Foraging ants affect community composition and diversity of phyllosphere fungi on a myrmecophilous plants, Mallotus japonicus.

Many microorganisms inhabit the aboveground parts of plants (i.e. the phyllosphere), which mainly comprise leaves. Understanding the structure of phyllosphere microbial communities and their drivers is important because they influence host plant fitness and ecosystem functions. Despite the high prevalence of ant-plant associations, few studies have used quantitative community data to investigate the effects of ants on phyllosphere microbial communities. In the present study, we investigated the effects of ants on the phyllosphere fungal communities of Mallotus japonicus using high-throughput sequencing. Mallotus japonicus is a myrmecophilous plants that bears extrafloral nectaries, attracting several ant species, but does not provide specific ant species with nest sites like myrmecophytes do. We experimentally excluded ants with sticky resins from the target plants and collected leaf discs to extract fungal DNA. The ribosomal DNA internal transcribed spacer 1 (ITS1) regions of the phyllosphere fungi were amplified and sequenced to obtain fungal community data. Our results showed that the exclusion of ants changed the phyllosphere fungal community composition; however, the effect of ants on OTU richness was not clear. These results indicate that ants can change the community of phyllosphere fungi, even if the plant is not a myrmecophyte.

The evolution of ectomycorrhizal symbiosis in the Late Cretaceous is a key driver of explosive diversification in Agaricomycetes.

Ectomycorrhizal (EcM) symbiosis, a ubiquitous plant-fungus interaction in forests, evolved in parallel in fungi. Why the evolution of EcM fungi did not necessarily increase ecological opportunities for explosive diversification remains unclear. This study aimed to reveal the driving mechanism of the evolutionary diversification in the fungal class Agaricomycetes, specifically by testing whether the evolution of EcM symbiosis in the Late Cretaceous increased ecological opportunities. The historical character transitions of trophic state and fruitbody form were estimated based on phylogenies inferred from fragments of 89 single-copy genes. Moreover, five analyses were used to estimate the net diversification rates (speciation rate minus extinction rate). The results indicate that the unidirectional evolution of EcM symbiosis occurred 27 times, ranging in date from the Early Triassic to the Early Paleogene. The increased diversification rates appeared to occur intensively at the stem of EcM fungal clades diverging in the Late Cretaceous, coinciding with the rapid diversification of EcM angiosperms. By contrast, the evolution of fruitbody form was not strongly linked with the increased diversification rates. These findings suggest that the evolution of EcM symbiosis in the Late Cretaceous, supposedly with coevolving EcM angiosperms, was the key drive of the explosive diversification in Agaricomycetes.

The Limited Establishment of Native Ectomycorrhizal Fungi in Exotic Eucalyptus spp. Stands in Japan.

Host specificity may potentially limit the distribution expansion of ectomycorrhizal (ECM) fungi into areas where their original host plants are absent. To test this hypothesis, we investigated whether populations of native ECM fungi may establish in stands of exotic host trees, namely those of the Eucalyptus species, in Japan. ECM fungal communities associated with eucalyptus and surrounding native host species (Pinus thunbergii and Fagaceae spp.) were investigated at two sites; one site in which eucalyptus and native trees were growing in isolation, and a second site in which these species were mixed. To identify fungal taxa, the nuclear ribosomal internal transcribed spacer region 1 was sequenced for the ECM fungi from the root tips and clustered into operational taxonomic units (OTUs). To confirm whether the retrieved OTUs were native to Japan, they were queried against the entire database of the National Center for Biotechnology Information, UNITE, and GlobalFungi, whereby sampling locations and associated hosts were obtained from sequences with ≥97% similarity. Eucalyptus trees were associated with seven and 12 ECM fungal OTUs, including putatively exotic OTUs in isolated and mixed sites, respectively. Among the 36 and 63 native ECM fungal OTUs detected from native hosts at isolated and mixed sites, only one OTU was shared with eucalyptus at the respective sites. This means that most native ECM fungi in Japan may be incapable of forming an association with exotic Eucalyptus spp. Notably, even ECM fungi associated with both Pinus and Quercus were not detected from eucalyptus, suggesting that host-fungus incompatibility is determined not only by host phylogenetic relatedness but also by host biogeographic affinities. Our findings show that the incompatibility with eucalyptus as well as dispersal limitation may prevent the distribution expansion of native ECM fungi in Japan into the distribution ranges of Eucalyptus spp., where the original hosts are absent.

