Progress report ARWSL EPFL ECOS Andy Siegenthaler Emanuela

Progress report AR-WSL, EPFL ECOS Andy Siegenthaler Emanuela Samaritani, Edward Mitchell, Alexandre Buttler RECIPE Meeting Scheyern 17 -19. 05. 2005 RECIPE meeting Scheyern 17 -20 May 2005

Achievements since last meeting Andy (A), Edward (Ed), Emanuela (Em) • A&Em: Methane emission in Chaux d’Abel, fall 2004 • Em: Photosynthesis and respiration measurements in Chaux d’Abel were started in May 2005 • A&Em: CH 4 -CO 2 depth profiles continues (peepers) - Data set now complete for 2004. • A: Development of a new method for measuring soil respiration under realistic conditions • A&Em: Soil respiration and methane production measurements in WPIII to test the new method developed by Andy • A&Em: Soil respiration and methane production measurements in WPI: Selective activities measured using specific inhibitors RECIPE Meeting Scheyern 17 -19. 05. 2005

Achievements - continued Andy (A), Edward (Ed), Emanuela (Em) • A&Em: Dendrochronological dating of the onset of regeneration • A&Em: Testing probes and protocols for analysing methanogenic Archea using FISH and CARD-FISH • Ed: WPI: Analyses of testate amoeba community structure in the Chaux d’Abel and Russey sites completed, ongoing for others sites • Ed: Establishment of the phylogenetic position of the Arcellinida (one of the two groups of testate amoebae) - Protist, in press • Ed, n. b. with Enrique Lara: Isolation of more Arcellinida species in view of continuing the phylogenetic work to increase the number of sequences in the data base. This will make it possible to analyse testate amoebae community profiles using molecular methods (c. f. also work by Antonis on the Euglyphid testate amoeba) RECIPE Meeting Scheyern 17 -19. 05. 2005

Dendrochronology Collection and preparation of birch cores, December 2004 Microscopic counting and measurement of tree rings, March 2005 RECIPE Meeting Scheyern 17 -19. 05. 2005

Birch recolonisation after abandonment La Chaux-d’Abel c Years (a) b a Regeneration stage Method: tree cores collected from the oldest birch trees found at the shortest distance from the centre of a selected stage Statistics: N=11, Kruskal-Wallis followed by Mann-Whitney RECIPE Meeting Scheyern 17 -19. 05. 2005

Gas profiles measurements Cylindrical peepers RECIPE Meeting Scheyern 17 -19. 05. 2005

Cylindrical peepers Pipe to let air get down when peepers are retrieved HT-Tuffryn membrane 50 mm (mesh: 0. 2 µm) sealed to the PE cap with drinkable-water Coltgum Transparent PET 0. 5 Removable handle mm sheet to reduce possible leaching from the wood Cylinder (1 m) PE cap Acryl 50 ml pot with water-tight PE cap Point 20 cm Insertion for the pot and the cap 9/17/2020 Power-tape A. Siegenthaler 7

Dissolved gases at different depths and stages (peepers) Chaux d’Abel, December 2004 RECIPE Meeting Scheyern 17 -19. 05. 2005

Dissolved gases at different depths and stages (peepers) Le Russey, autumn 2004 RECIPE Meeting Scheyern 17 -19. 05. 2005

Stable isotopes in peeper’s water: method summary 1) Samples were taken from the headspace of the degassed peat solutions found in the dialysis chambers (peepers). We used very fine needles (0. 55 mm diam. ) to sample through the 1 cm thick septae. 2) Separation between CH 4 and CO 2 was made using a infrared gas chromatograph equipped with a Varian poraplot Q coated column together with plot fused silica (25 m x 0, 32 mm). 3) The isotopic (12 C-13 C)/12 C ratios were measured separately for CH 4 and CO 2 with help of a continuous-flow isotope ratio mass-spectrometer coupled to the gas chromatograph (GC-MS). RECIPE Meeting Scheyern 17 -19. 05. 2005

Delta 13 C at various depths and stages (peepers) - Analyses by Andreas Chaux-d’Abel, autumn 2004 Note: the CH 4 concentrations were too low to measure d 13 C at H 3 and, for A and C at H 4. RECIPE Meeting Scheyern 17 -19. 05. 2005

