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.

Climate change induced declines in fuel moisture may turn currently fire-free Pyrenean mountain forests into fire-prone ecosystems.

Fuel moisture limits the availability of fuel to wildfires in many forest areas worldwide, but the effects of climate change on moisture constraints remain largely unknown. Here we addressed how climate affects fuel moisture in pine stands from Catalonia, NE Spain, and the potential effects of increasing climate aridity on burned area in the Pyrenees, a mesic mountainous area where fire is currently rare. We first quantified variation in fuel moisture in six sites distributed across an altitudinal gradient where the long-term mean annual temperature and precipitation vary by 6-15 °C and 395-933 mm, respectively. We observed significant spatial variation in live (78-162%) and dead (10-15%) fuel moisture across sites. The pattern of variation was negatively linked (r = |0.6|-|0.9|) to increases in vapor pressure deficit (VPD) and in the Aridity Index. Using seasonal fire records over 2006-2020, we observed that summer burned area in the Mediterranean forests of Northeast Spain and Southern France was strongly dependent on VPD (r = 0.93), the major driver (and predictor) of dead fuel moisture content (DFMC) at our sites. Based on the difference between VPD thresholds associated with large wildfire seasons in the Mediterranean (3.6 kPa) and the maximum VPD observed in surrounding Pyrenean mountains (3.1 kPa), we quantified the "safety margin" for Pyrenean forests (difference between actual VPD and that associated with large wildfires) at 0.5 kPa. The effects of live fuel moisture content (LFMC) on burned area were not significant under current conditions, a situation that may change with projected increases in climate aridity. Overall, our results indicate that DFMC in currently fire-free areas in Europe, like the Pyrenees, with vast amounts of fuel in many forest stands, may reach critical dryness thresholds beyond the safety margin and experience large wildfires after only mild increases in VPD, although LFMC can modulate the response.

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.

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.

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.

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.

Increase in membrane thickness during development compensates for eggshell thinning due to calcium uptake by the embryo in falcons.

We compared membrane thickness of fully developed eggs with those of non-developed eggs in different endangered falcon taxa. To our knowledge, membrane thickness variation during development has never been examined before in falcons or any other wild bird. Yet, the egg membrane constitutes an important protective barrier for the developing embryo. Because eggshell thinning is a general process that occurs during bird development, caused by calcium uptake by the embryo, eggs are expected to be less protected and vulnerable to breakage near the end of development. Thus, egg membranes could play an important protective role in the later stages of development by getting relatively thicker. We used linear mixed models to explore the variation in membrane thickness (n = 378 eggs) in relation to developmental stage, taxon, female age, mass and identity (73 females), egg-laying sequence (105 clutches) and the study zone. Our results are consistent with the prediction that egg membranes are thicker in fully developed eggs than in non-developed eggs, suggesting that the increase in membrane thickness during development may compensate for eggshell thinning. In addition, our data shown that thicker membranes are associated with larger, heavier and relatively wider eggs, as well as with eggs that had thinner eggshells. Egg-laying sequence, female age and the study zone did not explain the observed variation of membrane thickness in the falcon taxa studied. As we provide quantitative data on membrane thickness variation during development in falcons not subjected to contamination or food limitation (i.e. bred under captive conditions), our data may be used as a reference for studies on eggs from natural populations. Considering the large variation in membrane thickness and the multiple factors affecting on it and its importance in the protection of the embryo, we encourage other researchers to include measurements on membranes in studies exploring eggshell thickness variation.