Aboveground herbivores drive stronger plant species-specific feedback than belowground fungi to regulate tree community assembly.

Ectomycorrhizal (EcM) tree species often become more dominant than arbuscular mycorrhizal (AM) tree species in temperate forests, but they generally coexist. Theory predicts that ecological feedback mediated by aboveground herbivory and/or belowground microbes could explain these dominance/coexistence patterns. An experimental test of how aboveground/belowground organisms associated with AM/EcM trees mediate ecological feedbacks has been lacking at the community-level. By establishing AM and EcM tree sapling assemblages in mesocosms and then introducing seedlings of each type in a reciprocal planting experiment, we compared seedling performance under varying sapling species (conspecifics, heterospecifics within the same and different mycorrhizal types), using traits that reflect either aboveground herbivory-mediated feedback or belowground fungal-mediated feedback or both. When examining seedling traits that reflect aboveground herbivory-mediated feedbacks (i.e., foliar damage), AM plants tended to experience less foliar damage and EcM plants more damage under conspecific versus heterospecific saplings within the same mycorrhizal types, and aboveground herbivory-mediated feedback was species-specific rather than mycorrhizal type-specific. Conversely, when examining traits that reflect belowground fungal-mediated feedbacks, both AM and EcM plant species often exhibited mycorrhizal type-specific feedbacks (e.g., greater aboveground biomass under the same versus different mycorrhizal-type saplings) rather than species-specific feedbacks. Furthermore, tree species affected by herbivory-mediated feedback were less affected by belowground feedback, indicating that the relative importance of the feedbacks varied among plant species. Analysis of plant-associated organisms verified that the feedback outcomes corresponded with species accumulation of belowground fungi (but not of aboveground herbivores). Thus, aboveground herbivores drive stronger plant species-specific feedback than belowground fungi to regulate temperate tree diversity.

Molecular prospecting for cryptic species of the Hypholoma fasciculare complex: toward the effective and practical delimitation of cryptic macrofungal species.

Many macrofungal cryptic species remain unidentified. A possible solution is to increase the number of loci analyzed and use rigorous statistics for macrofungal species delimitation. To validate this assumption, cryptic species of the Hypholoma fasciculare complex, a group of common wood-decomposing fungi, were attempted to be delineated. Massively parallel sequencing of mitochondrial ribosomal RNA (mt_rRNA), nuclear ribosomal internal transcribed spacer (ITS) region, and 24 single-copy genes were performed for 96 specimens collected in Japan. Then, the species boundaries were inferred using comparative gene genealogies (mt_rRNA vs. ITS), Bayesian Poisson tree process (bPTP) model for the phylogeny of concatenated nuclear sequences, and analysis of molecular variance (AMOVA) for single nucleotide polymorphisms. In both the mt_rRNA and ITS phylogenies, the H. fasciculare complex was not divided into well-supported clades. Nevertheless, based on the bPTP, two mitochondrial haplotypes were inferred to represent distinct species (H. fasciculare and H. subviride). The results of AMOVA also indicated that the differentiation of nuclear loci can be explained mostly by differences between haplotype. These results suggest that it is necessary to increase the number of target loci to 20 or more and use both phylogeny-based and population genetics-based statistics for the accurate delimitation of macrofungal species.

Effects of sampling seasons and locations on fish environmental DNA metabarcoding in dam reservoirs.

