WHIM (UK)

OPERATING INSTITUTE: NERC, Centre for Ecology & Hydrology.

MAIN PURPOSE: Peatland flora, chemistry and processes.

ECOSYSTEM TYPE: Ombrotrophic bog.

EXPERIMENTAL TREATMENTS: Wet and dry N deposition.

LOCALISATION: 55.813036607527785 -3.2318115234375

FACILITIES: Whim bog offers a globally unique comparison of how the main N forms affect semi-natural vegetation, with meteorological data and treatment history since 2002. A quantified ammonia concentration/ deposition gradient, is provided, plus a wet deposition system, comparing oxidised (NaNO₃) and reduced N (NH₄Cl), where treatments are meteorology dependent. ie. wind direction and rainfall frequency define the treatment exposure (~120 events y^-1).  There are four large (13m²) wet plots (4). Opportunities exist to evaluate a whole range of ecosystem services from conservation to carbon sequestration, GHG emissions and water chemistry in addition to understanding how changes in vegetation affect the delivery of these services.
In 2012, a LICOR system was installed for continuous methane and CO₂ monitoring. This system has also been used for real time C flux monitoring on the plots. Flux data from the low N deposition plots have been collected, as well as CN data for soil and various species. Burial of t bags has allowed us to assess conditions for decomposition and see how N form and dose affects water soluble phenol in these materials. Measure-ment of DOC and DON in soil water collected in dipwells have also been resumed.

CONTACT: M. JONES (This email address is being protected from spambots. You need JavaScript enabled to view it.) This email address is being protected from spambots. You need JavaScript enabled to view it.S. OWEN (This email address is being protected from spambots. You need JavaScript enabled to view it.)

RECENT PUBLICATIONS:
●   Sheppard, L. J.,  Leith, I. D.,  Mizunuma, T., Cape J. N., Crossley, A.,  Leeson,  S., Sutton, M. A., Van Dijk, N., Fowler, D. (2011). Dry deposition of ammonia gas drives species change faster than wet deposition of ammonium ions: evidence from a long-term field manipulation. Global Change Biology 17, 3589-3607.
●   Sheppard, L. J., Leith I. D., Mizunuma, T., Van Dijk, N., Cape, J. N., Sutton, M. A., (2011). All forms of reactive nitrogen deposition to Natura 2000 sites should not be treated equally: effects of wet versus dry and reduced versus oxidised nitrogen deposition. In Nitrogen Deposition and Natura 2000: Science & practice in determining environmental impacts Eds Hicks WK, Whitfield CP, Bealey WJ, Sutton MA. COST office 2011, 181-189.
●   Currey, P. M., Johnson, D., Dawson, L., Van der Wal, R., Thornton, B., Sheppard, L., Leith, I., Artz RRE (2011). Five years of simulated atmospheric nitrogen deposition have only subtle effects on the fate of newly synthesized carbon in Calluna vulgaris and Eriophorum vaginatum. Soil Biology and Biochemistry 43, 495-502.

TA PROJECTS: Nitrogen cycling in a peat land: the effect of different forms and loads of nitrogen deposition on N₂, NO and N₂O losses to the atmosphere (NitroWHIM).

TA User (visit): Eva Van Den Elzen, Radboud University Nijmegen, The Netherlands (April, 2014 – 5 days) - “Fast Track” Application.
Project Description: Peatlands represent an important and at the same time fragile carbon sink that is important with respect to climate change. Given the strong link between the carbon (C) and the nitrogen (N) cycle, the consequences of increasing anthropogenic N deposition is of concern for the sustainability of these ecosystems. Peat bogs are likely to be highly sensitive to enhanced nitrogen inputs, since they have evolved under conditions of restricted nitrogen inputs (Bobbink et al 1998) and peat mosses (Sphagnum spec., the keystone genus of peatlands) are able to outcompete other species by monopolizing nutrients and carbon (Lamers et al 1999; 2000; Fritz et al 2014). High N loads were shown to counteract this competitive advantage of Sphagnum and can result in leaching of N and opportunities for vascular plants to take over.
The Whim bog field study (CEH) is a long-term (since 2002) nitrogen application experiment in an ombrotrophic bog site in Scotland. At a relatively low background N deposition a free air release of NH3 and real-time additions of NO^3- and NH⁴⁺ in rainwater have been applied. The effects of these different N forms on biogeochemistry and physiological processes in Sphagnum peat have been studied since 2005 in active collaboration between CEH Edinburgh (Dr. L Sheppard and Mr. I Leith) and Radboud University Nijmegen (Prof. J Roelofs and Dr. L van den Berg).
Especially dry N deposition (NH3) was shown to cause strong declines of Sphagnum mosses (Sheppard et al 2013), as this form can easily become toxic at high concentrations and lead to N losses to the atmosphere as the greenhouse gas N_^¬2O. It seems to take a much longer time for wet deposition (in the form of NO^3- or NH⁴⁺) to have negative effects on peat bog vegetation. Because different species in the bog respond differently to the various N forms, defining critical loads for this ecosystem is challenging (Sheppard et al 2014) and soil chemistry seems to play an important role as well.
We now aim to add to these studies by studying the effects of different forms of N-deposition on total nitrogen dynamics of Sphagnum peatlands.  As the total nitrogen cycle and budget remains obscure du tor the lack of N output data, we propose to investigate the fate of the additional N once it leaches into the peat soil, and how the biogeochemical processes of denitrification and anammox are affected by the different forms and loads of nitrogen.

