1. Deliverables D9 Genetic diversity of protistan groups 1. Develop 18S rRNA gene based detection of selected protistan groups (testate amoebae) 2. Develop and apply 18S rRNA gene based screening tool for testate amoebae

2. Phylogeny of Euglyphid Testate Amoebae 0.01 Euglypha rotunda Euglypha filifera Euglypha tuberculata Euglypha rotunda Euglypha acanthophorea Euglypha filifera Assulina muscorum Trachelocorythion pulchellum Trinema enchelys Tracheleuglypha dentata Cyphoderia ampulla Paulinella chromatophora Thaumatomonas sp. Cercomonas caudatus MONADOFILOSA Order EUGLYPHIDA 18S rRNA gene detection

3. Trinema cluster E.rotunda cluster Assulina cluster Trachelocorytion cluster Chaux-d`Abel A B C D 18S rRNA gene detection

5. PCR with fluorescently labeled primers Enzymatic digestion Fragment separation on sequencing gel DNA extraction Terminal Restriction Fragment Length Polymorphism (T-RFLP)

6. Bray-Curtis similarity of T-RFLP profiles from Chaux-d`Abel 1 2 3 4 5 6 7 8 9 0,1 0,20,30,40,50,60,70,80,9 1 336 337 338 303 302 314 326 325 A B C C D Correlation needed with plant cover, restoration stage and direct observation Profiles available for Schottland (A, C), France Russey (C, D) 18S rRNA gene diversity screening

7. PROBLEMS Only few DNA samples amplifiable No PCR-products at all with WP2 DNA samples > consequences for T-RFLP screening of most RECIPE sites > how to continue? Other important (more important) groups missing! Further deliverables decided during the RECIPE meetings Clone libraries using primers targeting all eukaryotes > not very useful; mostly „non-target“ (non-protistan) sequences Clone libraries using primers targeting all ciliates > done for Chaux-d`Abel > is information/additional screening on ciliate community structure usefull for functional microbial diversity analysis/which manuscripts could profit?

8. Deliverables D9 Genetic diversity of protistan groups 3. Develop FISH probes for the visualization, detection and enumeration of selected protistan groups (testate amoebae)

9. unstained cellsstained target cells -ACTGGCGTAAGTCAG- TGACCGCATTCAGTC Hybridization Fluorescence in situ Hybridization (FISH)

10. Accessible sites for FISH oligonucleotide probes Amplified region approx. 500 bp Detection by FISH

11. ...Make it accessible for FISH oligonucleotide probes!! > Use of unlabelled oligonucleotide “helper” probes (Fuchs et al., 2000) Detection by FISH

12. Trinema cluster E.rotunda cluster Assulina cluster Trachelocorytion cluster Chaux-d`Abel A B C D (E.strigosa/ciliata) ? Detection by FISH

13. Not yet delivered Screening of bulk samples Using picked organisms to test specificity Find organism behind abundant 18S rRNA gene sequences not available as cultures Top to bottom approach for the first screening > if necessary design of more specific probes with target sites on the complete 18S rRNA gene sequence FISH probed for selected sequenced testate amoebae... No probes yet for Assulina; potential sites for Tracheleuglypha dentata and Thaumatomonas clade Alternative to T-RFLP screening of RECIPE sites?