Habitat is more important than climate for structuring soil fungal communities associated in truffle sites.

The ectomycorrhizal fungi Tuber melanosporum Vittad. and Tuber aestivum Vittad. produce highly valuable truffles, but little is known about the soil fungal communities associated with these truffle species in places where they co-occur. Here, we compared soil fungal communities present in wild and planted truffle sites, in which T. melanosporum and T. aestivum coexist, in Mediterranean and temperate regions over three sampling seasons spanning from 2018 to 2019. We showed that soil fungal community composition and ectomycorrhizal species composition are driven by habitat type rather than climate regions. Also, we observed the influence of soil pH, organic matter content and C:N ratio structuring total and ectomycorrhizal fungal assemblages. Soil fungal communities in wild sites revealed more compositional variability than those of plantations. Greater soil fungal diversity was found in temperate compared to Mediterranean sites when considering all fungal guilds. Ectomycorrhizal diversity was significantly higher in wild sites compared to plantations. Greater mould abundance at wild sites than those on plantation was observed while tree species and seasonal effects were not significant predictors in fungal community structure. Our results suggested a strong influence of both ecosystem age and management on the fungal taxa composition in truffle habitats.

Silvicultural management and altitude prevail on soil properties and fungal community in shaping understorey plant communities in a Mediterranean pine forest.

Understorey vegetation plays a key role in Mediterranean forest ecosystem functioning. However, we still lack a thorough understanding of the patterns and drivers of understorey composition and diversity. As a result, understoreys are often ignored during assessments of forest functioning under climate change. Here we studied the effect of silvicultural management, topography, soil fungal community composition and soil physical and chemical properties on understorey community composition and diversity. The plant cover and number of individuals of understorey perennial plants, shrubs and non-dominant trees was recorded on 24 plots (paired: control-thinned) in a Mediterranean pine-dominated mountainous area in Northeast Spain. The study area represented a broad thinning intensity gradient (from 0 to 70 % in removed stand basal area) along a 400-m altitudinal range (from 609 m to 1013 m). Our results showed that thinning intensity and topography explained the greatest proportion of the total variance in the understorey species composition, i.e., 18 % and 16 %, respectively. Interestingly, the effects of the silvicultural treatments were significant only when considering the altitudinal effect, so that, the main impacts of thinning on the understorey community composition occurred at low altitudes (between 609 m and 870 m). Moreover, we found a significant decrease in both richness and abundance of understorey species in both the control and thinned plots with increasing altitude, with thinned plots being significantly richer in species compared to the control plots. The difference in the understorey community sensitivity to forest thinning along the altitudinal gradient suggests changes in factors that limit plant growth. Low elevation plots were restrained by light availability while high altitudes plots limited by winter freezing temperature.

Lack of Phylogenetic Differences in Ectomycorrhizal Fungi among Distinct Mediterranean Pine Forest Habitats.

Understanding whether the occurrences of ectomycorrhizal species in a given tree host are phylogenetically determined can help in assessing different conservational needs for each fungal species. In this study, we characterized ectomycorrhizal phylogenetic composition and phylogenetic structure in 42 plots with five different Mediterranean pine forests: i.e., pure forests dominated by P. nigra, P. halepensis, and P. sylvestris, and mixed forests of P. nigra-P. halepensis and P. nigra-P. sylvestris, and tested whether the phylogenetic structure of ectomycorrhizal communities differs among these. We found that ectomycorrhizal communities were not different among pine tree hosts neither in phylogenetic composition nor in structure and phylogenetic diversity. Moreover, we detected a weak abiotic filtering effect (4%), with pH being the only significant variable influencing the phylogenetic ectomycorrhizal community, while the phylogenetic structure was slightly influenced by the shared effect of stand structure, soil, and geographic distance. However, the phylogenetic community similarity increased at lower pH values, supporting that fewer, closely related species were found at lower pH values. Also, no phylogenetic signal was detected among exploration types, although short and contact were the most abundant types in these forest ecosystems. Our results demonstrate that pH but not tree host, acts as a strong abiotic filter on ectomycorrhizal phylogenetic communities in Mediterranean pine forests at a local scale. Finally, our study shed light on dominant ectomycorrhizal foraging strategies in drought-prone ecosystems such as Mediterranean forests.