The relation between physiological responses of Sphagnum capillifolium and nitrogen losses to the ground water due to elevated atmospheric nitrogen deposition (NPLOSS_WHIM).

TA User (visit): Leon van den Berg, Radboud University Nijmegen, the Netherlands (April, 2014 – 5 days).
Project Description: Earlier studies have shown that the living Sphagnum layer filters nitrogen (N) by retaining and storing substantial amounts of N in biomass and peat (e.g. Lamers et al 2000). It was shown that the N-filter ‘’fails’’ at high N-loads resulting in N leaching to deeper soil layers where it can stimulate peat breakdown and growth of vascular plants such as sedges and grasses. Assimilation of especially the reduced N forms (NH⁴⁺ and NH₃) can become problematic to many mosses as these forms are toxic in high concentrations. Recently strong declines of Sphagnum mosses have been related to atmospheric N deposition in a long term N-manipulation experiment (Sheppard et al 2014) and decline occurred most dramatically in the plots that received reduced N as the dominant N form. Earlier work by our groups has resulted in an identification of physiological responses of the Sphagnum mosses to different N forms and N loads. A recent experiment (Fritz et al 2014) showed that N uptake rates decline over longer exposure time, probably due to physiological restrictions, and that uptake rates are N-form dependent. During short exposure time, uptake rates were high and N-filtering was near to 100%. In a follow-up experiment, dominant N form in deposition was also linked to phosphorus (P) uptake rates (van den Berg et al in prep) showing higher P uptake rates when oxidised N was the dominant N form in deposition. These findings have important ecological and evolutionary implications: at high N input rates, the risk of N-toxicity seems to be reduced by lower uptake rates of Sphagnum, at the expense of its long-term filter capacity and related competitive advantage over vascular plants. This implies that N losses (leaching) to deeper groundwater layers are expected to be higher where N input is higher or where N uptake rates are reduced by the mosses (i.e. where oxidised N is dominant and deposited in high loads). In addition, in P-polluted bogs, P levels are high and P may not be retained in the mosses when reduced N is the dominant N form. This may result in P leaching and higher P availability in ground and pore water where it can stimulate growth of vascular plants. The environmentally relevant and scientifically interesting differences in assimilation rates and physiological responses of Sphagnum peat under different N deposition regimes in relation to N and P losses to deeper ground water have, until now, not been studied in long term experiments.

Inter- and intra-specific differences in decomposition of Sphagnum litter under exposure to reactive nitrogen compounds (DeSNi).