Environmental DNA (eDNA) analysis has seen rapid development in the last decade, as a novel biodiversity monitoring method. Previous studies have evaluated optimal strategies, at several experimental steps of eDNA metabarcoding, for the simultaneous detection of fish species. However, optimal sampling strategies, especially the season and the location of water sampling, have not been evaluated thoroughly. To identify optimal sampling seasons and locations, we performed sampling monthly or at two-monthly intervals throughout the year in three dam reservoirs. Water samples were collected from 15 and nine locations in the Miharu and Okawa dam reservoirs in Fukushima Prefecture, respectively, and five locations in the Sugo dam reservoir in Hyogo Prefecture, Japan. One liter of water was filtered with glass-fiber filters, and eDNA was extracted. By performing MiFish metabarcoding, we successfully detected a total of 21, 24, and 22 fish species in Miharu, Okawa, and Sugo reservoirs, respectively. From these results, the eDNA metabarcoding method had a similar level of performance compared to conventional long-term data. Furthermore, it was found to be effective in evaluating entire fish communities. The number of species detected by eDNA survey peaked in May in Miharu and Okawa reservoirs, and in March and June in Sugo reservoir, which corresponds with the breeding seasons of many of fish species inhabiting the reservoirs. In addition, the number of detected species was significantly higher in shore, compared to offshore samples in the Miharu reservoir, and a similar tendency was found in the other two reservoirs. Based on these results, we can conclude that the efficiency of species detection by eDNA metabarcoding could be maximized by collecting water from shore locations during the breeding seasons of the inhabiting fish. These results will contribute in the determination of sampling seasons and locations for fish fauna survey via eDNA metabarcoding, in the future.

Environmental DNA analysis shows high potential as a tool for estimating intraspecific genetic diversity in a wild fish population.

Environmental DNA (eDNA) analysis has recently been used as a new tool for estimating intraspecific diversity. However, whether known haplotypes contained in a sample can be detected correctly using eDNA-based methods has been examined only by an aquarium experiment. Here, we tested whether the haplotypes of Ayu fish (Plecoglossus altivelis altivelis) detected in a capture survey could also be detected from an eDNA sample derived from the field that contained various haplotypes with low concentrations and foreign substances. A water sample and Ayu specimens collected from a river on the same day were analysed by eDNA analysis and Sanger sequencing, respectively. The 10 L water sample was divided into 20 filters for each of which 15 PCR replications were performed. After high-throughput sequencing, denoising was performed using two of the most widely used denoising packages, unoise3 and dada2. Of the 42 haplotypes obtained from the Sanger sequencing of 96 specimens, 38 (unoise3) and 41 (dada2) haplotypes were detected by eDNA analysis. When dada2 was used, except for one haplotype, haplotypes owned by at least two specimens were detected from all the filter replications. Accordingly, although it is important to note that eDNA-based method has some limitations and some risk of false positive and false negative, this study showed that the eDNA analysis for evaluating intraspecific genetic diversity provides comparable results for large-scale capture-based conventional methods. Our results suggest that eDNA-based methods could become a more efficient survey method for investigating intraspecific genetic diversity in the field.

Environmental DNA metabarcoding for fish community analysis in backwater lakes: A comparison of capture methods.

The use of environmental DNA (eDNA) methods for community analysis has recently been developed. High-throughput parallel DNA sequencing (HTS), called eDNA metabarcoding, has been increasingly used in eDNA studies to examine multiple species. However, eDNA metabarcoding methodology requires validation based on traditional methods in all natural ecosystems before a reliable method can be established. To date, relatively few studies have performed eDNA metabarcoding of fishes in aquatic environments where fish communities were intensively surveyed using multiple traditional methods. Here, we have compared fish communities' data from eDNA metabarcoding with seven conventional multiple capture methods in 31 backwater lakes in Hokkaido, Japan. We found that capture and field surveys of fishes were often interrupted by macrophytes and muddy sediments in the 31 lakes. We sampled 1 L of the surface water and analyzed eDNA using HTS. We also surveyed the fish communities using seven different capture methods, including various types of nets and electrofishing. At some sites, we could not detect any eDNA, presumably because of the polymerase chain reaction (PCR) inhibition. We also detected the marine fish species as sewage-derived eDNA. Comparisons of eDNA metabarcoding and capture methods showed that the detected fish communities were similar between the two methods, with an overlap of 70%. Thus, our study suggests that to detect fish communities in backwater lakes, the performance of eDNA metabarcoding with the use of 1 L surface water sampling is similar to that of capturing methods. Therefore, eDNA metabarcoding can be used for fish community analysis but environmental factors that can cause PCR inhibition, should be considered in eDNA applications.

