Download Thierry Heger/Edward Mitchell

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
Lobose
pseudopodia
The Euglyphida
Filose 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:
N. carinata

Genus Hyalosphenia:
H. elegans
H. papilio

Genus Apodera
A. vas

Genus Heleopera
H. rosea
(2 geographical origins)
N. penardiana
N. tubulosa
N. tincta tincta
N. tincta major
(2 geographical origins)
N. flabellulum
N. lageniformis
Results


All species are
clearly genetically
distinct
Paraphyly of
genera Nebela and
Hyalosphenia
ML tree, 100 bootstraps, ln(L)=-2646, 918 sites
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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
& other habitats
Water Table
Depth
Conductivity
S. teres
S. fuscum
Fen
pH
Lagg
S. recurvum
Lawn
Hollow
1 . 8
- 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
Nebela carinata
CCA analysis
-0.81
3.2
-1.3
Lamentowicz & Mitchell
Microbial Ecology, 2005
Palaeoecology

Palaeoecological diagram and reconstruction of water
table depth & pH (Mitchell et al. 2001 Holocene)
Organisation
of the SSU rRNA gene
v4
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
„Core Nebelas“
- Rounded test base
Nebela carinata, N. penardiana
-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.