TA User (visit): Sirkku Manninen, University of Helsinki, Finland (September, 2013 – 25 days).
Project Description: Earlier studies performed at the Whim bog show that the red (open grown) and the green (shade grown) type of Sphagnum capillifolium differ in terms N concentration, C:N ratio, pH, and their responses to elevated wet deposition of NH⁴⁺-N and NO^3- -N. Moreover, there are differences in the responses of S. fallax (green) and S. papillosum (yellowish – greenish brown) to elevated wet deposition of reactive N at Whim bog.
Peatlands are very important for C sequestration, but only if decomposition rates are low. Sphagnum litter is resistant to microbial, because it is rich in phenolics. Decomposition of Sphagnum litter has been found to be enhanced under exposure to elevated wet N deposition. However, adding N and NPK decreased S. capillifolium litter mass loss at Whim bog. This may be because the red type contains an anthocyanin called sphagnorubin (polyphenolic compound). The effects of N availability on litter peat decomposition may be connected through the activity of phenol oxidase, one of the few enzymes able to degrade polyphenols.
The long-term Whim bog N manipulation experiment offers an excellent possibility: i) to show potential differences in litter decomposition rate and subsequent CO₂ emissions between red and green pigmented S. capillifolium as well as between Sphagnum species, in response to long-term (>11 years) N inputs, (ii) to study inter- and intra-specific differences in the litter decomposition rate and subsequent CO₂ emissions of Sphagna under elevated wet deposition of NH⁴⁺-N cf. NO^3- -N as well as NH₃, and iii) to study which characteristics of the moss litter play a key role in terms of its decomposition rate and subsequent CO₂ emissions.
The project includes: peat litter incubations and CO₂ measurements, analyses of litter total C and N concentrations, measurements of DOC released from peat litter and water soluble phenol concentration, assays for activity of phosphatase and oxidative phenol oxidase, and use litter bags to study liteer mass loss.
The Centre for Ecology and Hydrology in Edinburgh has all the instrumentation to perform the study. Data will be gathered and analysed by the applicant (supported by staff at the site).
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Effects of long term addition of reduced and oxidised nitrogen on the amino acid composition and nitrogen assimilation of Sphagnum capillifolium at Whim bog. – AminoWHIM.

TA User (visit): Leon van den Berg, Radboud University Nijmegen, The Netherlands (August, 2013 – 3 days).
Project Description:
Introduction: Bog vegetation reacts slowly to small increments of nitrogen (N) availability as the living Sphagnum layer filters N by retaining and storing substantial amounts of N in biomass and peat (e.g. Lamers et al. 2000). High total N loads were, however, found to have detrimental effects on biomass production of mosses (Arroniz-Crespo et al. 2008; Limpens et al. 2011) and result in direct physiological changes in Sphagnum mosses (e.g. Rudolph et al. 1993; Pearce et al. 2003). The relative effects of airborne ammonium (NH⁴⁺), ammonia (NH₃) and nitrate (NO^3-), have received much less attention.N uptake by Sphagnum mosses is accompanied by proton or base cation excretion, depending on the form in which N is assimilated (reduced or oxidised). Assimilation of especially the reduced N forms (NH⁴⁺ and NH₃) can become problematic to many plants and mosses as these forms are toxic in high concentrations, and result in a vegetation decline when loads are excessive (e.g Sheppard et al. 2009). In order to detoxify the reduced N forms, plants and Sphagnum mosses store excessive N in N-rich amino acids in the cells. N-rich amino acid accumulation may therefore act as a strong bio-indicator of stress in Sphagnum mosses due to increased N availability and may even act as an alarming signal for future declines of the mosses.Losses of base cations may be problematic for mosses that assimilate an excess of oxidised N forms, especially in the long term. Recently strong declines of Sphagnum mosses have been related to atmospheric deposition of nitrogen in a long term N-manipulation experiment (Sheppard et al. submitted). However, the environmentally relevant and scientifically interesting differences in assimilation rates, detoxifying mechanisms and deficiencies that may develop as a result of either reduced or oxidised N have, until now, not been studied in long term experiments.
Whim bog experimental site: The Whim-bog field study (CEH) on an ombrotrophic bog is a long term (since 2002), near natural, N-application experiment at relatively low background N deposition. The application involves a free air release of NH3 and additions of NO^3- and NH⁴⁺ in rainwater. It can therefore provide evidence of the effect of different N forms under near natural conditions on Sphagnum species.There has been active collaboration between CEH Edinburgh (Dr. L Sheppard and Mr. I Leith) and Radboud University Nijmegen (Prof. J Roelofs and Dr. L van den Berg) since 2005 to study the differential effects of reduced and oxidised N on biochemistry and physiological processes in Sphagnum peat. In 2005, and again in 2008, amino acid concentrations were measured in Sphagnum capillifolium and these provided considerable information concerning the vitality of these species. All three N forms enhanced the amino acid concentrations with the N-rich amino acid arginine (4 N to every C) as the most dominant amino acid to detoxifying N. In the wet plots, both the 64 and 32 kg N ha^-1 y^-1 treatments significantly enhanced N-rich amino acid concentrations, with reduced N having a bigger effect than would be predicted solely from the higher foliage N concentration. The elevated concentrations are symptomatic of impending damage to the Sphagnum in these treatments despite the fact that there were no visible damage symptoms in 2005 and 2008 in the wet plots. In contrast, along the NH3 transect S. capillifolium was in a very poor state, which was also reflected in enhanced amino acid concentrations.
Objectives: Amino acid concentrations in S. capillifolium were analysed in 2005 and 2008, respectively 3 and 6 years after the start of the experiment. Since 2008, measurements of amino acid concentrations, base cation content and total carbon and N content of the plants have unfortunately not been repeated. We propose to reanalyse the amino acid content, base cation content and C and N content of the Sphagnum mosses in 2013 (11 years after start of the experiment) in order to obtain a full understanding of the physiological responses to long term application of the 3 different N forms. We aim to relate the long term application of different N forms to S. capillifolium to N-rich aminoacid content, base cation content, C and N content and vitality of the species.