Timing of evolutionary innovation: scenarios of evolutionary diversification in a species-rich fungal clade, Boletales.

Acquisition of mutualistic symbiosis could provide hosts and/or symbionts with novel ecological opportunities for evolutionary diversification. Such a mechanism is one of the major components of coevolutionary diversification. However, whether the origin of mycorrhizal symbiosis promotes diversification in fungi still requires clarification. Here, we aimed to reveal evolutionary diversification in a clade comprising ectomycorrhizal (ECM) fungi. Based on a phylogenic tree inferred from the sequences of 87 single-copy genes, we reconstructed the origins of ECM symbiosis in a species-rich basidiomycetous order, Boletales. High-resolution phylogeny of Boletales revealed that ECM symbiosis independently evolved from non-ECM states at least four times in the group. Among them, only the second most recent event, occurring in the clade of Boletaceae, was inferred to involve an almost synchronous rapid diversification and rapid transition from non-ECM to ECM symbiosis. Our results contradict the hypothesis of evolutionary priority effect, which postulates the greatest ecological opportunities in the oldest lineages. Therefore, the novel resources that had not been pre-empted by the old ECM fungal lineages - supposedly the coevolving angiosperm hosts - could be available for the young ECM fungal lineages, which resulted in evolutionary diversification occurring only in the young ECM fungal lineages.

Structural diversity across arbuscular mycorrhizal, ectomycorrhizal, and endophytic plant-fungus networks.

BACKGROUND: Below-ground linkage between plant and fungal communities is one of the major drivers of terrestrial ecosystem dynamics. However, we still have limited knowledge of how such plant-fungus associations vary in their community-scale properties depending on fungal functional groups and geographic locations. METHODS: By compiling a high-throughput sequencing dataset of root-associated fungi in eight forests along the Japanese Archipelago, we performed a comparative analysis of arbuscular mycorrhizal, ectomycorrhizal, and saprotrophic/endophytic associations across a latitudinal gradient from cool-temperate to subtropical regions. RESULTS: In most of the plant-fungus networks analyzed, host-symbiont associations were significantly specialized but lacked "nested" architecture, which has been commonly reported in plant-pollinator and plant-seed disperser networks. In particular, the entire networks involving all functional groups of plants and fungi and partial networks consisting of ectomycorrhizal plant and fungal species/taxa displayed "anti-nested" architecture (i.e., negative nestedness scores) in many of the forests examined. Our data also suggested that geographic factors affected the organization of plant-fungus network structure. For example, the southernmost subtropical site analyzed in this study displayed lower network-level specificity of host-symbiont associations and higher (but still low) nestedness than northern localities. CONCLUSIONS: Our comparative analyses suggest that arbuscular mycorrhizal, ectomycorrhizal, and saprotrophic/endophytic plant-fungus associations often lack nested network architecture, while those associations can vary, to some extent, in their community-scale properties along a latitudinal gradient. Overall, this study provides a basis for future studies that will examine how different types of plant-fungus associations collectively structure terrestrial ecosystems.

Mycorrhizal fungi mediate the direction and strength of plant-soil feedbacks differently between arbuscular mycorrhizal and ectomycorrhizal communities.

Plants influence their soil environment, which affects the next generation of seedlings that can be established. While research has shown that such plant-soil feedbacks occur in the presence of mycorrhizal fungi, it remains unclear when and how mycorrhizal fungi mediate the direction and strength of feedbacks in tree communities. Here we show that arbuscular mycorrhizal and ectomycorrhizal fungal guilds mediate plant-soil feedbacks differently to influence large-scale patterns such as tree species coexistence and succession. When seedlings are grown under the same mycorrhizal type forest, arbuscular mycorrhizal plant species exhibit negative or neutral feedbacks and ectomycorrhizal plant species do neutral or positive feedbacks. In contrast, positive and neutral feedbacks dominate when seedlings are grown in associations within the same versus different mycorrhizal types. Thus, ectomycorrhizal communities show more positive feedbacks than arbuscular mycorrhizal communities, potentially explaining why most temperate forests are ectomycorrhizal.

