Timing of forest fine root production advances with reduced snow cover in northern Japan: implications for climate-induced change in understory and overstory competition.

To investigate the effect of reduced snow cover on fine root dynamics in a cool-temperate forest in northern Japan because of decreases in snowfall at high latitudes due to global warming, we monitored root length, production, and mortality before and after snow removal with an in-ground root scanner. We measured root dynamics of both overstory deciduous oak (Quercus crispula) and understory evergreen dwarf bamboo (Sasa nipponica), the two major species in the forest. Snow removal advanced the timing of peak root production by a month both in total and in Sasa, but not in oak. There was a significant interaction between snow removal and plant form on root production; this indicates that enhanced Sasa root production following snow removal might increase its ability to compete with oak. In contrast, snow removal did not enhance root mortality, suggesting that the roots of these species tolerate soil freezing. The earlier snow disappearance in the snow removal plot expanded the growing season in Sasa. We speculate that this change in the understory environment would advance the timing of root production by Sasa by extending the photosynthetic period in spring. We propose that different responses of root production to reduced snow cover between the two species would change the competitive interactions of overstory and understory vegetation, influencing net primary production and biogeochemistry (e.g., carbon and nitrogen cycles) in the forest ecosystem.

Metagenomic analysis reveals rapid development of soil biota on fresh volcanic ash.

Little is known of the earliest stages of soil biota development of volcanic ash, and how rapidly it can proceed. We investigated the potential for soil biota development during the first 3 years, using outdoor mesocosms of sterile, freshly fallen volcanic ash from the Sakurajima volcano, Japan. Mesocosms were positioned in a range of climates across Japan and compared over 3 years, against the developed soils of surrounding natural ecosystems. DNA was extracted from mesocosms and community composition assessed using 16S rRNA gene sequences. Metagenome sequences were obtained using shotgun metagenome sequencing. While at 12 months there was insufficient DNA for sequencing, by 24 months and 36 months, the ash-soil metagenomes already showed a similar diversity of functional genes to the developed soils, with a similar range of functions. In a surprising contrast with our hypotheses, we found that the developing ash-soil community already showed a similar gene function diversity, phylum diversity and overall relative abundances of kingdoms of life when compared to developed forest soils. The ash mesocosms also did not show any increased relative abundance of genes associated with autotrophy (rbc, coxL), nor increased relative abundance of genes that are associated with acquisition of nutrients from abiotic sources (nifH). Although gene identities and taxonomic affinities in the developing ash-soils are to some extent distinct from the natural vegetation soils, it is surprising that so many of the key components of a soil community develop already by the 24-month stage. In this system, however, rapid development may be facilitated by the relatively moderate pH of the Sakurajima ash, proximity of our mesocosms to propagule sources, and the rapid establishment of a productive bryophyte and lichen layer on the surface. Ash from other volcanoes richer in acids or more distant from propagule sources could show a different pattern and slower soil biota development.

Evaluation of host effects on ectomycorrhizal fungal community compositions in a forested landscape in northern Japan.

Community compositions of ectomycorrhizal (ECM) fungi are similar within the same host taxa. However, careful interpretation is required to determine whether the combination of ECM fungi and plants is explained by the host preference for ECM fungi, or by the influence of neighbouring heterospecific hosts. In the present study, we aimed to evaluate the effects of host species on the ECM community compositions in a forested landscape (approx. 10 km) where monodominant forest stands of six ECM host species belonging to three families were patchily distributed. A total of 180 ECM operational taxonomic units (OTUs) were detected with DNA metabarcoding. Quantitative multivariate analyses revealed that the ECM community compositions were primarily structured by host species and families, regardless of the soil environments and spatial arrangements of the sampling plots. In addition, 38 ECM OTUs were only detected from particular host tree species. Furthermore, the neighbouring plots harboured similar fungal compositions, although the host species were different. The relative effect of the spatial factors on the ECM compositions was weaker than that of host species. Our results suggest that the host preference for ECM fungi is the primary determinant of ECM fungal compositions in the forested landscape.

Arbuscular Mycorrhizal Community in Roots and Nitrogen Uptake Patterns of Understory Trees Beneath Ectomycorrhizal and Non-ectomycorrhizal Overstory Trees.

