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					 PEATBOG: CROSS-EUROPEAN SURVEY SAMPLING PROTOCOL


PEATBOG: impacts of environmental change on
peatland biodiversity and biogeochemistry

Objective
In approximately 40 locations across the European N deposition gradient, peatland floristic
diversity and biogeochemistry will be assessed. Sites are selected to span the range of
European       NHy    and     NOx    deposition    by   using    the     Unified   EMEP     Model
(http://www.emep.int/UniDoc/index.ht ml), compared where possible to national deposition
models (F ig. 1). We w ill ensure that there is a comparable range in SO 4 deposition values and
that climate co-correlations w ith at mospheric deposition are minimized. Each site will be
visited once during the growing season (June-August), for recording the vegetation and
collecting plant, water and soil samples for analysis and further experimentation (i.e. pH,
macro-ions in water, soil C:N and C:P, plant tissue C, N, and P, degree of peat humification).
Species composition and diversity will be recorded in hollows and hummock mic rohab itats
using five quadrants (50 c m × 50 c m) in each microhabitat. All vascular plants and bryophytes
will be identified to species as much as possible and their cover will be estimated. These values
will later be converted to species diversity and mean Elle nberg values for nutrients, acidity,
light and moisture. The water table w ill be measured with respect to the surface of the
hollows and hummocks. This sampling strategy will require approximately one to one -and-a-
half day per site.


Linking peatland plant and microbia l diversity to geogra phic climate and nitroge n
gradients
Generally, ecologists use two basic approaches for evaluating the effects of increased N
deposition on species richness and composition in descriptive field studies: historical and
spatial comparisons of plant diversity and composition of the vegetation. The spatial
comparison relies on spatial gradients in deposition levels across study sites, and change can
be regarded as a space-for-time substitution. In this study we will use this approach to
elucidate the effects of increases in temperature, precipitation and nitrogen deposition on
peatland plant species diversity and biogeochemistry. Moreover, vegetation surveys will be
made along geographically dispersed peatlands differing in their prevailing environmental
conditions (temperature, N deposition, precipitation).




                                                          Figure 1 Overview of the proposed field sites
                                                          for the cross-European field survey


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 PEATBOG: CROSS-EUROPEAN SURVEY SAMPLING PROTOCOL

Measureme nt protocol
1.   Logistics (e.g. meeting local authorities; find location) + plot selection (1 hour, 15 min)
2.   Quick site survey + description (1 hour)
     2.1. Geographical coordinates:
     2.2. Elevation:
     2.3. Area:
     2.4. Bog type:
     2.5. Surrounding:
     2.6. Distance to sea:
     2.7. Microhabitat characteristics
     2.8. Site quality:
     2.9. Environmental conditions (mean annual temp., mean annual precipitation, days of snow cover,
3.   Picture of the survey area + select plots (15 min)
4.   Pore water sampling (15 min)
     4.1. at site: pH, EC, DOC
     4.2. at lab: cations, anions

5.   Vegetation survey (6 hours)
     5.1. vascular plants: total cover; species specific cover (DOMIN scale); species diversity
     5.2. Sphagnum mosses: total cover; covers per section; species diversity
     5.3. lichens: total cover; cover per growth form; species diversity
     5.4. liverworts: present/absent (approximate cover if possible)
     5.5. Non-Sphagnum mosses: total cover; cover feather mosses/brown mosses (to species); species
         diversity

6.   Plant collection (45 min)
     6.1. Total vascular plant biomass (25 cm × 25 cm); Eriophorum vaginatum/Vaccinium oxycoccus
     6.2. Sphagnum rubellum or S. capillifolium (c. 25 shoots)
7.   Water tables (15 min)
8.   Bulk density (30 min)
9.   Microbial community (potentially incl. amoebe) (45 min)
10. Core for Pb dating (1 hour)


                                                                                    (= total 12 hours)