Optimising first- and second-line treatment strategies for untreated major depressive disorder - the SUN☺D study: a pragmatic, multi-centre, assessor-blinded randomised controlled trial.

BACKGROUND: For patients starting treatment for depression, current guidelines recommend titrating the antidepressant dosage to the maximum of the licenced range if tolerated. When patients do not achieve remission within several weeks, recommendations include adding or switching to another antidepressant. However, the relative merits of these guideline strategies remain unestablished. METHODS: This multi-centre, open-label, assessor-blinded, pragmatic trial involved two steps. Step 1 used open-cluster randomisation, allocating clinics into those titrating sertraline up to 50 mg/day or 100 mg/day by week 3. Step 2 used central randomisation to allocate patients who did not remit after 3 weeks of treatment to continue sertraline, to add mirtazapine or to switch to mirtazapine. The primary outcome was depression severity measured with the Patient Health Questionnaire-9 (PHQ-9) (scores between 0 and 27; higher scores, greater depression) at week 9. We applied mixed-model repeated-measures analysis adjusted for key baseline covariates. RESULTS: Between December 2010 and March 2015, we recruited 2011 participants with hitherto untreated major depression at 48 clinics in Japan. In step 1, 970 participants were allocated to the 50 mg/day and 1041 to the 100 mg/day arms; 1927 (95.8%) provided primary outcomes. There was no statistically significant difference in the adjusted PHQ-9 score at week 9 between the 50 mg/day arm and the 100 mg/day arm (0.25 point, 95% confidence interval (CI), - 0.58 to 1.07, P = 0.55). Other outcomes proved similar in the two groups. In step 2, 1646 participants not remitted by week 3 were randomised to continue sertraline (n = 551), to add mirtazapine (n = 537) or to switch to mirtazapine (n = 558): 1613 (98.0%) provided primary outcomes. At week 9, adding mirtazapine achieved a reduction in PHQ-9 scores of 0.99 point (0.43 to 1.55, P = 0.0012); switching achieved a reduction of 1.01 points (0.46 to 1.56, P = 0.0012), both relative to continuing sertraline. Combination increased the percentage of remission by 12.4% (6.1 to 19.0%) and switching by 8.4% (2.5 to 14.8%). There were no differences in adverse effects. CONCLUSIONS: In patients with new onset depression, we found no advantage of titrating sertraline to 100 mg vs 50 mg. Patients unremitted by week 3 gained a small benefit in reduction of depressive symptoms at week 9 by switching sertraline to mirtazapine or by adding mirtazapine. TRIAL REGISTRATION: ClinicalTrials.gov, NCT01109693 . Registered on 23 April 2010.

Network hubs in root-associated fungal metacommunities.

BACKGROUND: Although a number of recent studies have uncovered remarkable diversity of microbes associated with plants, understanding and managing dynamics of plant microbiomes remain major scientific challenges. In this respect, network analytical methods have provided a basis for exploring "hub" microbial species, which potentially organize community-scale processes of plant-microbe interactions. METHODS: By compiling Illumina sequencing data of root-associated fungi in eight forest ecosystems across the Japanese Archipelago, we explored hubs within "metacommunity-scale" networks of plant-fungus associations. In total, the metadata included 8080 fungal operational taxonomic units (OTUs) detected from 227 local populations of 150 plant species/taxa. RESULTS: Few fungal OTUs were common across all the eight forests. However, in each of the metacommunity-scale networks representing northern four localities or southern four localities, diverse mycorrhizal, endophytic, and pathogenic fungi were classified as "metacommunity hubs," which were detected from diverse host plant taxa throughout a climatic region. Specifically, Mortierella (Mortierellales), Cladophialophora (Chaetothyriales), Ilyonectria (Hypocreales), Pezicula (Helotiales), and Cadophora (incertae sedis) had broad geographic and host ranges across the northern (cool-temperate) region, while Saitozyma/Cryptococcus (Tremellales/Trichosporonales) and Mortierella as well as some arbuscular mycorrhizal fungi were placed at the central positions of the metacommunity-scale network representing warm-temperate and subtropical forests in southern Japan. CONCLUSIONS: The network theoretical framework presented in this study will help us explore prospective fungi and bacteria, which have high potentials for agricultural application to diverse plant species within each climatic region. As some of those fungal taxa with broad geographic and host ranges have been known to promote the survival and growth of host plants, further studies elucidating their functional roles are awaited.