Sampling forest soils to describe fungal diversity and composition. Which is the optimal sampling size in mediterranean pure and mixed pine oak forests?

Soil sampling is a critical step affecting perceived fungal diversity, however sampling optimization for high-throughput-DNA sequencing studies have never been tested in Mediterranean forest ecosystems. We identified the minimum number of pooled samples needed to obtain a reliable description of fungal communities in terms of diversity and composition in three different Mediterranean forests (pine, oak, and mixed-pine-oak). Twenty soil samples were randomly selected in each of the three plots per type. Samples obtained in 100 m2 plots were pooled to obtain mixtures of 3, 6, 10, 15, 20 samples, and sequenced using Illumina MiSeq of fungal ITS2 amplicons. Pooling three soil samples in Pinus and Quercus stands provided consistent richness estimations, while at least six samples were needed in mixed-stands. ß-diversity decreased with increasing sample pools in monospecific-stands, while there was no effect of sample pool size on mixed-stands. Soil sample pooling had no effect over species composition. We estimate that three samples would be already optimal to describe fungal richness and composition in Mediterranean pure stands, while at least six samples would be needed in mixed stands.

The relationships of present vegetation, bacteria, and soil properties with soil organic matter characteristics in moist acidic tundra in Alaska.

Soil organic matter (SOM) is related to vegetation, soil bacteria, and soil properties; however, not many studies link all these parameters simultaneously, particularly in tundra ecosystems vulnerable to climate change. Our aim was to describe the relationships between vegetation, bacteria, soil properties, and SOM composition in moist acidic tundra by integrating physical, chemical, and molecular methods. A total of 70 soil samples were collected at two different depths from 36 spots systematically arranged over an area of about 300 m × 50 m. Pyrolysis-gas chromatography/mass spectrometry and pyrosequencing of the 16S rRNA gene were used to identify the molecular compositions of the SOM and bacterial community, respectively. Vegetation and soil physicochemical properties were also measured. The sampling sites were grouped into three, based on their SOM compositions: Sphagnum moss-derived SOM, lipid-rich materials, and aromatic-rich materials. Our results show that SOM composition is spatially structured and linked to microtopography; however, the vegetation, soil properties, and bacterial community composition did not show overall spatial structuring. Simultaneously, soil properties and bacterial community composition were the main factors explaining SOM compositional variation, while vegetation had a residual effect. Verrucomicrobia and Acidobacteria were related to polysaccharides, and Chloroflexi was linked to aromatic compounds. These relationships were consistent across different hierarchical levels. Our results suggest that SOM composition at a local scale is closely linked with soil factors and the bacterial community. Comprehensive observation of ecosystem components is recommended to understand the in-situ function of bacteria and the fate of SOM in the moist acidic tundra.

White mulch and irrigation increase black truffle soil mycelium when competing with summer truffle in young truffle orchards.

The black truffle (Tuber melanosporum Vittad.) and the summer truffle (Tuber aestivum Vittad.) are two of the most appreciated edible fungi worldwide. The natural distributions of both species partially overlap. However, the interspecific interactions between these truffles and how irrigation and mulching techniques impact the dynamics between them are still unknown. Here, an experimental truffle plantation with Quercus ilex was established in Maials (Catalonia, Spain), combining three soil mulch treatments (white mulch, black mulch and bare soil as a control) and two irrigation regimes (irrigated and non-irrigated as a control) to investigate truffle mycelial dynamics in soil when both truffle species co-occur. The development of truffle mycelium in two different seasons (spring and autumn) in two consecutive years (2017 and 2018) was quantified using qPCR. Truffle mycelia of both species showed greatest development under white mulch. When mycelia of both truffle species co-occurred in soil, irrigation combined with white mulch resulted in greater quantities of T. melanosporum mycelial biomass, whereas the control irrigation treatment favoured the development of T. aestivum. Mulch treatments were also advantageous for seedling growth, which was expressed as root collar diameter and its increment during the study period. Significant relationships between root collar diameter and root growth and the amount of mycelial biomass in the soil were observed for both truffle species. Our results indicate the potential advantages of using white mulch to support irrigation in truffle plantations located in areas with dry Mediterranean climatic conditions to promote the development of Tuber mycelium.

