an intermingled distribution of genets can reduce the mating costs of clonal growth and enhance overall fitness, particularly female fitness.

Clonal structure, seed set, and self-pollination rate in mass-flowering bamboo species during off-year flowering events.

Bamboos are typical examples of highly synchronized semelparous species. Their mass-flowering events occur at supra-annual intervals but they sometimes flower on a small scale in off-years. If some bamboo ramets (culms) of a genet flower and die in off-years, whereas other culms of the same genet do not flower synchronously, the genet can still survive blooming in an off-year and could participate in the next mass-flowering event. At genet level, the effect might be similar to that achieved by synchronously reproducing iteroparous plants. In addition, if multiple genets flower simultaneously in off-years, cross-pollination will be promoted. However, it is not known whether all the culms in a genet flower synchronously and whether multiple genets flower in off-years. We determined the clonal structure of three temperate dwarf bamboo species, i.e., Sasa senanensis, S. kurilensis, and S. palmata, at 24 off-year flowering sites and the surrounding areas in northern Japan using seven microsatellite markers. We also estimated seed set at seven of the sites and self-pollination rates at five sites to determine off-year reproductive success. Next, we investigated whether seed sets at the culm level were related to flowering area and/or number of flowering genets, using generalized linear mixed-effect models (GLMMs). Multiple genets flowered at 9/24 flowering sites. We found that 40/96 of the genets identified had some flowering culms. Non-flowering culms were present in 24/40 flowering genets. Seed set was in the range 2.2%-12.5% and the self-pollination rate was 96.3%. In the best GLMM, seed set increased with flowering area. Seeds were produced in off-years, but cross-pollination was rare in off-years. We suggest that some dwarf bamboos may exhibit iteroparity or imperfectly synchronized semelparity at the genet level, a characteristic similar to that of other reproductively synchronous plants. We also found synchronous flowering of a few genets even in off-years.

[Efficacy and toxicity of lapatinib plus capecitabine therapy in HER2-positive metastatic breast cancer].

We retrospectively investigated the efficacy and toxicity of lapatinib plus capecitabine in 45 HER2-positive breast cancer patients. The median number of treatment courses was 6(1-22). Brain metastasis developed in 18 cases(40%), and 19 cases(42.2%)had received previous capecitabine treatment for metastatic breast cancer. The objective response rate(ORR=CR+PR)was 22.2%(10/45), and clinical benefit rate(CR+PR+long SD=24w)was 46.7%(21/45).The median time to progression(TTP)was 24.9 weeks(95% CI: 15.2 -34.6 ), and the median overall survival(OS)was 78.1 weeks(95% CI: 55.7 -100.5)in all 45 cases. The median TTP was significantly longer in patients who had not received capecitabine previously(30 vs 16 weeks, 95% CI: 16.3 -43.7, p=0.0051 ). There was no statistical difference in median OS associated with previous capecitabine exposure(42.7 weeks, 95% CI: 21.4 -64, p=0.057 ). The median TTP was significantly longer in patients who received less than 2 treatment regimens with trastuzumab for MBC rather than 3 regimens more(27.3 vs 16 weeks, p=0.0257 ), but there was no statistical difference in median OS(81 vs 40.9 weeks, p=0.26 ). Lapatinib in combination with capecitabine is likely more useful in patients who are naive to capecitabine, who received less than two regimens for metastatic breast cancer.

Japanese two-year-olds use morphosyntax to learn novel verb meanings.

Previous research has found that children who are acquiring argument-drop languages such as Turkish and Chinese make use of syntactic frames to extend familiar verb meanings (Göksun, Küntay & Naigles, 2008; Lee & Naigles, 2008). This article investigates whether two-year-olds learning Japanese, another argument-drop language, make use of argument number and case markings in learning novel verbs. Children watched videos of novel causative and non-causative actions via Intermodal Preferential Looking. The novel verbs were presented in transitive or intransitive frames; the NPs in the transitive frames appeared 'bare' or with case markers. Consistent with previous findings of Morphosyntactic Bootstrapping, children who heard the novel verbs in the transitive frame with case markers reliably assigned those verbs to the novel causative actions.