Composition and Diversity of Soil Fungi in Dipterocarpaceae-Dominated Seasonal Tropical Forests in Thailand.

Although fungi play essential roles in nutrient cycles and plant growth in forest ecosystems, limited information is currently available on the community compositions of soil fungi in tropical forests. Few studies have examined fungal community structures in seasonal tropical forests, in which forest fires potentially have a large impact on above- and belowground community processes. Based on high-throughput sequencing technologies, we herein examined the diversity and community structures of soil fungi in dry seasonal tropical forests in Sakaerat, northeast Thailand. We found that fungal community compositions diverged among dry evergreen, dry deciduous, and fire-protected dry deciduous forests within the region. Although tree species diversity did not positively correlate with soil fungal diversity, the coverage of an understory bamboo species (Vietnamosasa pusilla) showed a strong relationship with fungal community structures. Our community ecological analysis also yielded a list of fungi showing habitat preferences for either of the neighboring evergreen and deciduous forests in Sakaerat. The present results provide a basis for managing soil fungal communities and aboveground plant communities in seasonal tropical forests in Southeast Asia.

Root-Associated Fungi Shared Between Arbuscular Mycorrhizal and Ectomycorrhizal Conifers in a Temperate Forest.

Arbuscular mycorrhizal and ectomycorrhizal symbioses are among the most important drivers of terrestrial ecosystem dynamics. Historically, the two types of symbioses have been investigated separately because arbuscular mycorrhizal and ectomycorrhizal plant species are considered to host discrete sets of fungal symbionts (i.e., arbuscular mycorrhizal and ectomycorrhizal fungi, respectively). Nonetheless, recent studies based on high-throughput DNA sequencing technologies have suggested that diverse non-mycorrhizal fungi (e.g., endophytic fungi) with broad host ranges play roles in relationships between arbuscular mycorrhizal and ectomycorrhizal plant species in forest ecosystems. By analyzing an Illumina sequencing dataset of root-associated fungi in a temperate forest in Japan, we statistically examined whether co-occurring arbuscular mycorrhizal (Chamaecyparis obtusa) and ectomycorrhizal (Pinus densiflora) plant species could share non-mycorrhizal fungal communities. Among the 919 fungal operational taxonomic units (OTUs) detected, OTUs in various taxonomic lineages were statistically designated as "generalists," which associated commonly with both coniferous species. The list of the generalists included fungi in the genera Meliniomyces, Oidiodendron, Cladophialophora, Rhizodermea, Penicillium, and Mortierella. Meanwhile, our statistical analysis also detected fungi preferentially associated with Chamaecyparis (e.g., Pezicula) or Pinus (e.g., Neolecta). Overall, this study provides a basis for future studies on how arbuscular mycorrhizal and ectomycorrhizal plant species interactively drive community- or ecosystem-scale processes. The physiological functions of the fungi highlighted in our host-preference analysis deserve intensive investigations for understanding their roles in plant endosphere and rhizosphere.

Cognitive and Behavioral Skills Exercises Completed by Patients with Major Depression During Smartphone Cognitive Behavioral Therapy: Secondary Analysis of a Randomized Controlled Trial.