Effects of long-term removal of sheep-grazing in a series of British upland plant communities: Insights from plant species composition and traits.

Environmental and management pressures are considered a threat for preserving plant communities worldwide. Identification of long-term impacts of changing management practices on plant community composition must, therefore, be a major priority to ensure improvement in conservation value. Land abandonment/wilding is one topical area where there is little available information on long-term impacts. To address this, here, we describe plant species compositional change over relatively long-time scales in a range of four British upland communities (high-level grasslands, intermediate grasslands, blanket bogs and high-level bogs) and its relationships with a series of life-history and plant trait variables. Our aims were to determine whether the business-as-usual sheep grazing practice was maintaining these communities, and if grazing was to be removed as part of abandonment/wilding strategies, would there be a conservation benefit. We used a series of long-term experimental grazing-exclosure studies at twelve sites (>40 years) on the Moor House National Nature Reserve in northern England. Each site consisted of paired plots; i.e. sheep-grazed versus ungrazed. Our results showed that there was relatively little change in dominant plant species with most change occurring within sub-dominant species. Similarly, different temporal responses in plant species richness were detected between the experiments (richness was reduced in three experiments, only one increased). In any case, the vegetation temporal trajectories were moving in similar direction in grazed and ungrazed plots at most sites. Interestingly, blanket bog experiments showed a clear compositional convergence for both grazed and ungrazed plots, although, the between-site differences related to a combination of elevation and past burning testament. Finally, the bryophytes (especially liverworts) and lichens were the groups that contributed most to the reductions in species richness. Moreover, implementation of a no-stock grazing strategy under a land abandonment/wilding approach will not bring about much plant change in the short-term in the plant communities studied here.

Production and turnover of mycorrhizal soil mycelium relate to variation in drought conditions in Mediterranean Pinus pinaster, Pinus sylvestris and Quercus ilex forests.

In forests, ectomycorrhizal mycelium is pivotal for driving soil carbon and nutrient cycles, but how ectomycorrhizal mycelial dynamics vary in ecosystems with drought periods is unknown. We quantified the production and turnover of mycorrhizal mycelium in Mediterranean Pinus pinaster, Pinus sylvestris and Quercus ilex forests and related the estimates to standardised precipitation index (SPI), to study how mycelial dynamics relates to tree species and drought-moisture conditions. Production and turnover of mycelium was estimated between July and February, by quantifying the fungal biomass (ergosterol) in ingrowth mesh bags and using statistical modelling. SPI for time scales of 1-3 months was calculated from precipitation records and precipitation data over the study period. Forests dominated by Pinus trees displayed higher biomass but were seasonally more variable, as opposed to Q. ilex forests where the mycelial biomass remained lower and stable over the season. Production and turnover, respectively, varied between 1.4-5.9 kg ha-1  d-1 and 7.2-9.9 times yr-1 over the different forest types and were positively correlated with 2-month and 3-month SPI over the study period. Our results demonstrated that mycorrhizal mycelial biomass varied with season and tree species and we speculate that production and turnover are related to physiology and plant host performance during drought.

Insect defoliation is linked to a decrease in soil ectomycorrhizal biomass and shifts in needle endophytic communities.

Insect outbreaks of increasing frequency and severity in forests are predicted due to climate change. Insect herbivory is known to promote physiological changes in forest trees. However, little is known about whether these plant phenotypic adjustments have cascading effects on tree microbial symbionts such as fungi in roots and foliage. We studied the impact of defoliation by the pine processionary moth in two infested Pinus nigra forests through a multilevel sampling of defoliated and non-defoliated trees. We measured tree growth, nutritional status and carbon allocation to chemical defenses. Simultaneously, we analysed the putative impact of defoliation on the needle endophytes and on the soil fungal communities. Higher concentrations of chemical defenses were found in defoliated trees, likely as a response to defoliation; however, no differences in non-structural carbohydrate reserves were found. In parallel to the reductions in tree growth and changes in chemical defenses, we observed shifts in the composition of needle endophytic and soil fungal communities in defoliated trees. Defoliated trees consistently corresponded with a lower biomass of ectomycorrhizal fungi in both sites, and a higher alpha diversity and greater relative abundance of belowground saprotrophs and pathogens. However, ectomycorrhizal alpha diversity was similar between non-defoliated and defoliated trees. Specific needle endophytes in old needles were strongly associated with non-defoliated trees. The potential role of these endophytic fungi in pine resistance should be further investigated. Our study suggests that lower biomass of ectomycorrhizal fungi in defoliated trees might slow down tree recovery since fungal shifts might affect tree-mycorrhizal feedbacks and can potentially influence carbon and nitrogen cycling in forest soils.