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 PEATBOG: CROSS-EUROPEAN SURVEY SAMPLING PROTOCOL



Sampling procedures
1. Plot selection
       At site, a representative area in the bog will be searched. Next, five hummock, plus their
adjacent       lawns/hollows will be selected. At these five hummock, and five lawns/hollows, 50 cm ×
50 cm plots will be laid out. These plots (n=10) will be intensively sampled. The five hummocks and
       lawns/hollows will be selected by (blindly) throwing five clearly marked bamboo sticks. Those
       hummocks and lawns/hollows closest to the sticks will be incorporated in the survey. In those
       cases where the stick land on top of the hummock, the most adjacent hollow will be chosen in the
       direction where the stick points toward.
2. Site description:
       2.1 Geographical coordinates (latitude/longitude, in degrees and minutes):
               Provide the coordinates of the approximate centre of the site. If the site is composed of more than
               one separate area, provide coordinates for each of these areas

       2.2 Elevation from map (in metres: average and/or maximum & minimum)
       2.3 Area (in hectares)
       2.4 Peatland type:
               Provide as much possible details on the character of the site. Ombrotrophic ra ised bog, blanket bog,
               Aapa mire, etc.

       2.5 Surrounding:
               Provide as much possible details on the surrounding landscape. Describe the surface area, general
               geology and geomorphological features, general soil types. Is the site part of a National park , what is
               the general surrounding land use, etc. Can we identify potential local sources for Nitrogen (<
               1km distance from the site)?

       2.6 Distance to sea (in kilometers)
       2.7 Microhabitat characteristics
             Measure hummock heights of the hummock included in the survey; estimate hollows and
       hummock cover at the survey area. Estimate the cover of mud-bottoms.

       2.8 Site quality
               Does the survey area look healthy? Is the site gripped? Are there any signs of human activity (turf
               cutting, etc.)?

       2.9 Environmental conditions
               Provide as much as possible information on the local climate:
               - mean annual temp.
               - mean annual precipitation
               - days of snow cover
               - deposition levels (N, P)

3. Picture of the survey area
       At each survey area a digital picture will be taken. Additionally, each individual plot will be
       photographed (make sure site name and plot number are visible in the picture).

4. Pore water sampling
       4.1     The most important geochemical conditions (pH, EC, DOC) will be measured from
       each plot at each site. Water samples will be taken at 5-10 cm depth, using Rhizon SMS
       soil moisture samplers (Rhizon SMS-5cm; Eijkelkamp Agrisearch Equipment, NL). Water samples
       will be collected using 50 ml glass vials. pH and EC of the water samples will b e determined
       immediately using a standard pH meter (Ag/AgCl2 electrode) and EC meter. DOC will be
       measured collorimetrical (at 254, 400, 465, and 665 nm) using a handheld photo -spectrometer.
       4.2    Additional sample will be preserved using chloroform and analysed later for total
       concentrations of cations and anions using an inductively coupled plasma emission
       spectrophotometer (ICP, Spectroflame).

5. Vegetation survey
       In order to explore the relationship between peatbog floristic biodiversity and ecosys tem
       functioning at multiple regional environmental conditions (e.g. temperature, N deposition,
       precipitation/evapotranspiration), and along micro -gradients (i.e. water table), at all sites
       plant, Sphagnum moss, non-Sphagnum moss, lichen and liverwort diversity will be recorded over
       the five replicated hummocks and lawns/hollows. Samples of mosses, liverwort, and lichen
       species which cannot be determined in the field will be collected and identified with the use of
       binoculars or a microscope.

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 PEATBOG: CROSS-EUROPEAN SURVEY SAMPLING PROTOCOL


       Vascular plants
       Total plant cover (% area) will be estimated on each plot. Next, individual species will be noted
       after which individual species cover will be estimated. We will use the Domin scale to estimate
       vascular plant cover. These values will be trans formed to Domin 2.6 (Curall 1987), in order to
       enable statistical and mathematical handling of the data.

       Sphagnum, non-Sphagnum mosses, lichens and liverworts
       For Sphagnum and the non-Sphagnum mosses we will estimate ground cover, using the
       Domin scale. First, total ground cover will be estimated (including bare peat surface), after
       which cover will be estimated for each individual species. Species which cannot be identified in
       the field, will be given a reference name and identified later. Similarly, ground cover of lichens
       and liverworts will be estimated. These will further be separated in growth forms, from which
       growth form cover is estimated. Samples will be collected and identified to the species (as much
       as possible) at later stage.