BACKGROUND: A strong and growing body of evidence has demonstrated the effectiveness of cognitive behavioral therapy (CBT), either face-to-face, in person, or as self-help via the Internet, for depression. However, CBT is a complex intervention consisting of several putatively effective components, and how each component may or may not contribute to the overall effectiveness of CBT is poorly understood. OBJECTIVE: The aim of this study was to investigate how the users of smartphone CBT use and benefit from various components of the program. METHODS: This is a secondary analysis from a 9-week, single-blind, randomized controlled trial that has demonstrated the effectiveness of adjunctive use of smartphone CBT (Kokoro-App) over antidepressant pharmacotherapy alone among patients with drug-resistant major depressive disorder (total n=164, standardized mean difference in depression severity at week 9=0.40, J Med Internet Res). Kokoro-App consists of three cognitive behavioral skills of self-monitoring, behavioral activation, and cognitive restructuring, with corresponding worksheets to fill in. All activities of the participants learning each session of the program and completing each worksheet were uploaded onto Kokoro-Web, which each patient could use for self-check. We examined what use characteristics differentiated the more successful users of the CBT app from the less successful ones, split at the median of change in depression severity. RESULTS: A total of 81 patients with major depression were allocated to the smartphone CBT. On average, they completed 7.0 (standard deviation [SD] 1.4) out of 8 sessions of the program; it took them 10.8 (SD 4.2) days to complete one session, during which they spent 62 min (SD 96) on the app. There were no statistically significant differences in the number of sessions completed, time spent for the program, or the number of completed self-monitoring worksheets between the beneficiaries and the nonbeneficiaries. However, the former completed more behavioral activation tasks, engaged in different types of activities, and also filled in more cognitive restructuring worksheets than the latter. Activities such as "test-drive a new car," "go to a coffee shop after lunch," or "call up an old friend" were found to be particularly rewarding. All cognitive restructuring strategies were found to significantly decrease the distress level, with "What would be your advice to a friend who has a similar problem?" found more helpful than some other strategies. CONCLUSIONS: The CBT program offered via smartphone and connected to the remote server is not only effective in alleviating depression but also opens a new avenue in gathering information of what and how each participant may utilize the program. The activities and strategies found useful in this analysis will provide valuable information in brush-ups of the program itself and of mobile health (mHealth) in general. TRIAL REGISTRATION: Japanese Clinical Trials Registry UMIN CTR 000013693; https://upload.umin.ac.jp/cgi-open-bin/ctr_e/ ctr_view.cgi?recptno=R000015984 (Archived by WebCite at http://www.webcitation.org/6u6pxVwik).

Usefulness and limitations of sample pooling for environmental DNA metabarcoding of freshwater fish communities.

Environmental DNA (eDNA) metabarcoding has been used increasingly to assess biodiversity of aquatic vertebrates. However, there still remains to be developed a sampling design of eDNA metabarcoding that can ensure high detection rates of species with minimum total survey effort, especially for large-scale surveys of aquatic organisms. We here tested whether pooling of eDNA samples can be used to evaluate biodiversity of freshwater fishes in four satellite lakes of Lake Biwa, Japan. Fish communities detected by eDNA metabarcoding of the mitochondrial 12S region were compared between the individual and pooled samples. In the individual samples, 31, 22, 33, and 31 fish lineages (proxies for species) were observed at the respective sites, within which moderate spatial autocorrelation existed. In the pooled samples, 30, 20, 29, and 27, lineages were detected, respectively, even after 15 PCR replicates. Lineages accounting for < 0.05% of the total read count of each site's individual samples were mostly undetectable in the pooled samples. Moreover, fish communities detected were similar among PCR replicates in the pooled samples. Because of the decreased detection rates, the pooling strategy is unsuitable for estimating fish species richness. However, this procedure is useful potentially for among-site comparison of representative fish communities.

Not only in the temperate zone: independent gametophytes of two vittarioid ferns (Pteridaceae, Polypodiales) in East Asian subtropics.

Independent gametophyte ferns are unique among vascular plants because they are sporophyteless and reproduce asexually to maintain their populations in the gametophyte generation. Such ferns had been primarily discovered in temperate zone, and usually hypothesized with (sub)tropical origins and subsequent extinction of sporophyte due to climate change during glaciations. Presumably, independent fern gametophytes are unlikely to be distributed in tropics and subtropics because of relatively stable climates which are less affected by glaciations. Nonetheless, the current study presents cases of two independent gametophyte fern species in subtropic East Asia. In this study, we applied plastid DNA sequences (trnL-L-F and matK + ndhF + chlL datasets) and comprehensive sampling (~80%) of congeneric species for molecular identification and divergence time estimation of these independent fern gametophytes. The two independent gametophyte ferns were found belonging to genus Haplopteris (vittarioids, Pteridaceae) and no genetic identical sporophyte species in East Asia. For one species, divergence times between its populations imply recent oversea dispersal(s) by spores occurred during Pleistocene. By examining their ex situ and in situ fertility, prezygotic sterility was found in these two Haplopteris, in which gametangia were not or very seldom observed, and this prezygotic sterility might attribute to their lacks of functional sporophytes. Our field observation and survey on their habitats suggest microhabitat conditions might attribute to this prezygotic sterility. These findings point to consideration of whether recent climate change during the Pleistocene glaciation resulted in ecophysiological maladaptation of non-temperate independent gametophyte ferns. In addition, we provided a new definition to classify fern gametophyte independences at the population level. We expect that continued investigations into tropical and subtropical fern gametophyte floras will further illustrate the biogeographic significance of non-temperate fern gametophyte independence.