Similar diurnal, seasonal and annual rhythms in radial root expansion across two coexisting Mediterranean oak species.

Dendrometers are being increasingly used to measure stem radius changes in trees and to unravel the mechanisms underlying stem daily rhythms of radial expansion and contraction. Nevertheless, automated dendrometers have not been often used to measure root radius dynamics, their relationship with environmental variables and the influence of endogenous processes, especially in drought-prone Mediterranean areas. Here, we measured root radius dynamics of two coexisting oak species (the evergreen Quercus ilex L. and the deciduous Quercus faginea Lam). Our goals were to describe annual, seasonal and diurnal scale root radius patterns and to disentangle the role of different environmental parameters as drivers. Long-term high-resolution measurements (every 15 min over 7 years) were collected with automated point dendrometers on the main tree roots of five individuals per species. Root radius annual change patterns were bimodal and similar for both oak species. Quercus faginea Lam showed three times larger root increment in the spring than Q. ilex, but the bimodal pattern was stronger in Q. ilex, which showed a larger root increment in autumn. Quercus faginea Lam showed an earlier root phenological activation in the spring and in late summer compared with Q. ilex. The effects of environmental drivers across species were similar at daily scales: root radius increased with air temperature and soil moisture, and it decreased with rising vapor pressure deficit. Furthermore, daily root radius variations for both oak species were maintained after extracting statistically the environmental effects, which points toward a significant role of endogenous drivers. These differences in root radius change patterns at seasonal to daily scales likely result from the differences in leaf phenology and growth strategy. Quercus faginea Lam is deciduous and has a faster growing rate in spring than the evergreen Q. ilex, which can grow more in summer.

Plant species colonization in newly created road habitats of South Korea: Insights for more effective restoration.

Despite the advances in restoration methods for newly created road habitats such as roadcuts and embankments, implementation in different parts of the world is limited by high cost and lack of knowledge of naturally formed plant communities. However, a cheaper alternative is to relay in natural successional process in sites under optimal conditions. Thus, the first steps should focus on identifying plant species that colonize roadways and road habitats as well as optimal colonization sites. Our study aimed to describe species composition, exotic species presence, and diversity among four roadways (Jeongok-Youngjung, JG; Seolma-Gueup, SM; Singal, SG; and Samga-Daechon, DC) and three habitat types (embankments, plain areas, and roadcuts) in South Korean peninsula. The effect of some environmental factors on plant composition was also examined (soil type, soil slope, and surrounding landscape). Our results showed that established plant species composition was influenced by the interaction between roadways and habitats types, which was also the main interaction affecting plant richness and evenness. Surprisingly, environmental variables had no effect on plant species composition, with a residual amount of explained variation. A total of 48 plant species were described as indicator of different roadways and habitat types, and 50% of them were invasive or cultivated species. It appeared that different regional-dependent processes, such as northern vs. southern roadways, interact with local process in new-road habitats, producing complex patterns of plant species colonization and composition. Thus, ecological restoration solutions should be targeted at site-specific needs (local) while taking into consideration the differences between northern and southern roadways (regional). Here, regional-pool and local-constraints interaction controls plant composition and diversity during road construction in South Korea. Finally, new restoration actions should be based on plant species that have been established spontaneously in these degraded areas.

Marine protected areas are more effective but less reliable in protecting fish biomass than fish diversity.

