WPI: How to sample in the field and dispatch. (after Charquemont, vers ISTO C, N, S (LECO) takes 0.05 g dry material of MLURI Micromorphology 2-3 cm3 of bulk in small beakers (no mix) Samples 3 to 8 Fixing in GA (fill with 2% conc) Shipping Monosach. + Lignin 100 g fwt Samples 2 to 8 Shipping in small containers with isolated ice pack wrapping Deadline: 1-2 weeks at 40C First, for density: vol. of slice (known) + weight of slice + dry weight 105 0C of subsample 1 MLURI CLPP + FTIR 60-100 g fwt Samples 2 to 8 For FTIR, drying in Aberdeen 2x 0.05 g dwt Shipping in PE bags with isolated ice packing wrapping (no direct contact to prevent freezing) Deadline:40C during 14 days TUM-BO PLFA + DNA -80 g fwt + 10 g fwt in separate PE bags - Samples 2 to 8 deep freezing immediately after sampling, shipping under frozen conditions on dry ice DNA extractions 2 3 x Mix of 3 parts within each slice for each purpose 3 x 3 4 6 8 5 5 and 7 are spared samples 2 may not always occur, 3 may be scarce, so take enough nearby 7 UFZ, formerly EPFL DNA Takes the extractions of GSF Only sample 2 and 3 for protists FISH-protists Sample 2/3: reversed beakers of 75 ml (scissors and grapefruit knife), fixing onsite with PFA (50 ml. and conc ?) Shipping immediately with isolated ice pack wrapping FISH-bacteria Samples 3 to 8 for bacteria 3 g fwt Fixing in PFA (vol and conc.?) Shipping in centrifugation tubes with screw cap. Take outside the core in same situation ECOBIO C, N, fumigation + in vitro incubations 275 g of 3 to 8 Shipping in PE bags with isolated ice pack wrapping (no direct contact to prevent freezing) Deadline:40C during 14 days Rotifers Reversed beaker of 75 ml for samples 2,/3 and 4 (scissors and grapefruit knife) Keep the water and cap Shipping immediately with isolated ice pack wrapping UFC-CE, AR-WSL Bacteria, protists, fungi, micrometazoa Sample 2/3: reversed beaker of 75 ml (scissors and grapefruit knife), fixing onsite with 50 ml GA 2% final conc. Samples 4 to 8: 1 cm3 in center of slice, in small containers, fixing in 10 ml GA 2% final conc. Keep in dark Shipping immediately with isolated ice pack wrapping

Field procedure: Choose for each gaz collar (maximum for 3, the number of replicates), in comparable vegetation, the spot for the core drilling, and the spot for the surface sampling. Mind the trampling, keep away from the collar and the surface where the vegetation will have to be recorded (approx. 2m or more) On the spot for surface sampling, take samples for rotifers (ECOBIO), bacteria and others (UFC-CE, AR-WSL), and FISH-protists (UFZ) (arrows in violet) Collect around more litter (sample 2) for adding in next steps (PE bags brought to the laboratory), if necessary also an intact piece of moss carpet (sample 3) Take out the core with the corer (>50 cm length, including the living vegetation), bring it to the laboratory in the transparent plastic tube, after having caped tight the lower end.Mind to carry the core in natural position. Slice the core, according to the slicing guidelines (samples 3 to 8). Be precise for volume: 5 cm trenches Weigh the whole slice Take a small fwt subsample (pool of 3) for dry weight measurement (gives the % water content and allows to calculate the apparente density of the slice) In the next steps, the samples 5 and 7 will be spared without analysing Take 1 cm3 as bulk peat in center of slice for bacteria and others (UFC-CE, AR-WSL), weigh the sample Take 3 g fwt (pool of 3 x 1 g) for FISH-bacteria (UFZ) Take 2-3 cm3 as bulk peat for micromorphology (ISTO) Take 100 g fwt (pool of 3 x 33 g) for monosach+lignin (ISTO) Take 60-100 g fwt (pool of 3 x 20-33 g) for CLPP+FTIR (MLURI) Take 80 g fwt (pool of 3 x 27 g) for PLFA (TUM-BO) Take 10 g fwt (pool of 3 x 3.3 g) for DNA+DNA (TUM-BO) Take 275 g (pool of 3 x 90 g) for C, N, fumigation and incubation (ECOBIO) Sampling for microorganisms (point 2, surface samples) will be repeated: autumn 03, spring, summer and autumn 04