Effects of long term addition of reduced and oxidised nitrogen on dinitrogen fixation and uptake of phosphorus and potassium by Sphagnum – microbe associations in peatlands.

TA User (visit): Eva van den Elzen, Radboud University Nijmegen, The Netherlands (12 August, 2013 – 3 days).
Project Description: Peatlands provide a very important, but at the same time fragile terrestrial carbon sink, and are therefore important with respect to global change. Research at the IWWR in Nijmegen has shown that the keystone genus (ecosystem engineer) Sphagnum in peatlands is able to outcompete other species by monopolizing nutrients and carbon in a very efficient way (Lamers et al., 1999; 2000; Fritz et al., submitted). This unique capacity seems, to a large part, be caused by its complex mutualisms with microbial partners, present on the surface and in the hyaline cells of the mosses.The mutualisms include photosynthetic carbon acquisition from methane, internally oxidised to carbon dioxide by methanotrophes (Smolders et al., 2001; Raghoebarsing et al., 2005; Kip et al., 2010), and dinitrogen (N₂) fixation from the atmosphere by cyanobacteria (Kip, 2011) and a large array of other bacteria that are capable of N₂ fixation. How this association between Sphagnum and N₂ fixing bacteria works, which environmental factors affect it, and what the effects are on the acquisition of other nutrients such as phosphorus (P) and potassium (K), is largely unknown.Peat bog ecosystems are likely to be highly sensitive to enhanced nitrogen (N) inputs, since their stability depends on their vegetation including symbiotic microbes evolved under conditions of restricted nitrogen inputs (Bobbink et al., 1998). Recent studies in pristine Canadian boreal peatland showed that nitrogen deposition decreases N₂ fixation of the microbial community (Vile et al., subm.; Popma, 2012).The Whim bog field study (CEH) is a long term (since 2002) nitrogen application experiment in an ombrotrophic bog site. At a relatively low background N deposition a free air release of NH3 and additions of NO3- and NH4+ in rainwater have been applied. In addition, and unlike in many other experiments focusing on airborne N effects, effects of total NPK addition have also been tested. The effects of these different N forms on biogeochemistry and physiological processes in Sphagnum peat have been studied since 2005 in active collaboration between CEH Edinburgh (Dr. L Sheppard and Mr. I Leith) and Radboud University Nijmegen (Prof. J Roelofs and Dr. L van den Berg).We now aim to add to these studies by studying the effects of different forms of N-deposition and additional PK on total nutrient dynamics of Sphagnum peatlands, including dinitrogen fixation ability, phosphate and potassium uptake rates, and the composition of the microbial community that lives in association with Sphagnum.

Cellular and enzymatic activity and proteomics in N-treated lichens.

TA User (visit):Silvana Munzi, University of Lisbon, Portugal (April/August, 2013).
Project Description: Although several studies exist about lichen physiology in relation to nitrogen (N) availability, few of them investigated the consequences of short- and long-term exposure to N at cellular and molecular level. The main aim of this project is to contribute to fill this gap.
The Whim Bog, with N manipulation experiment which has been providing wet and dry deposition to ombrotrophic bog vegetation containing the matt forming lichen Cladonia portentosa, showed to offer suitable conditions to study both long-term and short-term effects. Our previous experiments carried out at the site allowed the ecophysiological consequences of long-term exposure on in situ thalli of C. portentosa and of short-term exposure on transplanted thalli of the N-sensitive Evernia prunastri and the N-tolerant Xanthoria parietina to be studied. Results provided important clues mainly on the relation between N availability and pH changes in lichens, the different effects of dry and wet N depositions, and the role of phosphorus and potassium in alleviating N toxicity (papers in preparation). We retain that a more exhaustive comprehension of the cascade of reactive N through the ecosystem cannot leave molecular and metabolic analysis out of consideration. Consequently, in order to answer the question: what are the cellular processes that determine the physiological response to and the ecological impacts of increased availability of reactive N, we propose:

• to repeat the collection of in situ material for long-term and the lichen transplants for short-term exposure,
• to verify the physiological performance of the samples,
• to observe (transmission electron microscopy) the ultrastructure of the two symbionts,
• to perform enzymatic essays,
• to make proteomic profiling of selected samples, and
• to integrate the data linking cellular and molecular observations, physiological response and ecological consequences.