Marine protected areas (MPAs) provide multiple conservation benefits, thus raising the question of how good and consistent they are at their roles. Here, we quantified three components, namely, diversity, biomass, and other relevant variables, in numerous protected and unprotected areas across four marine ecoregions in south-western Europe. We created a "global conservation status index" (CSIglobal) as the sum of CSIdiversity, CSIbiomass, and CSIrelevant. We then tested whether CSI and its three components varied as a function of protection and marine ecoregion. MPA efficiency, defined as the effect size of protection on CSIglobal, was unreliable and varied with geography. CSIbiomass and CSIrelevant contributed to the unreliability of MPA efficiency, while CSIdiversity was reliable. CSIbiomass showed the major efficiency in protected areas (60%). Biomass of threatened species was the single largest variable that contributed to MPA efficiency. Our easy-to-use approach can identify high- and low-efficient MPAs and help to clarify their actual roles.

Soil microclimate changes affect soil fungal communities in a Mediterranean pine forest.

Soil microclimate is a potentially important regulator of the composition of plant-associated fungal communities in climates with significant drought periods. Here, we investigated the spatio-temporal dynamics of soil fungal communities in a Mediterranean Pinus pinaster forest in relation to soil moisture and temperature. Fungal communities in 336 soil samples collected monthly over 1 year from 28 long-term experimental plots were assessed by PacBio sequencing of ITS2 amplicons. Total fungal biomass was estimated by analysing ergosterol. Community changes were analysed in the context of functional traits. Soil fungal biomass was lowest during summer and late winter and highest during autumn, concurrent with a greater relative abundance of mycorrhizal species. Intra-annual spatio-temporal changes in community composition correlated significantly with soil moisture and temperature. Mycorrhizal fungi were less affected by summer drought than free-living fungi. In particular, mycorrhizal species of the short-distance exploration type increased in relative abundance under dry conditions, whereas species of the long-distance exploration type were more abundant under wetter conditions. Our observations demonstrate a potential for compositional and functional shifts in fungal communities in response to changing climatic conditions. Free-living fungi and mycorrhizal species with extensive mycelia may be negatively affected by increasing drought periods in Mediterranean forest ecosystems.

Mushroom Emergence Detected by Combining Spore Trapping with Molecular Techniques.

Obtaining reliable and representative mushroom production data requires time-consuming sampling schemes. In this paper, we assessed a simple methodology to detect mushroom emergence by trapping the fungal spores of the fruiting body community in plots where mushroom production was determined weekly. We compared the performance of filter paper traps with that of funnel traps and combined these spore trapping methods with species-specific quantitative real-time PCR and Illumina MiSeq to determine the spore abundance. Significantly more MiSeq proportional reads were generated for both ectomycorrhizal and saprotrophic fungal species using filter traps than were obtained using funnel traps. The spores of 37 fungal species that produced fruiting bodies in the study plots were identified. Spore community composition changed considerably over time due to the emergence of ephemeral fruiting bodies and rapid spore deposition (lasting from 1 to 2 weeks), which occurred in the absence of rainfall events. For many species, the emergence of epigeous fruiting bodies was followed by a peak in the relative abundance of their airborne spores. There were significant positive relationships between fruiting body yields and spore abundance in time for five of seven fungal species. There was no relationship between fruiting body yields and their spore abundance at plot level, indicating that some of the spores captured in each plot were arriving from the surrounding areas. Differences in fungal detection capacity by spore trapping may indicate different dispersal ability between fungal species. Further research can help to identify the spore rain patterns for most common fungal species.IMPORTANCE Mushroom monitoring represents a serious challenge in economic and logistical terms because sampling approaches demand extensive field work at both the spatial and temporal scales. In addition, the identification of fungal taxa depends on the expertise of experienced fungal taxonomists. Similarly, the study of fungal dispersal has been constrained by technological limitations, especially because the morphological identification of spores is a challenging and time-consuming task. Here, we demonstrate that spores from ectomycorrhizal and saprotrophic fungal species can be identified using simple spore traps together with either MiSeq fungus-specific amplicon sequencing or species-specific quantitative real-time PCR. In addition, the proposed methodology can be used to characterize the airborne fungal community and to detect mushroom emergence in forest ecosystems.

Mushroom biomass and diversity are driven by different spatio-temporal scales along Mediterranean elevation gradients.

