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

2. Cloning and 18S rRNA gene sequence analysis • Samples 336, 337, 338 (D intact) 312, 313, 314, 315 (B intermediate) • 1µl from each triplicate pooled > 1:20 > 1 µl for PCR (30c) • Each pooled DNA amplified 4x • Separate clone libraries • Clones screened by Amplified Ribosomal DNA Restriction Analysis (ARDRA) 49.1 49.9 51 52.4 54.2 56.2 57.9 59.2 61

3. Screening clones and 18S rRNA gene PCR-products • Amplified Ribosomal DNA Restriction Analysis (ARDRA) Enzymatic digestion • Terminal Restriction Fragment Length Polymorphism (T-RFLP) Enzymatic digestion 100 200 300 400

4. Euglypha rotunda Euglypha rotunda Euglypha filifera Euglypha tuberculata Euglypha rotunda Euglypha rotunda Euglypha acanthophorea Euglypha filifera Assulina muscorum Trachelocorythion pulchellum Trinema enchelys Separating different organisms by ARDRA MspI HinfI

5. Sequence analysis of clone libraries from 336-338 Sequence identity between clones 91-100% Sequence identity between clones and closest related organisms 96-97% (91%) Cluster A n = 11 n = 5

6. Ongoing work • Finishing comparative sequence analysis for site B (intermediate) and D (intact) in Chaux-d’Abel • Testing signature sites for their suitability to design probes for fluorescence in situ hybridization (FISH) > quantification in fixed samples of e.g • Assulina muscorum Group • Trinema enchelys Group • Euglypha acanthophora/Euglypha filifera • Cluster A • Evaluation of enzymes for T-RFLP analysis Future work • Sequence analysis of Edward’s samples > design FISH probes • Using clones to evaluate alternative fingerprinting technique: DGGE or SSCP