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

The Arcellinida

Filose pseudopodia

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

The Euglyphida

Testate amoebae are polyphyletic

Lobose pseudopodia

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 (2 geographical origins)

N. penardianaN. tubulosaN. tincta tinctaN. tincta major (2 geographical origins)

N. flabellulumN. lageniformis

Genus Hyalosphenia:H. elegansH. papilio

Genus ApoderaA. vas

Genus HeleoperaH. rosea

Results

All species are clearly genetically distinct

Paraphyly of genera Nebela and Hyalosphenia

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

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

QuickTime™ et un décompresseurPhoto - JPEG sont requis pour visualiser

cette image.

A hard to sequence insertion yields precious phylogenetic information

Evaluation of the identification criteria used for Hyalospheniidae taxa

CONCLUSIONS

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

Water Table

Depth

pH

Conductivity

LaggFen

Other mosses& other habitats

S. teres

- 1 . 5

1 . 8

- 0 . 7 4 3 . 8

S. magellanicum

Hummock

S. fuscum

S. recurvum

LawnHollow

S. cuspidatum

S. capillifolium

CCA plotting the different Sphagnum species against

environmental data in peat bogs

Nebela bohemica

Nebela militaris

Hyalosphenia papilio

Nebela lageniformis

Heleopera rosea

Nebela flabellulum

-1.3

2.6-0.81 3.2

Axis 1

Axis 2

Nebela carinata

The Hyalosphaeniidae as bioindicators

Nebela tincta tincta

Hyalosphenia elegans

Ecological preferences of some Hyalospheniidae in Sphagnum peatlands

Lamentowicz & Mitchell Microbial Ecology, 2005

CCA analysis

Palaeoecology

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

Organisation of the SSU rRNA gene

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

v4

Insertion of 450 bp

Saccharomyces

Nebela

Phylogenetic relationships within Hyalosphaeniidae

Mixotrophic, proteinaceous test: Hyalosphenia papilio

Small, rounded species:Nebela tincta tincta, N. tincta major, N. flabellulum

- Large species - Rounded test baseNebela carinata, N. penardiana

-Large species-Pointed test base Nebela tubulosa, N. marginata

Elongated neck, more or less constricted:

Nebela lageniformis, Apodera vas

Phagotrophic, proteinaceous test: Hyalosphenia elegans

Outgroup: Heleopera rosea

Symbiotic Chlorella-like algae

„Core Nebelas“

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.