Molecular phylogeny of

the Arcellinida

Enrique LARA, Thierry J. HEGER, Flemming EKELUND,

Mariusz LAMENTOWICZ, Edward A. D. MITCHELL
Why work on the phylogeny of
testate amoebae in RECIPE?

 Initial plan: to study the diversity of protists

using molecular methods
– Focus on testate amoebae, the dominant group of
heterotrophic protists in peatlands
 Problem: almost no molecular data (DNA
sequences) on testate amoebae
 => Need for baseline data: sequencing
dominant species and establishing the
phylogeny based on molecular data
General characteristics of testate
amoebae

 Size: 10-300 µm

 Produce a shell (proteinaceous material or

agglutinated mineral particles)

 Feed on bacteria, fungi, micro-algae, rotifers, etc.

 Often narrow ecological tolerance

=> useful for ecology and paleoecology
 Testate amoebae are polyphyletic

The Arcellinida The Euglyphida

Lobose Filose pseudopodia

pseudopodia

(1) Adl, S.M. (2005), J. of Eukaryotic Microbiol.

Family Hyalospheniidae
(sensu Schultze, 1877)

 Includes 6 genera among

them Nebela (sensu lato),
Hyalosphenia and
Heleopera

 Are especially abundant and

diverse in peatlands
 Methods

 Isolation of 10-20 living amoebae from each

species under inverted microscope
 DNA extraction
 PCR with newly designed Arcellinida – and
Hyalospheniidae specific primers

 Sequencing of SSU rRNA gene

11 studied species from 4 genera

 Genus Nebela:  Genus Hyalosphenia:

N. carinata H. elegans
(2 geographical origins) H. papilio
N. penardiana
N. tubulosa  Genus Apodera
N. tincta tincta A. vas
N. tincta major
(2 geographical origins)  Genus Heleopera
N. flabellulum H. rosea
N. lageniformis
Results

 All species are

clearly genetically
distinct
 Paraphyly of
genera Nebela and
Hyalosphenia

ML tree, 100 bootstraps, ln(L)=-2646, 918 sites

 QuickTime™ et un décompresseur
Photo - JPEG sont requis pour visualiser
cette image.

A hard to sequence insertion yields

precious phylogenetic information

 An insertion of about 450

bp is present in the SSU
rRNA gene of the
Hyalospheniidae.
 By aligning the sequences
with the insertion, it is
possible to resolve the
phylogenetic position of
closely related taxa.

ML tree, 500 bootstraps, ln(L)=-2621, 1406 sites

 CONCLUSIONS
Evaluation of the identification criteria used for
Hyalospheniidae taxa

 All morphospecies have so far proven to be genetically

distinct.
 Even subspecies are distinct!
=> what is the true diversity of testate amoebae?
 No evidence for geographical genetic variation
… at least in the SSU rRNA gene
Perspectives

 Ecology and paleoecology

– Further work on the phylogeny with other genera and
species:
=> resolve remaining taxonomic uncertainties

 Biogeography and evolution

– Variable genetic markers are needed to infer the
dispersal potential of testate amoebae
=> cosmopolitanism versus endemism of protists?
Acknowledgements

 Colleagues from Copenhagen University

 Colleagues from EPFL (Switzerland): Pierre Rossi, Christof Holliger

and Andy Siegenthaler

 Funding: EU project RECIPE, University of Copenhagen

 Many thanks also to the people who brought mosses samples from
all over the world (from Machu Pichu to Northern Sweden and
Marion Island!!!) and made this work possible
Position of the Hyalosphaeniidae inside the Arcellinida

ML tree, 100 bootstraps, ln(L)=-2744, 542 sites

 CCA plotting the different Sphagnum
species against environmental data in
peat bogs
S. capillifolium

S. magellanicum
Hummock
Other mosses
Water Table & other habitats
Depth

Conductivity
S. teres
S. fuscum

Fen
pH Lagg

S. recurvum
1 . 8
Lawn
Hollow - 0 . 7 4 3 . 8

- 1 . 5
S. cuspidatum
 The Hyalosphaeniidae as bioindicators

Ecological preferences of some

Hyalospheniidae in Sphagnum peatlands
Axis 2

Nebela militaris

Heleopera rosea

Nebela tincta tincta

Nebela flabellulum
Axis 1
Nebela bohemica
Hyalosphenia papilio

Hyalosphenia elegans

Nebela lageniformis
2.6
Lamentowicz & Mitchell
-0.81 3.2 Microbial Ecology, 2005
Nebela carinata -1.3

CCA analysis
Palaeoecology

 Palaeoecological diagram and reconstruction of water

table depth & pH (Mitchell et al. 2001 Holocene)
 Organisation
v4 of the SSU rRNA gene
Saccharomyces

Nebela
Insertion of 450 bp
 An insertion of about 450 bp is present in the SSU rRNA gene of the
Hyalospheniidae.
 This insertion is located at position 1200 in Saccharomyces pombe
SSU rRNA sequence (X58056)
 Also present at least in Bullinularia indica, probably in other species
as well
 Highly variable, therefore informative among closely related
species...
 Is extremely difficult to sequence, probably because of a complex
secondary structure
Phylogenetic relationships within
Hyalosphaeniidae
- Large species
- Rounded test base
Nebela carinata, N. penardiana
„Core Nebelas“ -Large species
-Pointed test base
Nebela tubulosa, N. marginata
Small, rounded species:
Nebela tincta tincta, N. tincta major,
N. flabellulum
Mixotrophic, proteinaceous test:
Hyalosphenia papilio
Symbiotic Chlorella-like
algae
Phagotrophic, proteinaceous test:
Hyalosphenia elegans

Elongated neck, more or less

constricted:
Nebela lageniformis, Apodera vas

Outgroup: Heleopera rosea

 CONCLUSIONS
Evaluation of the identification criteria used for
Hyalospheniidae taxa

 Shell shape most reliable for classification into major groups…

… but shell composition is NOT sufficient for defining a genus (here
genus Hyalosphenia)
• The presence of a carenated ridge is a criterion which
could separate very closely related species
– ... if this is not a case of phenotypic plasticity
(ex: N. marginata/N. tubulosa);
– Parallel example: the spines of the cercozoan
testate amoebae from the genus Euglypha.