Questions: Sample 1 and 2 seem not to be attraktive to people! My guess is that Fatima would like to have them for C, H, N elemental analysis and for biochemical markers May be Edward would like to see what organisms are in there GA or FA or PFA for microorganims? If all could use PFA, then we would be sure to work exactly on the same subsamples Needed quantitiees in each layer (samples 3 to 8): The total needed material is approx. 500 g and 80 cm3 of fresh peat. Andy has calculated that with the smallest sampler (slices of 5 cm height and 134 mm diam.), one would get either 705 cm3 or 634 g (with a wet volumic mass of 0.9 g/cm3) This means that we will have to be precise in the shipped quantities

For me, the fixative used does not make much difference For me, the fixative used does not make much difference. I know it does for other protists or bacteria (UFC-CE0) and (UFZ, formerly EPFL). I would I expect to find only few species if any of testate amoebae in the standing biomass and fresh litter. I expect to find much clearer differences in sample 3, where most of the living individuals and highest diversity is to be expected. One last thing for those in charge of preparing the samples and sending them. Please make sure the samples arrive to me by late November. I will be absent six weeks (in Switerland) until mid-January and I am not sure I can trust people to put the samples in the cold room in my absence. Around new-year the US mail seems to decide to switch back to the ponney express delivery system and it is not rare even for letters to take 7 (yes seven) weeks to get to us. So please post the samples by priority mail and enquire about how long it will take for the samples to reach me. Also if you have an option for tracking the parcels, it might be worth doing this. Finally work on the basis that people are going to play football with the parcels. I think they really do quite often. Best wishes Edward

Hallo Alexendre, vielen Dank für deine Mühen um eine einheitliche, sinnvolle "sampling and dispatching strategy". Nachdem wir nun Hochmoorbohrkerne aus Schottland getestet haben, muss ich meine zuvor gemachten Angaben revidieren, ich hoffe, dass läßt sich mengenmäßig machen: for PLFA: 80 g fresh matter in a single PE bag for DNA: 10 g fresh matter in a single PE bag deep freezing immediately after sampling, shipping in PE bags under frozen conditions on dry ice!!! Vielen Dank nochmals und bis die Tage, Andreas Gattinger & Michael Schloter Team TUM-BIO

Alexandre raises an important point. Having more replicates could be very valuable if we find that we have a high variability among samples, which would not be surprising at all. Since we will homogenize the different samples before sub sampling we should avoid the problem of very fine scale heterogeneity, but we may still find a lot of variations that may or may not be explainable with the variables we will measure. However, we will only know this once we will have analyzed the samples, looked at other variables, and made numerical analyses, at which time it will be too late to take more samples in the same conditions. One approach might be, as Alexandre suggests, to take more cores. If we go this way I suggest that we keep them intact (frozen?) until we decide that we need to analyze them, if we do. If the data looks good with the first set of cores then we will not need to work on them, if not, then we have a "plan b" that give us a second chance to have better data. Of course this implies some more sampling work and storage space, which may be a problem. Best wishes Edward

Rebekka Artz a *crit : Dear all, re: Alexandre's question: I have done some preliminary fungal diversity analysis on some replicate cores I took from our Scottish site to look at exactly the variability between cores and found quite substantial differences in DGGE band patterns (in some cases less than 70% similarity!). These were most pronounced (as one might have expected) in the upper layers of the cores (living moss and slightly decaying moss layer) whereas older peat horizons showed much more similar band patterns. I will show the data set at Charquemont. I think it is very important to have at least an idea of the variability between cores. In addition, if we are considering differences between keystone species in terms of their effects on gas fluxes, I guess it would also make sense to look for such effects on carbon sequestration and diversity aspects too. We had discussed the other point Alexandre has made (about preserving the anoxic conditions in peat) here at Aberdeen, too. We felt here, that it would be worthwhile to try and adopt the CLPP methods to anoxic conditions to obtain a much more realistic picture of carbon utilisation. This may be a more long-term process as it would involve a potentially lengthy period of method development. I would be very interested in other people's views on this matter. Best wishes and looking forward to meeting you all, Rebekka Dear Alexandre, many thanks for the updated sampling scheme. Steve asked me to point out that we would be requiring 0.05 g dry weight for the FTIR analyses (just in case somebody wishes to have FTIR but no CLPP analysis performed on a sample).

Dear partners, We also agree to the suggestion made by Ed Mitchell, taking more samples and keeping/storing them as potential replicates. Best wishes from München, Andreas Gattinger & Michael Schloter