2 D-clustering d 13 C-CH 4 vs d 13 C-CO 2 A C B RECIPE Meeting Scheyern 17 -19. 05. 2005

Repartition of delta 13 C from dissolved gases (peepers), pooled depths D 13 C-CO 2(0/00) Chaux d’Abel - December 2004 a b c Stats: N = 20, error bars = +/- 1 std error, Kruskal-Wallis followed by Mann -Whitney RECIPE Meeting Scheyern 17 -19. 05. 2005

Soil respiration profiles : A new method of laboratory incubation RECIPE Meeting Scheyern 17 -19. 05. 2005

Development of a new method to study soil respiration in mixed conditions Some problems with standard basal respirations: Because of the difficulties to separate anaerobiosis from aerobiosis in cores that are an assemblage of micro-sites and because it is also artificial to incubate peat in either N 2 or air, we developed a method that allowed us to directly incubate the samples taken on-site. We measured all replicates of Jura sites at three different depths over a period of 15 days at 4 -6 days interval. The advantages of this method are: 1) reduced incubation times, 2) get as close as possible to peat atmosphere conditions, 3) no core disturbance to get reed of the roots, 4) no need of extreme incubations in either air or N 2, 5) drastic reduction in the time of exposition to atmospheric conditions different than in-situ ones, RECIPE Meeting Scheyern 17 -19. 05. 2005 6) increase of efficiency and cost effectiveness especially with further “selective” respirations using various microbial inhibitors.

The Monovette system RECIPE Meeting Scheyern 17 -19. 05. 2005

New setup for basal respiration Remove roots at one end of the Monovette 1. 7 cm peat cores sampling at 3 depths, from 3 replicates and in 3 stages Transfer into a 9 ml Monovette leave no air space Create artificial 1 ml headspace and 2 days incubation Flushing 1 ml of the triggered respiration Take 1 ml of gas for GC analysis, consider that loss Incubate the cores for 2, 6, 12, 18 days at 6°C Create an artificial 30% headspace seal properly Inject 2 ml of water or of inhibitor cocktail Short term, anaerobic and aerobic respirations RECIPE Meeting Scheyern 17 -19. 05. 2005

Selective inhibition cocktails RECIPE Meeting Scheyern 17 -19. 05. 2005

Preliminary tests for the WPIII basal respiration at depths in the control plots RECIPE Meeting Scheyern 17 -19. 05. 2005

WP 1: Anaerobic respiration of methanogens Chaux-d’Abel a b b N. b. Interestingly, surface respiration measured in fall 2004 showed greater respiration in stage C (advanced) ! RECIPE Meeting Scheyern 17 -19. 05. 2005

Depth profiles: origin of the difference in the overall anaerobic respiration - Chaux-d’Abel Twice as much respiration in site A at 25 and 45 cm depths, very significant. RECIPE Meeting Scheyern 17 -19. 05. 2005

Trend for the autotrophic methanogens Chaux-d’Abel (MA+) ° There seems to be less autotrophic methanogens activity in the intermediate stage. RECIPE Meeting Scheyern 17 -19. 05. 2005

Significantly lower basal respiration in the advanced stage Trend towards lower eukaryotic (E*) (all but fungi) respiration in the advanced stage Approx. -14% RECIPE Meeting Scheyern 17 -19. 05. 2005

Steady increase of the methanogens’ respiration - Le Russey b a a/b • Respiration is higher in the advanced stage and mainly occurring near the surface! • Not many significant results are found at le Russey, probably RECIPE Meeting Scheyern 17 -19. 05. 2005 because of the reduced number of replicates (3 x).

WPIII. Basal respiration at various depths and for specific litters - preliminary results Depths CO 2 respiration differs significantly among treatments only at one depth (H 6)! Respiration is lower for Sphagnum as compared to either Eriophorum treatments RECIPE Meeting Scheyern 17 -19. 05. 2005

Litterbag experiment - species and water table effects on D 13 C-CO 2 a b c a/b Position Method: surface gases collection with syringe after 8 min and 3 h 36 in the same dark chambers used for respiration. D 13 C analysed by A. Gattinger. Statistics: N = 9, error bars = +/- 1 std error(s), Kruskal-Wallis followed by Mann-Whitney RECIPE Meeting Scheyern 17 -19. 05. 2005

