Amphitrema flavum 3 no 18S rDNA sequenceArcella sp. 1 no 18S rDNA sequenceAssulina muscorum 55 AJ418791 Assulina seminulum 41 no 18S rDNA sequenceBullinularia indica 28 no 18S rDNA sequenceCetropyxis aerophila 1 no 18S rDNA sequenceCentropyxis laevigata 39 no 18S rDNA sequenceCorythion dubium 18 no 18S rDNA sequenceEuglypha compressa 50 no 18S rDNA sequence (G)Euglypha laevis 1 no 18S rDNA sequence (G)Euglypha strigosa 8 no 18S rDNA sequence (G)Heleopera sylvatica 2 no 18S rDNA sequenceHyalosphenia elegans 55 no 18S rDNA sequenceHyalosphenia minuta 2 no 18S rDNA sequenceHyalosphenia papilio 56 no 18S rDNA sequenceNebela griseola 1 no 18S rDNA sequenceNebela miltaris 3 no 18S rDNA sequenceNebela tincta 57 no 18S rDNA sequencePhryganella acropodia 3 no 18S rDNA sequencePseudodifflugia gracilis 42 AJ418794

(Mitchell et al. 2000)

Phylogeny of Euglyphid Testate Amoebae

0.01

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

MO

NA

DO

FILOS

AOrderEUGLYPHIDA

DNA samples and PCR with EUK-primers

300-303 (triplicates) A312-315 (triplicates) B intermediate324-327 (triplicates) C tendency ombrotrophic336-337 (triplicates) D intact

300 312 324 336 N 300 312 324 336

1 µl DNA (1:10) 2 µl DNA (1:10)

Design of Euglyphida-specific PCR primers for 18S rRNA gene sequences

Filosea100fFilosea1170rFilosea1630rFilosea1700r DNA 336 (D) 1:20, 1µl, 35 cycles

300 312 324 336 N 300 312 324 336

1 µl DNA 2 µl DNA

Filosea 100f-1170r Filosea 100f-1630r Filosea 100f-1700r51 54.2 56.2

57.351 54.2 56.2 57.3

51 54.2 56.2 57.3

PCR on DNA from different sites: 100f-1630r

Samples 312 (B intermediate) 1µl from each triplicate pooled > 1:20 > 1ml for PCR (30c)

Samples 336 (D intact)1µl from each triplicate pooled > 1:20 > 1ml for PCR (30c)

300 312 324 336 N 300 312 324 336

1 µl DNA 2 µl DNA

49.9 51 52.4 54.2 56.2 57.9 59.2 61 49.9 51 52.4 54.2 56.2 57.9 59.2 61

312 B 336 D

PCR on DNA from different sites: 100f-1700r

Samples 336 (D intact)1µl from each triplicate pooled > 1:20 > 1ml for PCR (35c)

49.1 49.9 51 52.4 54.2 56.2 57.9 59.2 61

Cloning and sequence analysis

• Samples 336, 337, 338 (D intact)• 1µl from each triplicate pooled > 1:20 > 1ml for PCR

(30c)• Each pooled DNA amplified (4x) with both primer sets• Separate clone libraries

First Results• Primer sets seem to be specific• Sequence identities of clone sequences (500bp)

– Trinema enchelys 95-98% – Euglypha rotunda 93-98% – Assulina muscorum 94%– Euglypha tuberculata 94-95%

Next steps

• Cloning DNA from all sampling points A-D in Chaux-d’Abel

• Use sequence data to– Perform phylogenetic analysis of the Euglyphida-

communities– Design probes for Fluorescence in situ Hybridization– Compare sequence data with sequences from isolates

(?)– Compare molecular and direct observation approach

• Use sequence data from non-Euglyphida isolates for new primers (?)

• Develop a fingerprint screening method