Mushrooms are important non-wood-forest-products in many Mediterranean ecosystems, being highly vulnerable to climate change. However, the ecological scales of variation of mushroom productivity and diversity, and climate dependence has been usually overlooked due to a lack of available data. We determined the spatio-temporal variability of epigeous sporocarps and the climatic factors driving their fruiting to plan future sustainable management of wild mushrooms production. We collected fruiting bodies in Pinus sylvestris stands along an elevation gradient for 8 consecutive years. Overall, sporocarp biomass was mainly dependent on inter-annual variations, whereas richness was more spatial-scale dependent. Elevation was not significant, but there were clear elevational differences in biomass and richness patterns between ectomycorrhizal and saprotrophic guilds. The main driver of variation was late-summer-early-autumn precipitation. Thus, different scale processes (inter-annual vs. spatial-scale) drive sporocarp biomass and diversity patterns; temporal effects for biomass and ectomycorrhizal fungi vs. spatial scale for diversity and saprotrophic fungi. The significant role of precipitation across fungal guilds and spatio-temporal scales indicates that it is a limiting resource controlling sporocarp production and diversity in Mediterranean regions. The high spatial and temporal variability of mushrooms emphasize the need for long-term datasets of multiple spatial points to effectively characterize fungal fruiting patterns.

Endogenous circadian rhythms in pigment composition induce changes in photochemical efficiency in plant canopies.

There is increasing evidence that the circadian clock is a significant driver of photosynthesis that becomes apparent when environmental cues are experimentally held constant. We studied whether the composition of photosynthetic pigments is under circadian regulation, and whether pigment oscillations lead to rhythmic changes in photochemical efficiency. To address these questions, we maintained canopies of bean and cotton, after an entrainment phase, under constant (light or darkness) conditions for 30-48 h. Photosynthesis and quantum yield peaked at subjective noon, and non-photochemical quenching peaked at night. These oscillations were not associated with parallel changes in carbohydrate content or xanthophyll cycle activity. We observed robust oscillations of Chl a/b during constant light in both species, and also under constant darkness in bean, peaking when it would have been night during the entrainment (subjective nights). These oscillations could be attributed to the synthesis and/or degradation of trimeric light-harvesting complex II (reflected by the rhythmic changes in Chl a/b), with the antenna size minimal at night and maximal around subjective noon. Considering together the oscillations of pigments and photochemistry, the observed pattern of changes is counterintuitive if we assume that the plant strategy is to avoid photodamage, but consistent with a strategy where non-stressed plants maximize photosynthesis.

Modelling Carbon Emissions in Calluna vulgaris-Dominated Ecosystems when Prescribed Burning and Wildfires Interact.

A present challenge in fire ecology is to optimize management techniques so that ecological services are maximized and C emissions minimized. Here, we modeled the effects of different prescribed-burning rotation intervals and wildfires on carbon emissions (present and future) in British moorlands. Biomass-accumulation curves from four Calluna-dominated ecosystems along a north-south gradient in Great Britain were calculated and used within a matrix-model based on Markov Chains to calculate above-ground biomass-loads and annual C emissions under different prescribed-burning rotation intervals. Additionally, we assessed the interaction of these parameters with a decreasing wildfire return intervals. We observed that litter accumulation patterns varied between sites. Northern sites (colder and wetter) accumulated lower amounts of litter with time than southern sites (hotter and drier). The accumulation patterns of the living vegetation dominated by Calluna were determined by site-specific conditions. The optimal prescribed-burning rotation interval for minimizing annual carbon emissions also differed between sites: the optimal rotation interval for northern sites was between 30 and 50 years, whereas for southern sites a hump-backed relationship was found with the optimal interval either between 8 to 10 years or between 30 to 50 years. Increasing wildfire frequency interacted with prescribed-burning rotation intervals by both increasing C emissions and modifying the optimum prescribed-burning interval for minimum C emission. This highlights the importance of studying site-specific biomass accumulation patterns with respect to environmental conditions for identifying suitable fire-rotation intervals to minimize C emissions.

Circadian rhythms have significant effects on leaf-to-canopy scale gas exchange under field conditions.

