Light drives nitrogen fixation in tropical montane cloud forests in Costa Rica.

Tropical montane cloud forests are high altitude ecosystems characterized by very high ambient humidity, which favors organisms that depend on the environment for their water status, such as bryophytes and their nitrogen-fixing symbionts. Bryophyte-associated N2 fixation is a major source of new N in several northern environments, but their contributions to the N cycle in other ecosystems is still poorly understood. In this work, we evaluated N2 fixation rates associated with epiphytic bryophytes growing along the stems of pumpwood trees (Cecropia sp.) as well as in surrounding litter and soil from a primary and a secondary cloud forests in the Talamanca Mountain Range, Costa Rica. Nitrogen fixation was significantly higher in substrates from the secondary forest compared to those from the primary forest. Overall, N2 fixation rates associated with epiphytic bryophytes were 57 times those of litter and 270 times what was measured in soil. Further, light intensity was the major factor influencing N2 fixation rates in all substrates. Increased access to light in disturbed cloud forests may therefore favor bryophyte-associated N2 fixation, potentially contributing to the recovery of these ecosystems.

The Feather Moss Hylocomium splendens Affects the Transcriptional Profile of a Symbiotic Cyanobacterium in Relation to Acquisition and Turnover of Key Nutrients.

Moss-cyanobacteria symbioses were proposed to be based on nutrient exchange, with hosts providing C and S while bacteria provide N, but we still lack understanding of the underlying molecular mechanisms of their interactions. We investigated how contact between the ubiquitous moss Hylocomium splendens and its cyanobiont affects nutrient-related gene expression of both partners. We isolated a cyanobacterium from H. splendens and co-incubated it with washed H. splendens shoots. Cyanobacterium and moss were also incubated separately. After 1 week, we performed acetylene reduction assays to estimate N2 fixation and RNAseq to evaluate metatranscriptomes. Genes related to N2 fixation and the biosynthesis of several amino acids were up-regulated in the cyanobiont when hosted by the moss. However, S-uptake and the biosynthesis of the S-containing amino acids methionine and cysteine were down-regulated in the cyanobiont while the degradation of selenocysteine was up-regulated. In contrast, the number of differentially expressed genes in the moss was much lower, and almost no transcripts related to nutrient metabolism were affected. It is possible that, at least during the early stage of this symbiosis, the cyanobiont receives few if any nutrients from the host in return for N, suggesting that moss-cyanobacteria symbioses encompass relationships that are more plastic than a constant mutualist flow of nutrients.

Global N2O emissions from our planet: Which fluxes are affected by man, and can we reduce these?

In some places, N2O emissions have doubled during the last 2-3 decades. Therefore, it is crucial to identify N2O emission hotspots from terrestrial and aquatic systems. Large variation in N2O emissions occur in managed as well as in natural areas. Natural unmanaged tropical and subtropical wet forests are important N2O sources globally. Emission hotspots, often coupled to human activities, vary across climate zones, whereas N2O emissions are most often a few kg N ha-1 year-1 from arable soils, drained organic soils in the boreal and temperate zones often release 20-30 kg N ha-1 year-1. Similar high N2O emissions occur from some tropical crops like tea, palm oil and bamboo. This strong link between increased N2O emissions and human activities highlight the potential to mitigate large emissions. In contrast, water where oxic and anoxic conditions meet are N2O emission hotspots as well, but not possible to reduce.

Nitrogen fixation associated with two cohabiting moss species expresses different patterns under Cu and Zn contamination.

Nitrogen (N2) fixation by moss-associated cyanobacteria is an important N source in pristine ecosystems. Previous studies have shown that moss-associated N2 fixation is sensitive to anthropogenic N pollution. However, we still lack understanding of the effects of other factors derived from anthropogenic sources, such as heavy metal pollution on N2 fixation. To test this, we collected two dominant mosses (Pleurozium schreberi and Spaghnum palustre) from a temperate bog in Denmark and assessed their N2 fixation responses to simulated heavy metal pollution by adding 5 levels (plus a control) of copper (Cu, 0-0.05 mg g dw-1) and zinc (Zn, 0-0.1 mg g dw-1). Metal concentrations in both mosses increased linearly with Cu and Zn addition, but N2 fixation activity associated with S. palustre was to a greater extent negatively affected by both Cu and Zn additions than that associated with P. schreberi. Copper additions even promoted N2 fixation in P. schreberi. Hence, the heavy metal sensitivity of N2-fixing cyanobacteria is dependent on the host moss-species, and the vulnerability of ecosystems towards heavy metal pollution could vary depending on the dominant moss species.