Nitrogen (N) is an essential plant nutrient, and plants can take up N from several sources, including via mycorrhizal fungal associations. The N uptake patterns of understory plants may vary beneath different types of overstory trees, especially through the difference in their type of mycorrhizal association (arbuscular mycorrhizal, AM; or ectomycorrhizal, ECM), because soil mycorrhizal community and N availability differ beneath AM (non-ECM) and ECM overstory trees (e.g., relatively low nitrate content beneath ECM overstory trees). To test this hypothesis, we examined six co-existing AM-symbiotic understory tree species common beneath both AM-symbiotic black locust (non-ECM) and ECM-symbiotic oak trees of dryland forests in China. We measured AM fungal community composition of roots and natural abundance stable isotopic composition of N (δ15N) in plant leaves, roots, and soils. The root mycorrhizal community composition of understory trees did not significantly differ between beneath non-ECM and ECM overstory trees, although some OTUs more frequently appeared beneath non-ECM trees. Understory trees beneath non-ECM overstory trees had similar δ15N values in leaves and soil nitrate, suggesting that they took up most of their nitrogen as nitrate. Beneath ECM overstory trees, understory trees had consistently lower leaf than root δ15N, suggesting they depended on mycorrhizal fungi for N acquisition since mycorrhizal fungi transfer isotopically light N to host plants. Additionally, leaf N concentrations in the understory trees were lower beneath ECM than the non-ECM overstory trees. Our results show that, without large differences in root mycorrhizal community, the N uptake patterns of understory trees vary between beneath different overstory trees.

Consequences of microbial diversity in forest nitrogen cycling: diverse ammonifiers and specialized ammonia oxidizers.

We tested the ecosystem functions of microbial diversity with a focus on ammonification (involving diverse microbial taxa) and nitrification (involving only specialized microbial taxa) in forest nitrogen cycling. This study was conducted on a forest slope, in which the soil environment and plant growth gradually changed. We measured the gross and net rates of ammonification and nitrification, the abundance of predicted ammonifiers and nitrifiers, and their community compositions in the soils. The abundance of predicted ammonifiers did not change along the soil environmental gradient, leading to no significant change in the gross ammonification rate. On the other hand,  the abundance of nitrifiers and the gross nitrification rate gradually changed. These accordingly determined the spatial distribution of net accumulation of ammonium and nitrate available to plants. The community composition of predicted ammonifiers gradually changed along the slope, implying that diverse ammonifiers were more likely to include taxa that were acclimated to the soil environment and performed ammonification at different slope locations than specialized nitrifiers. Our findings suggest that the abundance of ammonifiers and nitrifiers directly affects the corresponding nitrogen transformation rates, and that their diversity affects the stability of the rates against environmental changes. This study highlights the role of microbial diversity in biogeochemical processes under changing environments and plant growth.

Soil nitrogen cycling is determined by the competition between mycorrhiza and ammonia-oxidizing prokaryotes.

Mycorrhizal fungi have considerable effects on soil carbon (C) storage, as they control the decomposition of soil organic matter (SOM), by modifying the amount of soil nitrogen (N) available for free-living microbes. Through their access to organic N, ectomycorrhizal (ECM) fungi compete with free-living soil microbes; this competition is thought to slow down SOM decomposition. However, arbuscular mycorrhizal (AM) fungi cannot decompose SOM, and therefore must wait for N to first be processed by free-living microbes. It is unclear what form of N the ECM fungi and free-living microbes compete for, or which microbial groups compete for N with ECM fungi. To investigate this, we focused on the N transformation steps (i.e., the degradation of high-molecular-weight organic matter, mineralization, and nitrification) and the microbes driving each step. Simple comparisons between AM forests and ECM forests are not sufficient to assert that mycorrhizal types would determine the N transformation steps in soil, because soil physiochemistry, which strongly affects N transformation steps, differs between the forests. We used an aridity gradient with large differences in soil moisture, pH, and SOM quantity and quality, to distinguish the mycorrhizal and physicochemical effects on N transformation. Soil samples (0-10 cm depth) were collected from AM-symbiotic black locust forests under three aridity levels, and from ECM-symbiotic oak forests under two aridity levels. Soil physicochemical properties, extractable N dynamics and abundance, composition, and function of soil microbial communities were measured. In ECM forests, the ammonia-oxidizing prokaryotic abundance was low, whereas that of ECM fungi was high, resulting in lower nitrate N content than in AM forests. Since ECM forests did not have lower saprotrophic fungal abundance and prokaryotic decompositional activity than the AM forests, the hypothesis that ECM fungi could reduce SOM decay and ammonification by free-living microbes, might not hold in ECM forests. However, the limitation of ECM fungi on nitrate N production would result in a feedback that will accelerate plant dependence on these fungi, thereby raising soil C storage through an increase in the ECM biomass and plant C investment in soils.