Host shifts enhance diversification of ectomycorrhizal fungi: diversification rate analysis of the ectomycorrhizal fungal genera Strobilomyces and Afroboletus with an 80-gene phylogeny.

Mutualisms with new host lineages can provide symbionts with novel ecological opportunities to expand their geographical distribution, thereby leading to evolutionary diversification. Because ectomycorrhizal (ECM) fungi provide ideal opportunities to test the relationship between host shifts and diversification, we tested whether mutualism with new host lineages could increase the diversification rates of ECM fungi. Using a Bayesian tree inferred from 23 027-base nucleotide sequences of 80 single-copy genes, we tested whether the diversification rate had changed through host-shift events in the monophyletic clade containing the ECM fungal genera Strobilomyces and Afroboletus. The results indicated that these fungi were initially associated with Caesalpinioideae/Monotoideae in Africa, acquired associations with Dipterocarpoideae in tropical Asia, and then switched to Fagaceae/Pinaceae and Nothofagaceae/Eucalyptus. Fungal lineages associated with Fagaceae/Pinaceae were inferred to have approximately four-fold and two-fold greater diversification rates than those associated with Caesalpinioideae/Monotoideae and Dipterocarpoideae or Nothofagaceae/Eucalyptus, respectively. Moreover, the diversification rate shift was inferred to follow the host shift to Fagaceae/Pinaceae. Our study suggests that host-shift events, particularly those occurring with respect to Fagaceae/Pinaceae, can provide ecological opportunities for the rapid diversification of Strobilomyces-Afroboletus. Although further studies are needed for generalization, we propose a possible diversification scenario of ECM fungi.

Histamine H1 receptor occupancy by the new-generation antipsychotics olanzapine and quetiapine: a positron emission tomography study in healthy volunteers.

RATIONALE: Histamine H1 antagonists have hypnotic, appetite-promoting, and sedative side effects. Most second-generation antipsychotics have potent antagonistic effects on histamine H1 receptor (H1R). Positron emission tomography (PET) can measure the H1R occupancy (H1RO) in vivo, although there are no reports regarding antipsychotics. OBJECTIVES: We studied the H1RO of olanzapine and quetiapine in vivo with respect to their plasma concentrations and subjective drowsiness by performing human PET imaging studies with [(11)C]doxepin, a potent PET ligand of H1R. METHODS: Six healthy Japanese male volunteers were enrolled. Cross-randomized PET imaging was performed after a single oral administration of olanzapine (2.5 mg), quetiapine (25 mg), or placebo. PET data were analyzed by region of interest and voxel-by-voxel analysis. We concurrently measured plasma drug concentrations by liquid chromatography/tandem mass spectrometry and evaluated subjective sleepiness. RESULTS: The binding potential ratios of olanzapine and quetiapine in the cerebral cortex were significantly lower than that of the placebo. The H1RO values of olanzapine and quetiapine in the cortex were approximately 61-80 and 56-81%, respectively. The binding potential ratios of the drugs were significantly lower than that of the placebo in the dorsolateral prefrontal and lateral temporal cortices, and anterior and posterior cingulate gyri. The H1RO values in the cortex were significantly correlated with subjective sleepiness but not plasma drug concentrations. CONCLUSIONS: Olanzapine and quetiapine have high H1RO values in the human brain under their clinical minimum doses. This study provides a foundation of the properties by which new-generation antipsychotics block the central histaminergic system in humans.