Do Nitrogen and Phosphorus Additions Affect Nitrogen Fixation Associated with Tropical Mosses?

Tropical cloud forests are characterized by abundant and biodiverse mosses which grow epiphytically as well as on the ground. Nitrogen (N)-fixing cyanobacteria live in association with most mosses, and contribute greatly to the N pool via biological nitrogen fixation (BNF). However, the availability of nutrients, especially N and phosphorus (P), can influence BNF rates drastically. To evaluate the effects of increased N and P availability on BNF in mosses, we conducted a laboratory experiment where we added N and P, in isolation and combined, to three mosses (Campylopus sp., Dicranum sp. and Thuidium peruvianum) collected from a cloud forest in Peru. Our results show that N addition almost completely inhibited BNF within a day, whereas P addition caused variable results across moss species. Low N2 fixation rates were observed in Campylopus sp. across the experiment. BNF in Dicranum sp. was decreased by all nutrients, while P additions seemed to promote BNF in T. peruvianum. Hence, each of the three mosses contributes distinctively to the ecosystem N pool depending on nutrient availability. Moreover, increased N input will likely significantly decrease BNF associated with mosses also in tropical cloud forests, thereby limiting N input to these ecosystems via the moss-cyanobacteria pathway.

Chemical Stimulation of Heterocyte Differentiation by the Feather Moss Hylocomium splendens: a Potential New Step in Plant-Cyanobacteria Symbioses.

Cyanobacteria associated with mosses play a key role in the nitrogen (N) cycle in unpolluted ecosystems. Mosses have been found to release molecules that induce morphophysiological changes in epiphytic cyanobionts. Nevertheless, the extent of moss influence on these microorganisms remains unknown. To evaluate how mosses or their metabolites influence N2 fixation rates by cyanobacteria, we assessed the nitrogenase activity, heterocyte frequency and biomass of a cyanobacterial strain isolated from the feather moss Hylocomium splendens and a non-symbiotic strain when they were either growing by themselves, together with H. splendens or exposed to H. splendens water, acetone, ethanol, or isopropanol extracts. The same cyanobacterial strains were added to another moss (Taxiphyllum barbieri) and a liverwort (Monosolenium tenerum) to assess if these bryophytes affect N2 fixation differently. Although no significant increases in nitrogenase activity by the cyanobacteria were observed when in contact with H. splendens shoots, both the symbiotic and non-symbiotic cyanobacteria increased nitrogenase activity as well as heterocyte frequency significantly upon exposure to H. splendens ethanol extracts. Contact with T. barbieri shoots, on the other hand, did lead to increases in nitrogenase activity, indicating low host-specificity to cyanobacterial activity. These findings suggest that H. splendens produces heterocyte-differentiating factors (HDFs) that are capable of stimulating cyanobacterial N2 fixation regardless of symbiotic competency. Based on previous knowledge about the chemical ecology and dynamics of moss-cyanobacteria interactions, we speculate that HDF expression by the host takes place in a hypothetical new step occurring after plant colonization and the repression of hormogonia.

Enhanced foliar 15 N enrichment with increasing nitrogen addition rates: Role of plant species and nitrogen compounds.

Determining the abundance of N isotope (δ15 N) in natural environments is a simple but powerful method for providing integrated information on the N cycling dynamics and status in an ecosystem under exogenous N inputs. However, whether the input of different N compounds could differently impact plant growth and their 15 N signatures remains unclear. Here, the response of 15 N signatures and growth of three dominant plants (Leymus chinensis, Carex duriuscula, and Thermopsis lanceolata) to the addition of three N compounds (NH4 HCO3 , urea, and NH4 NO3 ) at multiple N addition rates were assessed in a meadow steppe in Inner Mongolia. The three plants showed different initial foliar δ15 N values because of differences in their N acquisition strategies. Particularly, T. lanceolata (N2 -fixing species) showed significantly lower 15 N signatures than L. chinensis (associated with arbuscular mycorrhizal fungi [AMF]) and C. duriuscula (associated with AMF). Moreover, the foliar δ15 N of all three species increased with increasing N addition rates, with a sharp increase above an N addition rate of ~10 g N m-2  year-1 . Foliar δ15 N values were significantly higher when NH4 HCO3 and urea were added than when NH4 NO3 was added, suggesting that adding weakly acidifying N compounds could result in a more open N cycle. Overall, our results imply that assessing the N transformation processes in the context of increasing global N deposition necessitates the consideration of N deposition rates, forms of the deposited N compounds, and N utilization strategies of the co-existing plant species in the ecosystem.

A research agenda for nonvascular photoautotrophs under climate change.