The Impacts of Soil Fertility and Salinity on Soil Nitrogen Dynamics Mediated by the Soil Microbial Community Beneath the Halophytic Shrub Tamarisk.

Nitrogen (N) is one of the most common limiting nutrients for primary production in terrestrial ecosystems. Soil microbes transform organic N into inorganic N, which is available to plants, but soil microbe activity in drylands is sometimes critically suppressed by environmental factors, such as low soil substrate availability or high salinity. Tamarisk (Tamarix spp.) is a halophytic shrub species that is widely distributed in the drylands of China; it produces litter enriched in nutrients and salts that are thought to increase soil fertility and salinity under its crown. To elucidate the effects of tamarisks on the soil microbial community, and thus N dynamics, by creating "islands of fertility" and "islands of salinity," we collected soil samples from under tamarisk crowns and adjacent barren areas at three habitats in the summer and fall. We analyzed soil physicochemical properties, inorganic N dynamics, and prokaryotic community abundance and composition. In soils sampled beneath tamarisks, the N mineralization rate was significantly higher, and the prokaryotic community structure was significantly different, from soils sampled in barren areas, irrespective of site and season. Tamarisks provided suitable nutrient conditions for one of the important decomposers in the area, Verrucomicrobia, by creating "islands of fertility," but provided unsuitable salinity conditions for other important decomposers, Flavobacteria, Gammaproteobacteria, and Deltaproteobacteria, by mitigating salt accumulation. However, the quantity of these decomposers tended to be higher beneath tamarisks, because they were relatively unaffected by the small salinity gradient created by the tamarisks, which may explain the higher N mineralization rate beneath tamarisks.

Changes in the anatomy, morphology and mycorrhizal infection of fine root systems of Cryptomeria japonica in relation to stand ageing.

Biomass allocation to fine roots often increases under soil nutrient deficiency, but the fine root biomass does not often increase in old stands, even under nutrient limitation. Therefore, in old stands, the morphology, anatomy, branching architecture and mycorrhization of fine roots may compensate efficiently for nutrient acquisition by the low fine root biomass. In this study, changes in the morphology, anatomy and arbuscular mycorrhizal infection at each branching position of fine root clusters were evaluated in relation to stand age. A chronosequence (6­90 years of age) of stands in a Cryptomeria japonica D. Don plantation was used for these analyses. The fine root size parameters, such as length, weight and tip numbers of fine root clusters, increased with stand age. The specific root tip length (SRTL) decreased with increasing stand age, suggesting that the allocation to root active portions decreased with stand age. From the anatomical observation, the ephemeral root tips increased with stand age, suggesting that root tip turnover within a root cluster was high in old stands. The proportions of proto-xylem groups among branching positions indicated that the life cycles in branching hierarchy should be clearer in old stands than that in younger stands. The increasing in the mycorrhizal infection of root tips in old stands should enhance the root tip absorptive functions. The SRTL was correlated with the wood/needle ratio, suggesting that carbon limitation as the stand ages may result in decline of carbon allocation to maintain active root tips. However, increasing of the ephemeral tips and mycorrhizal infection rates may compensate the declines of tip allocation in old stands.

Development of Soil Bacterial Communities in Volcanic Ash Microcosms in a Range of Climates.

There is considerable interest in understanding the processes of microbial development in volcanic ash. We tested the predictions that there would be (1) a distinctive bacterial community associated with soil development on volcanic ash, including groups previously implicated in weathering studies; (2) a slower increase in bacterial abundance and soil C and N accumulation in cooler climates; and (3) a distinct communities developing on the same substrate in different climates. We set up an experiment, taking freshly fallen, sterilized volcanic ash from Sakurajima volcano, Japan. Pots of ash were positioned in multiple locations, with mean annual temperature (MAT) ranging from 18.6 to -3 °C. Within 12 months, bacteria were detectable by qPCR in all pots. By 24 months, bacterial copy numbers had increased by 10-100 times relative to a year before. C and N content approximately doubled between 12 and 24 months. HiSeq and MiSeq sequencing of the 16S rRNA gene revealed a distinctive bacterial community, different from developed vegetated soils in the same areas, for example in containing an abundance of unclassified bacterial groups. Community composition also differed between the ash pots at different sites, while showing no pattern in relation to MAT. Contrary to our predictions, the bacterial abundance did not show any relation to MAT. It also did not correlate to pH or N, and only C was statistically significant. It appears that bacterial community development on volcanic ash can be a rapid process not closely sensitive to temperature, involving distinct communities from developed soils.

