Phylogeny of Euglyphid Testate Amoebae

1
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 gt 120 gt 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
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-dAbel
• Testing signature sites for their suitability to
  design probes for fluorescence in situ
  hybridization (FISH) gt 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 Edwards samples gt design
  FISH probes
• Using clones to evaluate alternative
  fingerprinting technique DGGE or SSCP