Soil solution chemistry weak response to long-term N addition points towards a strong resilience of northeastern American forests to past and future N deposition.

Northern temperate and boreal forests are large biomes playing crucial ecological and environmental roles, such as carbon sequestration. Despite being generally remote, these forests were exposed to anthropogenic nitrogen (N) deposition over the last two centuries and may still experience elevated N deposition as human activities expand towards high latitudes. However, the impacts of long-term high N deposition on these N-limited forest ecosystems remain unclear. For 18 years, we simulated N deposition by chronically adding ammonium nitrate at rates of 3 (LN treatment) and 10 (HN treatment) times the ambient N deposition estimated at the beginning of the experiment at a temperate sugar maple and a boreal balsam fir forest site, both located in northeastern America. LN and HN treatments corresponded respectively to addition of 26 kgN·ha-1·yr-1 and 85 kgN·ha-1·yr-1 at the temperate site and 17 kgN·ha-1·yr-1 and 57 kgN·ha-1·yr-1 at the boreal site. Between 2002 and 2018, soil solution was collected weekly during summer and concentrations of NO3-, NH4+, Ca2+ and pH were measured, totalling ~12,700-13,500 observations per variable on the study period. N treatments caused soil solution NO3-, NH4+ and Ca2+ concentrations to increase while reducing its pH. However, ion responses manifested through punctual high concentration events (predominantly on the HN plots) that were very rare and leached N quantity was extremely low at both sites. Therefore, N addition corresponding to 54 years (LN treatment) and 180 years (HN treatment) of accelerated ambient N deposition had overall small impacts on soil solution chemistry. Our results indicate an important N retention of northeastern American forests and an unexpected strong resilience of their soil solution chemistry to long-term simulated N deposition, potentially explained by the widespread N-limitation in high latitude ecosystems. This finding can help predict the future productivity of N-limited forests and improve forest management strategies in northeastern America.

Eastern Canadian boreal forest soil and foliar chemistry show evidence of resilience to long-term nitrogen addition.

The boreal forest is one of the world's largest terrestrial biome and plays crucial roles in global biogeochemical cycles, such as carbon (C) sequestration in vegetation and soil. However, the impacts of decades of N deposition on N-limited ecosystems, like the eastern Canadian boreal forest, remain unclear. For 13 years, N deposition was simulated by periodically adding ammonium nitrate on soils of two boreal coniferous forests (i.e., balsam fir and black spruce) of eastern Canada, at low (LN) and high (HN) rates, corresponding to 3 and 10 times the ambient N deposition, respectively. We show that more than a decade of N addition had no strong effects on mineral soil C, N, P, and cation concentrations and on foliar total Ca, K, Mg, and Mn concentrations. In organic soil, C stock was not affected by N addition while N stock increased, and exchangeable Ca2+ and Mg2+ decreased at the balsam fir site under HN treatment. At both sites, LN treatment had nearly no impact on foliage and soil chemistry but foliar N and N:P significantly increased under HN treatment, potentially leading to foliar nutrient imbalance. Overall, our work indicates that, in the eastern Canadian boreal forest, soil and foliar nutrient concentrations and stocks are resilient to increasing N deposition potentially because, in the context of N limitation, extra N would be rapidly immobilized by soil micro-organisms and vegetation. These findings could improve modeling future boreal forest soil C stocks and biomass growth and could help in planning forest management strategies in eastern Canada.

Unraveling global and diazotrophic bacteriomes of boreal forest floor feather mosses and their environmental drivers at the ecosystem and at the plant scale in North America.

Feather mosses are abundant cryptogams of the boreal forest floor and shelter a broad diversity of bacteria who have important ecological functions (e.g., decomposition, nutrient cycling). In particular, nitrogen (N2-) fixation performed by feather moss-associated diazotrophs constitutes an important entry of nitrogen in the boreal forest ecosystem. However, the composition of the feather moss bacteriome and its environmental drivers are still unclear. Using cDNA amplicon sequencing of the 16S rRNA and nifH genes and cyanobacterial biomass quantification, we explored the active global and diazotrophic bacterial communities of two dominant feather moss species (i) at the ecosystem scale, along a 500-km climatic and nutrient deposition gradient in the North American boreal forest, and (ii) at the plant scale, along the moss shoot senescence gradient. We found that cyanobacteria were major actors of the feather moss bacteriome, accounting for 33% of global bacterial communities and 65% of diazotrophic communities, and that several cyanobacterial and methanotrophic genera were contributing to N2-fixation. Moreover, we showed that bacteria were occupying ecological niches along the moss shoot, with phototrophs being dominant in the apical part and methanotrophs being dominant in the basal part. Finally, climate (temperature, precipitation), environmental variables (moss species, month, tree density) and nutrients (nitrogen, phosphorus, molybdenum, vanadium, iron) strongly shaped global and diazotrophic bacteriomes. In summary, this work presents evidence that the feather moss bacteriome plays crucial roles in supporting moss growth, health, and decomposition, as well as in the boreal forest carbon and nitrogen cycles. This study also highlights the substantial effects of climate and nutrients on the feather moss bacteriome, suggesting the importance of understanding the impacts of global change on moss-associated bacterial growth and activity.

Quantification of Moss-Associated Cyanobacteria Using Phycocyanin Pigment Extraction.