Nitrogen uptake and nitrogen use efficiency above and below ground along a topographic gradient of soil nitrogen availability.

Nitrogen (N) uptake and nitrogen use efficiency (NUE) are closely related through feedback mechanisms to soil N availability and N cycling in forested ecosystems. We investigated N uptake and NUE not only at the leaf, litterfall, and aboveground levels but also belowground and whole stand levels along a topographic gradient of soil N availability in a cool temperate deciduous forest in Japan. In this study, we addressed how whole stand level N uptake and NUE affect C and N cycling in forested ecosystems. At the leaf, litterfall, and aboveground levels, N uptake decreased and NUE increased with decreasing soil N availability. This pattern resulted from decreasing leaf N concentrations and increasing N resorption efficiencies as soil N availability declined. Low N concentrations in litterfall may have resulted in little soil N being available to plants, due to microbial immobilization. In contrast, when belowground components were included, N uptake and NUE were not correlated with soil N availability. This was mainly due to higher levels of fine root production when soil N availability was low. Higher fine root allocation can result in a high input of detritus to decomposer systems and, thus, contribute to accumulation of soil organic matter and immobilization by microbes, which may result in further soil N availability decline. Our results suggest that allocation to the fine root rather than whole stand level NUE is important for C and N cycling in forested ecosystems, as is the feedback mechanism in which litterfall level NUE shifts with changes in the N concentration of litterfall.

Pollination efficiencies of flower-visiting insects as determined by direct genetic analysis of pollen origin.

The amount and genetic composition of pollen grains that are transported to flowers influence the reproduction and fitness of plants. Despite the importance of insect-pollination systems, an understanding of those systems is still lacking due to the absence of a genetic analysis of pollen grains that are transported to flowers. We evaluated the pollination efficiencies of bumblebees (Apidae, Bombus spp.), flower beetles (Scarabaeidae, subfamily Cetoniinae, Protaetia and Eucetonia sp.), and small beetles (Lagriidae, Arthromacra sp.) that visited the flowers of Magnolia obovata (Magnoliaceae) using quantitative (flower visitation frequency, amount of adherent pollen per insect) and qualitative (origin and genetic diversity of adherent pollen per insect) criteria. Most of the pollen adhering to bumblebees and small beetles was self-pollen. This result suggests that visitation by these insects may cause geitonogamous pollen flow and negatively affect the reproduction of M. obovata, causing inbreeding depression. In contrast, flower beetles transported large amounts of genetically diverse outcross pollen. Our results suggest that certain beetle species contribute quantitatively and qualitatively to the pollination of M. obovata. Direct genetic analysis of pollen grains will advance our understanding of plant mating systems and may shed light on the mutualism and coevolution of plants and flower visitors.

Changes in crown development patterns and current-year shoot structure with light environment and tree height in Fagus crenata (Fagaceae).

The relative effects of light and tree height on the architecture of leader crowns (i.e., the leading section of the main trunk, 100 cm in length) and current-year shoots for a canopy species, Fagus crenata, occupying both the ridge top and the valley bottom in a cool-temperate forest in Japan were investigated. For leader crowns, the number of current-year shoots and leaves increased with increasing tree height, whereas the mean length of current-year shoots increased with increasing relative photon flux density (PFD). The leader crown area decreased, and the depth and leaf area index of leader crowns increased, with increasing relative PFD. The mass of current-year shoots increased with relative PFD. However, this total mass was allocated differently between stems and leaves depending on tree height, such that the relative allocation to stems increased with increasing tree height. Furthermore, stem structures within current-year shoots also changed with height, such that taller trees produced thicker and shorter stems of the same volume. In contrast, leaf structure and leaf biomass allocations changed with relative PFD. Specific leaf area decreased with increasing relative PFD. In addition, leaf number increased more rapidly with increasing individual leaf mass for trees exposed to greater relative PFD. Consequently, the total leaf area supported by a stem of a given diameter decreased with increasing tree height and relative PFD. Thus, the architecture of leader crowns and current-year shoots were related differently to light and tree height, which are considered important for efficient light capture and the growth of small and tall trees in different environments.