Isoprene emissions and pigment content in Sphagna from peatlands (ICARO)

TA User (visit): Carlos Raul Ochoa Hueso, Rey Juan Carlos University, SPAIN (August, 2012).
Project Description: Anthropogenic reactive nitrogen (N) deposition can be detrimental to the sustainability and functioning of semi-natural terrestrial ecosystems across the globe. Ombrotrophic peatlands, dominated by mosses such as Sphagnum spp. represent major carbon (C) reserves, which play a very important role in mitigating the effects of climate change through C storage, a consequence of decomposition rates being slower than rates of C fixation. Increasing the N availability in such ecosystems can compromise their ability to store C. For example, it has been demonstrated that increased N deposition can reduce the phenol content in peatland mosses, accelerating litter decomposition and, therefore, reducing C sequestration. Increased N deposition can also increase photosynthetic and photoprotective pigment content in mosses, including chlorophylls and carotenoids. Carotenoids are photoprotective pigments made of isoprene monomers. The early stages of the carotenoid biosynthesis pathway also lead to the synthesis of isoprene in some plant species, including Sphagnum spp. When emitted to the atmosphere, isoprene can significantly contribute to the net C balance between a given ecosystem and the atmosphere and also interact with the local climate. In this context, we are interested in analyzing the pigment content (including carotenoids) of Sphagna in relation to isoprene emissions at Whim, an ombrotrophic peatland in the UK, where realistic doses of simulated N deposition are being regularly applied. Currently Dr. Lucy Sheppard’s group is measuring isoprene emissions (Dr Sue Owen) at Whim but have no facilities (HPLC) to look at different pigments. It will be complementary to measure the pigment content in Sphagnum species present at the field site to see if the variations in isoprene emissions and C balance can be partially related to changes in Sphagna pigment content. The plan would be to visit Whim for one week to do the field survey and then bring the samples back to my lab in Spain to run the samples with HPLC. This project would be highly beneficial for me as young scientist and for the group of Dr. Lucy Sheppard given that the measurements I am planning to do at Whim totally complement their research.
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Long-term effects of Nitrogen deposition in lichen ecophysiology.

TA User (visit): Silvana Munzi, University of Lisbon, PORTUGAL (June, 2012).
Project Description:The excess of reactive forms of nitrogen (N) from anthropogenic sources in the environment has been shown to have dramatic consequences for the structure and functioning of ecosystems and recently claimed to be at a global scale. Lichens are among the most sensitive communities to N pollution at the ecosystem level and several studies have investigated the relationships between lichens and N. However, the ecophysiological long-term consequences of the exposure of both sensitive and tolerant lichen species to N are not completely understood. The Whim Bog, with N manipulation experiment, which has been providing wet and dry deposition to ombrotrophic bog vegetation containing the matt forming lichen Cladonia portentosa, offers the potential to study both long term and short term effects.
We intend to answer the following questions:
1) What are the ecophysiological differences between long-term and short-term lichens exposed to N? We will approach this question by putting lichen transplants next to long-term exposed ones and make a series of ecophysiological performance tests in samples exposed for different periods: 1, 3, 6, 12 months and > 9 years. This will entail an initial visit to the site to set up the transplants and remove samples while the site operators will send us subsequent samples.
Some information derived from short-term laboratory studies have shown that intracellular K concentration might alleviate the N toxicity symptoms in some tolerant lichens. Moreover, other observations at the Whim site have shown that lichens tolerate more N when they are simultaneously exposed to K and/or P. Thus, another question will be:
2) Are K and/or P alleviating the N toxicity symptoms? For this, we will investigate the ecophsyiological performance of lichens (in situ lichens, growing on board walks, and/or lichen transplants) exposed to similar N concentrations but with an external supply of potential alleviators (K and P).
The ecophysiological performance will be measured by evaluating: the Fv/Fm ratio, N location by sequential elution, intra- and extra-cellular K and P and other parameters that will be found to be relevant during the study.
Expected outputs are scientific papers involving both the teams from Portugal and UK.
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Output: Proceedings of the annual congress of the Italian Lichen Society, published on the Notiziario della Società Lichenologica Italiana, ISSN: 1121-9165 (page 53).
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