BACKGROUND: Molecular clocks drive oscillations in leaf photosynthesis, stomatal conductance, and other cell and leaf-level processes over ~24 h under controlled laboratory conditions. The influence of such circadian regulation over whole-canopy fluxes remains uncertain; diurnal CO2 and H2O vapor flux dynamics in the field are currently interpreted as resulting almost exclusively from direct physiological responses to variations in light, temperature and other environmental factors. We tested whether circadian regulation would affect plant and canopy gas exchange at the Montpellier European Ecotron. Canopy and leaf-level fluxes were constantly monitored under field-like environmental conditions, and under constant environmental conditions (no variation in temperature, radiation, or other environmental cues). RESULTS: We show direct experimental evidence at canopy scales of the circadian regulation of daytime gas exchange: 20-79 % of the daily variation range in CO2 and H2O fluxes occurred under circadian entrainment in canopies of an annual herb (bean) and of a perennial shrub (cotton). We also observed that considering circadian regulation improved performance by 8-17 % in commonly used stomatal conductance models. CONCLUSIONS: Our results show that circadian controls affect diurnal CO2 and H2O flux patterns in entire canopies in field-like conditions, and its consideration significantly improves model performance. Circadian controls act as a 'memory' of the past conditions experienced by the plant, which synchronizes metabolism across entire plant canopies.

Soil drying procedure affects the DNA quantification of Lactarius vinosus but does not change the fungal community composition.

Drying soil samples before DNA extraction is commonly used for specific fungal DNA quantification and metabarcoding studies, but the impact of different drying procedures on both the specific fungal DNA quantity and the fungal community composition has not been analyzed. We tested three different drying procedures (freeze-drying, oven-drying, and room temperature) on 12 different soil samples to determine (a) the soil mycelium biomass of the ectomycorrhizal species Lactarius vinosus using qPCR with a specifically designed TaqMan® probe and (b) the fungal community composition and diversity using the PacBio® RS II sequencing platform. Mycelium biomass of L. vinosus was significantly greater in the freeze-dried soil samples than in samples dried at oven and room temperature. However, drying procedures had no effect on fungal community composition or on fungal diversity. In addition, there were no significant differences in the proportions of fungi according to their functional roles (moulds vs. mycorrhizal species) in response to drying procedures. Only six out of 1139 operational taxonomic units (OTUs) had increased their relative proportions after soil drying at room temperature, with five of these OTUs classified as mould or yeast species. However, the magnitude of these changes was small, with an overall increase in relative abundance of these OTUs of approximately 2 %. These results suggest that DNA degradation may occur especially after drying soil samples at room temperature, but affecting equally nearly all fungi and therefore causing no significant differences in diversity and community composition. Despite the minimal effects caused by the drying procedures at the fungal community composition, freeze-drying resulted in higher concentrations of L. vinosus DNA and prevented potential colonization from opportunistic species.

Natural forest expansion on reclaimed coal mines in Northern Spain: the role of native shrubs as suitable microsites.

The characterization of suitable microsites for tree seedling establishment and growth is one of the most important tasks to achieve the restoration of native forest using natural processes in disturbed sites. For that, we assessed the natural Quercus petraea forest expansion in a 20-year-old reclaimed open-cast mine under sub-Mediterranean climate in northern Spain, monitoring seedling survival, growth, and recruitment during 5 years in three contrasting environments (undisturbed forest, mine edge, and mine center). Seedling density and proportion of dead branches decreased greatly from undisturbed forest towards the center of the mine. There was a positive effect of shrubs on Q. petraea seedling establishment in both mine environments, which increase as the environment undergoes more stress (from the mine edge to the center of the mine), and it was produced by different shrub structural features in each mine environment. Seedling survival reduction through time in three environments did not lead to a density reduction because there was a yearly recruitment of new seedlings. Seedling survival, annual growth, and height through time were greater in mine sites than in the undisturbed forest. The successful colonization patterns and positive neighbor effect of shrubs on natural seedlings establishment found in this study during the first years support the use of shrubs as ecosystem engineers to increase heterogeneity in micro-environmental conditions on reclaimed mine sites, which improves late-successional Quercus species establishment.