Nonvascular photoautotrophs (NVP), including bryophytes, lichens, terrestrial algae, and cyanobacteria, are increasingly recognized as being essential to ecosystem functioning in many regions of the world. Current research suggests that climate change may pose a substantial threat to NVP, but the extent to which this will affect the associated ecosystem functions and services is highly uncertain. Here, we propose a research agenda to address this urgent question, focusing on physiological and ecological processes that link NVP to ecosystem functions while also taking into account the substantial taxonomic diversity across multiple ecosystem types. Accordingly, we developed a new categorization scheme, based on microclimatic gradients, which simplifies the high physiological and morphological diversity of NVP and world-wide distribution with respect to several broad habitat types. We found that habitat-specific ecosystem functions of NVP will likely be substantially affected by climate change, and more quantitative process understanding is required on: (1) potential for acclimation; (2) response to elevated CO2 ; (3) role of the microbiome; and (4) feedback to (micro)climate. We suggest an integrative approach of innovative, multimethod laboratory and field experiments and ecophysiological modelling, for which sustained scientific collaboration on NVP research will be essential.

Biotic and abiotic controls of nitrogen fixation in cyanobacteria-moss associations.

Most mosses are colonized by nitrogen (N)-fixing cyanobacteria. This discovery is relatively recent, which can explain the large knowledge gaps the field is now tackling. For instance, while we have a good understanding of the abiotic controls (e.g. nutrient availability, increased temperature), we still do not know much about the biotic controls of N2 fixation in mosses. I propose here that we should endeavour to position moss-cyanobacteria associations along the mutualism-parasitism continuum under varying abiotic conditions (e.g. nutrient availability). This would finally unravel the nature of the relationship between the partners and will be a big leap in our understanding of the evolution of plant-bacteria interactions using moss-cyanobacteria associations as a model system.

Unraveling host-microbe interactions and ecosystem functions in moss-bacteria symbioses.

Mosses are non-vascular plants usually found in moist and shaded areas, with great ecological importance in several ecosystems. This is especially true in northern latitudes, where mosses are responsible for up to 100% of primary production in some ecosystems. Mosses establish symbiotic associations with unique bacteria that play key roles in the carbon and nitrogen cycles. For instance, in boreal environments, more than 35% of the nitrogen fixed by diazotrophic symbionts in peatlands is transferred to mosses, directly affecting carbon fixation by the hosts, while moss-associated methanotrophic bacteria contribute 10-30% of moss carbon. Further, half of ecosystem N input may derive from moss-cyanobacteria associations in pristine ecosystems. Moss-bacteria interactions have consequences on a global scale since northern environments sequester 20% of all the carbon generated by forests in the world and stock at least 32% of global terrestrial carbon. Different moss hosts influence bacteria in distinct ways, which suggests that threats to mosses also threaten unique microbial communities with important ecological and biogeochemical consequences. Since their origin ~500 Ma, mosses have interacted with bacteria, making these associations ideal models for understanding the evolution of plant-microbe associations and their contribution to biogeochemical cycles.

The moss traits that rule cyanobacterial colonization.

BACKGROUND AND AIMS: Cyanobacteria associated with mosses represent a main nitrogen (N) source in pristine, high-latitude and -altitude ecosystems due to their ability to fix N2. However, despite progress made regarding moss-cyanobacteria associations, the factors driving the large interspecific variation in N2 fixation activity between moss species remain elusive. The aim of the study was to identify the traits of mosses that determine cyanobacterial colonization and thus N2 fixation activity. METHODS: Four moss species varying in N2 fixation activity were used to assess cyanobacterial abundance and activity to correlate it with moss traits (morphological, chemical, water-balance traits) for each species. KEY RESULTS: Moss hydration rate was one of the pivotal traits, explaining 56 and 38 % of the variation in N2 fixation and cyanobacterial colonization, respectively, and was linked to morphological traits of the moss species. Higher abundance of cyanobacteria was found on shoots with smaller leaves, and with a high frequency of leaves. High phenol concentration inhibited N2 fixation but not colonization. These traits driving interspecific variation in cyanobacterial colonization, however, are also affected by the environment, and lead to intraspecific variation. Approximately 24 % of paraphyllia, filamentous appendages on Hylocomium splendens stems, were colonized by cyanobacteria. CONCLUSIONS: Our findings show that interspecific variations in moss traits drive differences in cyanobacterial colonization and thus, N2 fixation activity among moss species. The key traits identified here that control moss-associated N2 fixation and cyanobacterial colonization could lead to improved predictions of N2 fixation in different moss species as a function of their morphology.

