Molecular phylogeny of the Arcellinida

1. Molecularphylogeny of the Arcellinida Enrique LARA, Thierry J. HEGER, Flemming EKELUND, Mariusz LAMENTOWICZ, Edward A. D. MITCHELL

2. 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

3. 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

4. Testate amoebae are polyphyletic The Arcellinida The Euglyphida Filose pseudopodia Lobose pseudopodia (1) Adl, S.M. (2005), J. of Eukaryotic Microbiol.

5. Family Hyalospheniidae (sensu Schultze, 1877) • Includes 6 genera among them Nebela (sensu lato), Hyalosphenia and Heleopera • Are especially abundant and diverse in peatlands

6. 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

7. 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 11 studied species from 4 genera

8. Results • All species are clearly genetically distinct • Paraphyly of generaNebela and Hyalosphenia ML tree, 100 bootstraps, ln(L)=-2646, 918 sites

9. 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

10. 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

11. 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 testateamoebae=>cosmopolitanism versus endemism of protists?

12. 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

13. Position of the Hyalosphaeniidae inside the Arcellinida ML tree, 100 bootstraps, ln(L)=-2744, 542 sites

14. 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 1 . 8 - 0 . 7 4 3 . 8 - 1 . 5 Fen Lagg pH S. recurvum Lawn Hollow S. cuspidatum

15. 2.6 -0.81 3.2 -1.3 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 Lamentowicz & Mitchell Microbial Ecology, 2005 Nebela carinata CCA analysis

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

17. Organisation of the SSU rRNA gene v4 Saccharomyces • 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 Nebela Insertion of 450 bp

18. 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

19. 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.