Molecular%20phylogeny%20of%20the%20Arcellinida

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Molecular phylogeny of the Arcellinida

• Enrique LARA, Thierry J. HEGER, Flemming EKELUND,
  Mariusz LAMENTOWICZ, Edward A. D. MITCHELL

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Outline

• Introduction
• Why study the molecular phylogeny of testate
  amoebae?
• Characteristics of the Arcellinida and open
  questions
• Methods
• Results
• Conclusions and perspectives

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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
• gt Need for baseline data sequencing dominant
  species and establishing the phylogeny based on
  molecular data

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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 gt useful for
  ecology and paleoecology

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The Arcellinida
Testate amoebae are polyphyletic
The Euglyphida
Filose pseudopodia
Lobose pseudopodia
(1) Adl, S.M. (2005), J. of Eukaryotic Microbiol.
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Family Hyalospheniidae (sensu Schultze, 1877)

• Includes 6 genera among them Nebela (sensu lato),
  Hyalosphenia and Heleopera
• Are especially abundant and diverse in peatlands

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Current challenges in the phylogeny and taxonomy
of testate amoebae

• Current classification is based on morphology
• The phylogenetic value of the morphological
  characters is uncertain.
• Phenotypic plasticity demonstrated for some
  species (1)
• Many taxa have uncertain value
• (1) Wanner M., Meisterfeld, R. (1994), Europ.
  J. Protistol. 30 (191-195)

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Ecological and palaeoecological implications of
taxonomic uncertainties

• Ecological studies require sound taxonomy to
  maximize the indicator value of the amoebae (2)
• gt Taxonomic uncertainties undermine the use of
  testate amoebae in ecology and paleoecology
• gt Need for a molecular phylogeny
• (2) Bobrov, A. A., Charman, D. J., Warner, B.
  G. (2002) Biology Bulletin29 605617

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Ecological and palaeoecological implications of
taxonomic uncertainties

• Similar morphology
• similar ecological
  tolerance?

Nebela tincta var. major
Nebela flabellulum
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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

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11 studied species from 4 genera

• Genus Nebela
• N. carinata
• (2 geographical origins)
• N. penardiana
• N. tubulosa
• N. tincta tincta
• N. tincta major
• (2 geographical origins)
• N. flabellulum
• N. lageniformis

• Genus Hyalosphenia
• H. elegans
• H. papilio
• Genus Apodera
• A. vas
• Genus Heleopera
• H. rosea

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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
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Evaluation of the identification criteria used
for Hyalospheniidae taxa
CONCLUSIONS

• All morphospecies have so far proven to be
  genetically distinct.
• Even subspecies are distinct!
• gt what is the true diversity of testate amoebae?
• No evidence for geographical genetic variation
• at least in the SSU rRNA gene

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Perspectives

• Ecology and paleoecology
• Further work on the phylogeny with other genera
  and speciesgt resolve remaining taxonomic
  uncertainties
• Biogeography and evolution
• Variable genetic markers are needed to infer the
  dispersal potential of testate amoebaegt
  cosmopolitanism versus endemism of protists?

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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

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Position of the Hyalosphaeniidae inside the
Arcellinida
ML tree, 100 bootstraps, ln(L)-2744, 542 sites
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CCA plotting the different Sphagnum species
against environmental data in peat bogs
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Ecological preferences of some Hyalospheniidae
in Sphagnum peatlands
The Hyalosphaeniidae as bioindicators
Axis 2
Nebela militaris
Heleopera rosea
Nebela tincta tincta
Nebela flabellulum
Axis 1
Nebela bohemica
Hyalosphenia papilio
Hyalosphenia elegans
Nebela lageniformis
Lamentowicz Mitchell Microbial Ecology, 2005
Nebela carinata
CCA analysis
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Palaeoecology

• Palaeoecological diagram and reconstruction of
  water table depth pH (Mitchell et al. 2001
  Holocene)

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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

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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
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CONCLUSIONSEvaluation 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.