6. Plant collection
       6.1 Total vascular plant biomass: From each plot we will harvest all vascular plants from a 25 cm
       × 25 cm area. All plant material will be separated to plant functional type level, put in paper
       bags, and air dried for further analyses (see below).
       6.1 (add) form the collected plant biomass, we will separate Eriophorum vaginatum/Vaccinium
       oxycoccus for further analyses (C:N:P:K; see below). These analyses will be performed on leaf
       material only
       6.4 Sphagnum rubellum/S. cappilifolium: At          each site, we will collect 6 × 25 shoots of S.
       rubellum (or if absent; S. capillifolium). These    samples will be separated in three replicates
       which will be air dried and stored for analyses    C, N, and P. The other three samples will stored in
       cooling boxes and frozen within a week after       collection. These samples will be used to measure
       Chlorophyll concentrations.
       In the laboratory, all air-dried plant material (total vascular plant biomass, E. vaginatum, V.
       oxycoccus, and S. rubellum), will be dried at 70 °C for at least 72 h, ground and digested using
       sulphuric and salicylic acid. P an d K will be analysed using ICP. C and N will be analysed on this
       dried and ground plant material using a flash combustion technique (C/N analyser).
       The thawed S. rubellum samples will be ground. Chlorophyl will be extracted in MgCO 3-saturated
       dimethyl sulphoxide (DMSO) at 70 °C for 40 min. Absorption will be measured at 665.1 and
       649.1 nm, after which chlorophyll a and b concentrations will be calculated. To express
       chlorophyll concentrations per unit dry weight, the solvent will be evaporated and the plant
       samples will be dried at 70 °C for 72 h.
       Additionally, soluble amino acids (N AA : N stored a amino acids) will be analysed as they reflect
       excess N better than total nitrogen (Wiedermann et al. 2009). Amino acids will be extracted from
       fresh (thawed), ground Sphagnum samples using 10 mM HCl. N AA will then be analysed on a
       HLPC following Nordin & Gunnarsson ( 2000).

7. Water tables
       Water table will be measured at each plot, using the bore -holes for the Pb-dating (see 10).

8. Bulk density
       At each plot, a small core (5 cm diameter, 10 cm deep) will be collected. These cores will be dried
       and weighed in order to calculate peat bulk density. These cores can also be used to measure
       peat chemistry (C, N, P, K; see 6).

9. Microbial community
       From each site, we will collect five samples from the top peat after removal of the active moss
       layer, and five “close to the water table” samples. These samples will be taken at the same
       location were vegetation surveys have been performed earlier. Sample size: 5 cm depth and with
       a diameter size 4-10 cm (not so important). The samples will be brought to the laboratory
       (a.s.a.p.), while kept at as low temperature as possible , and frozen at -20°C. The samples will be
       sent to Linköping University for microbial community analysis.

10. Pb dating
       Peat cores (10 cm diameter, 50 cm depth) will be collected from 20 sites along the WP1 transect
       for dating by use of 210Pb. The peat co res (one hollow and hummock at each site; 40 cores in
       total; alternatively 40 hollow, i.e. at all sites, cores will be taken) will be collected using a

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  PEATBOG: CROSS-EUROPEAN SURVEY SAMPLING PROTOCOL

        Finish corer and shipped to the laboratory of the PEATBOG partner in Bayreuth (Germany),
        where they w ill be sectioned at 5 cm intervals and the sections sent to Flett Research Ltd.,
        Winnipeg, Canada, for dating. After spiking a dry 0.5 g subsample with a 209Pb yield tracer,
        210Pb activity will be determined by the measurement of the α -emitting 210Po. To determine the
        age of the samples, the constant rate of supply (CRS) model will be used. With this method, an
        age resolution of ±10 years can be achieved. By measuring the carbon and nitrogen pools and
        the age of the cores, carbon and nitrogen accumulation rates will be calculated.




Credit
--
Dr. Bjorn JM Robroek
Ecology and Biodiversity Group
Institute of Environmental Biology, Utrecht University
Padualaan 8, 3584 CH Utrecht, The Netherlands
Email address: b.j.m.robroek@uu.nl
Tel: +31-(0)30-253 6190
http://www.researcherid.com/rid/C-4379-2008




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