In the boreal forest, cyanobacteria can establish associations with feather moss and realize the biological nitrogen fixation (BNF) reaction, consisting in the reduction of atmospheric dinitrogen into bioavailable ammonium. In this ecosystem, moss-associated cyanobacteria are the main contributors to BNF by contributing up to 50% of new N input. Current environmental changes driven by anthropogenic activities will likely affect cyanobacteria activity (i.e., BNF) and populations inhabiting mosses, leading to potential important consequences for the boreal forest. Several methods are available to efficiently measure BNF activity, but quantifying cyanobacteria biomass associated with moss is challenging because of the difficulty to separate bacteria colonies from the host plant. Attempts to separate cyanobacteria by shaking or sonicating in water were shown to be poorly efficient and repeatable. The techniques commonly used, microscopic counting and quantitative PCR (qPCR) are laborious and time-consuming. In aquatic and marine ecosystems, phycocyanin (PC), a photosynthesis pigment produced by cyanobacteria, is commonly used to monitor cyanobacteria biomass. In this study, we tested if PC extraction and quantification can be used to estimate cyanobacteria quantity inhabiting moss. We report that phycocyanin can be easily extracted from moss by freeze/thaw disturbance of cyanobacteria cells and can be quickly and efficiently measured by spectrofluorometry. We also report that phycocyanin extraction is efficient (high recovery), repeatable (relative SD < 13%) and that no significant matrix effects were observed. As for aquatic systems, the main limitation of cyanobacteria quantification using phycocyanin is the difference of cellular phycocyanin content between cyanobacteria strains, suggesting that quantification can be impacted by cyanobacteria community composition. Nonetheless, we conclude that phycocyanin extraction and quantification is an easy, rapid, and efficient tool to estimate moss-associated cyanobacteria number.

Molybdenum threshold for ecosystem scale alternative vanadium nitrogenase activity in boreal forests.

Biological nitrogen fixation (BNF) by microorganisms associated with cryptogamic covers, such as cyanolichens and bryophytes, is a primary source of fixed nitrogen in pristine, high-latitude ecosystems. On land, low molybdenum (Mo) availability has been shown to limit BNF by the most common form of nitrogenase (Nase), which requires Mo in its active site. Vanadium (V) and iron-only Nases have been suggested as viable alternatives to countering Mo limitation of BNF; however, field data supporting this long-standing hypothesis have been lacking. Here, we elucidate the contribution of vanadium nitrogenase (V-Nase) to BNF by cyanolichens across a 600-km latitudinal transect in eastern boreal forests of North America. Widespread V-Nase activity was detected (∼15-50% of total BNF rates), with most of the activity found in the northern part of the transect. We observed a 3-fold increase of V-Nase contribution during the 20-wk growing season. By including the contribution of V-Nase to BNF, estimates of new N input by cyanolichens increase by up to 30%. We find that variability in V-based BNF is strongly related to Mo availability, and we identify a Mo threshold of ∼250 ng·glichen-1 for the onset of V-based BNF. Our results provide compelling ecosystem-scale evidence for the use of the V-Nase as a surrogate enzyme that contributes to BNF when Mo is limiting. Given widespread findings of terrestrial Mo limitation, including the carbon-rich circumboreal belt where global change is most rapid, additional consideration of V-based BNF is required in experimental and modeling studies of terrestrial biogeochemistry.

The coastal environment affects lead and sodium uptake by the moss Hypnum cupressiforme used as an air pollution biomonitor.

Several studies suggest that potential competition exists between marine cations and heavy metals for binding sites on the cell wall of mosses. This competition would impact the heavy metal concentration measured in mosses by biomonitoring programs, which may underestimate air pollution by heavy metals in a coastal environment. In the present study, we aim to identify possible mechanisms affecting lead uptake by mosses in a coastal environment, specifically, the competition between lead (Pb2+) and sodium (Na+) for binding sites in Hypnum cupressiforme (Hc). We also compared the response of continental and coastal Hc populations to Pb2+ exposure by immersing the moss samples in artificial solutions that comprised six experimental treatments and subsequently locating and quantifying Pb2+ and Na+ using the sequential elution technique and X-ray microanalyses with a scanning electron microscope. We demonstrated that high concentrations of Pb2+ prevented Na+ from binding to the cell wall. We also examined the effect of the salt acclimation of Hc on Pb2+ and Na+ accumulation. Coastal Hc populations accumulated more Na and less Pb than continental Hc populations in all treatments. Moreover, our results showed treatment effects on the intra/extracellular distribution of Na+, as well as site. This feedback on the influence of salt stress tolerance on Pb2+ uptake by mosses requires further study and can be investigated for other heavy metals, leading to a better use of mosses as biomonitoring tools.

Urbanization drives the evolution of parallel clines in plant populations.

Urban ecosystems are an increasingly dominant feature of terrestrial landscapes. While evidence that species can adapt to urban environments is accumulating, the mechanisms through which urbanization imposes natural selection on populations are poorly understood. The identification of adaptive phenotypic changes (i.e. clines) along urbanization gradients would facilitate our understanding of the selective factors driving adaptation in cities. Here, we test for phenotypic clines in urban ecosystems by sampling the frequency of a Mendelian-inherited trait-cyanogenesis-in white clover (Trifolium repens L.) populations along urbanization gradients in four cities. Cyanogenesis protects plants from herbivores, but reduces tolerance to freezing temperatures. We found that the frequency of cyanogenic plants within populations decreased towards the urban centre in three of four cities. A field experiment indicated that spatial variation in herbivory is unlikely to explain these clines. Rather, colder minimum winter ground temperatures in urban areas compared with non-urban areas, caused by reduced snow cover in cities, may select against cyanogenesis. In the city with no cline, high snow cover might protect plants from freezing damage in the city centre. Our study suggests that populations are adapting to urbanization gradients, but regional climatic patterns may ultimately determine whether adaptation occurs.