Indirect effects of climate change inhibit N2 fixation associated with the feathermoss Hylocomium splendens in subarctic tundra.

Mosses can be responsible for up to 100% of net primary production in arctic and subarctic tundra, and their associations with diazotrophic cyanobacteria have an important role in increasing nitrogen (N) availability in these pristine ecosystems. Predictions about the consequences of climate change in subarctic environments point to increased N mineralization in soil and higher litter deposition due to warming. It is not clear yet how these indirect climate change effects impact moss-cyanobacteria associations and N2 fixation. This work aimed to evaluate the effects of increased N and litter input on biological N2 fixation rates associated with the feathermoss Hylocomium splendens from a tundra heath. H. splendens samples were collected near Abisko, northern Sweden, from a field experiment with annual additions of ammonium chloride and dried birch litter and the combination of both for three years. Samples were analyzed for N2 fixation, cyanobacterial colonization, C and N content and pH. Despite the high N additions, no significant differences in moss N content were found. However, differences between treatments were observed in N2 fixation rates, cyanobacterial colonization and pH, with the combined ammonium+litter treatment causing a significant reduction in the number of branch-colonizing cyanobacteria and N2 fixation, and ammonium additions significantly lowering moss pH. A significant, positive relationship was found between N2 fixation rates, moss colonization by cyanobacteria and pH levels, showing a clear drop in N2 fixation rates at lower pH levels even if larger cyanobacterial populations were present. These results suggest that increased N availability and litter deposition resulting from climate change not only interferes with N2 fixation directly, but also acidifies moss microhabitats and reduces the abundance of associated cyanobacteria, which could eventually impact the N cycle in the Subarctic.

Simulated rhizosphere deposits induce microbial N-mining that may accelerate shrubification in the subarctic.

Climate change is exposing high-latitude systems to warming and a shift towards more shrub-dominated plant communities, resulting in increased leaf-litter inputs at the soil surface, and more labile root-derived organic matter (OM) input in the soil profile. Labile OM can stimulate the mineralization of soil organic matter (SOM); a phenomenon termed "priming." In N-poor subarctic soils, it is hypothesized that microorganisms may "prime" SOM in order to acquire N (microbial N-mining). Increased leaf-litter inputs with a high C/N ratio might further exacerbate microbial N demand, and increase the susceptibility of N-poor soils to N-mining. We investigated the N-control of SOM mineralization by amending soils from climate change-simulation treatments in the subarctic (+1.1°C warming, birch litter addition, willow litter addition, and fungal sporocarp addition) with labile OM either in the form of glucose (labile C; equivalent to 400 µg C/g fresh [fwt] soil) or alanine (labile C + N; equivalent to 400 µg C and 157 µg N/g fwt soil), to simulate rhizosphere inputs. Surprisingly, we found that despite 5 yr of simulated climate change treatments, there were no significant effects of the field-treatments on microbial process rates, community structure or responses to labile OM. Glucose primed the mineralization of both C and N from SOM, but gross mineralization of N was stimulated more than that of C, suggesting that microbial SOM use increased in magnitude and shifted to components richer in N (i.e., selective microbial N-mining). The addition of alanine also resulted in priming of both C and N mineralization, but the N mineralization stimulated by alanine was greater than that stimulated by glucose, indicating strong N-mining even when a source of labile OM including N was supplied. Microbial carbon use efficiency was reduced in response to both labile OM inputs. Overall, these findings suggest that shrub expansion could fundamentally alter biogeochemical cycling in the subarctic, yielding more N available for plant uptake in these N-limited soils, thus driving positive plant-soil feedbacks.

High nitrogen resorption efficiency of forest mosses.

BACKGROUND AND AIMS: Nutrient resorption from senescing tissue is a key mechanism for plants to conserve nutrients, and can affect the nutrient dynamics of ecosystems. Yet, our limited knowledge of nitrogen (N) resorption and release from mosses hampers our understanding of the role of mosses as N sources and, thereby, N cycling in moss-dominated ecosystems. The aims of this study were to estimate N resorption efficiency (NRE) of two moss species, identify the pathways of N release from the mosses and to provide a better understanding of N cycling and budgeting strategies of mosses. METHODS: The dynamics of N allocation along annual moss segments of two dominant moss species (Actinothuidium hookeri and Hylocomium splendens) were assessed in old-growth fir forests using an in situ15N tracer experiment. KEY RESULTS: The NRE of A. hookeri and H. splendens was 61 and 52 %, respectively. While the mosses lost 23 and 33 % N from live tissues via leaching, 15 and 14 % of N remained in senesced tissues (>3 years old) in A. hookeri and H. splendens, respectively. CONCLUSIONS: Both mosses resorbed the majority of their tissue N, but a considerable amount of N was lost from live segments. Our results highlight the crucial role mosses play as N sinks in ecosystems, since N retention (resorbed and sequestered in senescent tissue) outweighed N loss via leaching. However, the sink strength depends on temperature and precipitation, which will change in a future climate. The values for NRE, leaching, etc. estimated here can help improve biogeochemical models aiming to complete N budgets for moss-abundant ecosystems.