Description of microbial communities • Because of the variability throughout time, we must terminate the spring 2004 re-sampling for WPI community counts. • As soon as our data-set is complete, we will link microbial diversity/abundance with physico-chemical and gas production measurements and integrate them in multivariate statistical analyses. • CARD-FISH measurements should be finished by end of July. • According to the preliminary C-balance results, I would look for the important nominal variables (treatments) and reduce the number of samples for the experimental sites. • Greetings from India ! RECIPE Meeting Scheyern 17 -19. 05. 2005

Arcellinida phylogeny • Illustrations of the seven testate amoebae taxa analyzed : a & b : Heleopera sphagni (small green spheres inside the cyst are endosymbiotic algae), c : Nebela tincta var. major, d & e : Hyalosphenia papilio , f : Bullinularia indica, g : Trigonopyxis arcula, h : Arcella artocrea, i : Centropyxis laevigata. Scale bars indicate 50 µm. RECIPE Meeting Scheyern 17 -19. 05. 2005

Partial SSU r. RNA data Arcella artocrea Centropyxis laevigata Trigonopyxis arcula Bullinularia indica Hyalosphenia papilio Nebela tincta major • Mr. Bayes tree of partial SSU r. RNA genes, showing the monophyly of Arcellinida. Numbers at nodes indicate posterior probabilities for Mr. Bayes analysis (upper) and bootstrap values for ML analysis (lower). Heleopera sphagni RECIPE Meeting Scheyern 17 -19. 05. 2005

Tree of the Actin gene data Heleopera sphagni RECIPE Meeting Scheyern 17 -19. 05. 2005 Arcellinida

Euglyphid testate amoebae Arcellinida RECIPE Meeting Scheyern 17 -19. 05. 2005

Testate amoeba species richness in the Jura sites RECIPE Meeting Scheyern 17 -19. 05. 2005

CA of testate amoebae samples RECIPE Meeting Scheyern 17 -19. 05. 2005

Testate amoebae How will the community structure of different taxonomic group compare among the sites? RECIPE Meeting Scheyern 17 -19. 05. 2005

Publications • In press: – Nikolaev SI, Mitchell EAD, Petrov NB, Berney C, Fahrni J, Pawlowski J, 2005. The testate lobose amoebae (order Arcellinida Kent, 1880) finally find their home within Amoebozoa. Protist, 156(2): (In press). • In prep, data available: – Emilie, Daniel, Hung, Andy, Edward: Microbial community methodological paper – Andy: Respiration methodological paper – Andy: WPI - Respiration, CH 4 measurements with inhibitors – Andy: WPIII - Respirations and methanogenesis profiles – P. Steinmann, Andy: WPI - Modelling of gas profiles in peat – Fatima, Edward, Alexandre, Daniel G, etc. : WPI preliminary - peat OM, Bacteria, and testate amoebae in Jura sites RECIPE Meeting Scheyern 17 -19. 05. 2005

Publications (continued) • In prep, data partly available: – Andy, Emanuela: WPI Peepers gas profiles, delta 13 C – Andy, Emanuela: WPI Archaea community profiles FISH/CARDFISH – Emanuela, Andy, Mika: WPI Surface gas fluxes, photosynthesis, respiration, methanogenesis – Andy, Daniel Gilbert, etc. : Microbial community structure in WPI at all sites - Alone or with testate amoebae, vegetation and other data? – Andy, Daniel Gilbert etc. : WPI Microbial communities in the Jura sites - relationship with other data gases, FISH, vegetation… – Antonis, Edward: WPI Euglyphida diversity in Chaux d’Abel - comparison molecular and microscopic data RECIPE Meeting Scheyern 17 -19. 05. 2005

Publications (continued) • Planned – Emanuela: WPI Growth of Sphagnum and vascular plant biomass production at Chaux d’Abel – Edward, Enrique? ? ? : Further phylogenetic data on the Arcellinida • Possible / optional – Edward: WPI Paleoecology using testate amoebae RECIPE Meeting Scheyern 17 -19. 05. 2005

RECIPE Meeting Scheyern 17 -19. 05. 2005

RECIPE Meeting Scheyern 17 -19. 05. 2005

RECIPE Meeting Scheyern 17 -19. 05. 2005

Does under-pressure have a differential effect on microbial activity? “Mont-Blanc” 4808 m “Nanda Devi” 7828 m N=9. Peat samples were incubated in under-pressured 9 ml air tight syringes for a period of 22 days. RECIPE Meeting Scheyern 17 -19. 05. 2005