Faster nitrogen cycling and more fungal and root biomass in cold ecosystems under experimental warming: a meta-analysis.

Warming can alter the biogeochemistry and ecology of soils. These alterations can be particularly large in high northern latitude ecosystems, which are experiencing the most intense warming globally. In this meta-analysis, we investigated global trends in how experimental warming is altering the biogeochemistry of the most common limiting nutrient for biological processes in cold ecosystems of high northern latitudes (>50°): nitrogen (N). For comparison, we also analyzed cold ecosystems at intermediate and high southern latitudes. In addition, we examined N-relevant genes and enzymes, and the abundance of belowground organisms. Together, our findings suggest that warming in cold ecosystems increases N mineralization rates and N2 O emissions and does not affect N fixation, at least not in a consistent way across biomes and conditions. Changes in belowground N fluxes caused by warming lead to an accumulation of N in the forms of dissolved organic and root N. These changes seem to be more closely linked to increases in enzyme activity that target relatively labile N sources, than to changes in the abundance of N-relevant genes (e.g., amoA and nosZ). Finally, our analysis suggests that warming in cold ecosystems leads to an increase in plant roots, fungi, and (likely in an indirect way) fungivores, and does not affect the abundance of archaea, bacteria, or bacterivores. In summary, our findings highlight global trends in the ways warming is altering the biogeochemistry and ecology of soils in cold ecosystems, and provide information that can be valuable for prediction of changes and for management of such ecosystems.

High heavy metal load does not inhibit nitrogen fixation in moss-cyanobacteria associations.

Nitrogen (N2) fixation by moss-associated cyanobacteria is one of the main sources of new N input in pristine ecosystems such as boreal forests and arctic tundra. Given the non-vascular physiology of mosses, they are especially sensitive to e.g. increased N input and heavy metal deposition. While the effects of increased N input on moss-associated N2 fixation has been comprehensively assessed, hardly any reports exist on the effects of increased heavy metal load on this key ecosystem function. To address this knowledge gap, we made use of an extreme metal pollution gradient in boreal forests of Northern Sweden originating from a metal mine and its associated smelters. We collected the common moss Pleurozium schreberi, known to host cyanobacteria, along a distance gradient away from the metal source of pollution and measured moss-metal content (Fe, Cu, Zn, Pb) as well as N2 fixation. We found a strong distance gradient in moss-metal content for all investigated metals: a sharp decline in metal content with distance away from the metal pollution source. However, we found a similarly steep gradient in moss-associated N2 fixation, with highest activity closest to the metal source of pollution. Hence, while mosses may be sensitive to increased heavy metal inputs, the activity of colonising cyanobacteria seem to be unaffected by heavy metals, and consequently, ecosystem function may not be compromised by elevated metal input.

Railroad derived nitrogen and heavy metal pollution does not affect nitrogen fixation associated with mosses and lichens at a tundra site in Northern Sweden.

Traffic derived nitrogen (N) and heavy metal pollution is a well-known phenomenon, but little explored in otherwise pristine ecosystems such as subarctic tundra. Here, the main source of N input to the ecosystem is via N2 fixation by moss- and lichen-associated bacteria. While inhibitory effects of N deposition on moss-associated N2 fixation have been reported, we still lack understanding of the effects of traffic derived N and heavy metal deposition on this ecosystem function in an otherwise pristine setting. To test this, we established a distance gradient (0-1280 m) away from a metal pollution source -a railway transporting iron ore that passes through a subarctic birch forest. We assessed the effects of railway-derived pollution on N2 fixation associated with two moss species Pleurozium schreberi, Hylocomium splendens and with the lichen Peltigera aphthosa. Deposition and availability of N and heavy metals (Fe, Cu, Zn, Pb) as well as the respective contents in moss, lichen and soil was assessed. While we found a steep gradient in metal concentration in moss, lichen and soil with distance away from the pollution source, N deposition did not change, and with that, we could not detect a distance gradient in moss- or lichen-associated N2 fixation. Hence, our results indicate that N2 fixing bacteria are either not inhibited by heavy metal deposition, or that they are protected within the moss carpet and lichen tissue.