Design and Evaluation of DNA-Barcoding High Throughput Methods for Analyzing Diatom Diversity: a Test Case Along a South-North Gradient in Central Europe (Rivers Neiße/Oder)
Zusammenfassung der Projektergebnisse
The microscopical analysis of diatom diversity is a well established approach in the assessment of the ecological status of water bodies and implemented e.g. in the European Water Framework Directive (WFD). This dissertation presents environmental DNA (eDNA) barcoding of diatom communities as an alternative methodology to the routine use of light microscopy. eDNA barcoding relates unidentified sequences from an environmental sample to identified sequences in a reference database to obtain an assignment of the environmental sequences to a respective taxon. This project demonstrated that eDNA barcoding in diatoms provides a more refined taxon detection than the microscopical approach according to the WFD. As a simple approach using the BLASTn algorithm for the assignment of sequences to taxonomic entities proved to be insufficient, the phylogenetic-based coalescent model approach (PCMA) has been introduced. The PCMA combines the general mixed Yule-coalescent (GMYC) model - a tree based approach - with the statistic evaluation of genetic clusters via bootstrap support. The results of this project influenced the decision of the CBOL Protist Working Group (ProWG) to declare the 18S V4 locus as pre-barcode for the assessment of general protist diversity. The quality of sequence assignment to taxonomic entities is directly related to the quality of the taxonomic treatment of the reference sequences. This project therefore provides best practice guidelines for the deposition of sequence information in taxonomic reference libraries. In diatoms reference sequences are almost always obtained from clonal cultures permitting good documentation possibilities. Along with the reference sequences, physical vouchers in form of herbarium specimen and the DNA extract need to be deposited in scientifically curated collections. Also minimum requirements for metadata are suggested: these include e.g. collection data, cultivation data, primer and amplification details, pherograms, as well as photographic documentation of microstructures important for identification. Even though these specifications are proposed for diatoms, they are transferable to other protist groups and also beyond that. The publications from this project contribute towards the establishment of the standard application of eDNA barcoding in water quality assessments via diatom community analysis by introducing (a) a methodological approach and (b) best practice guidelines for the deposition of reference sequences. Furthermore it is shown that DNA barcoding and the establishment of well documented reference databases have a large impact on diatom taxonomy and phylogenetics. Report in career special in the Berliner Morgenpost 2012 Report on Reference Libraries and eDNA barcoding on the science section in the Berliner Zeitung expected early 2015 Press release on “Revolutioniert DNA Barcoding die Gewässergüteanalyse?” und “Vier neue Kieselalgen-Arten in Berlin zufällig entdeckt” resulted in a VBIO report and an interview request from biotechnologie.tv on behalf of the Bundesministeriums für Bildung und Forschung (BMBF) Two technical reports to CEN (European Committee for Standardisation): Water quality - Technical report for the management of diatom barcodes for ecological status assessment, and: Water quality - Technical report for the routine sampling of benthic diatoms from rivers and lakes adapted for metabarcoding analyses
Projektbezogene Publikationen (Auswahl)
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(2012). CBOL Protist Working Group: Barcoding Eurakyotic Richness beyond the Animal, Plant, and Fungal Kingdoms. PLoS Biol.;10(11):e1001419
Pawlowski, J., Audic, S., Adl, S., Bass, D., Belbahri, L., Berney, C., Bowser, S.S., Cepicka, I., Decelle, J., Dunthorn, M., Fiore-Donno, A.M., Gile, G.H., Holzmann, M., Jahn, R., Jirků, M., Keeling, P.J., Kostka, M., Kudryavtsev, A., Lara, E., Lukeš, J., Mann, D.G., Mitchell, E.A., Nitsche, F., Romeralo, M., Saunders, G.W., Simpson, A.G., Smirnov, A.V., Spouge, J.L., Stern, R.F., Stoeck, T., Zimmermann, J., Schindel, D., de Vargas, C.
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(2014). An investigation into the morphology and genetics of Cyclotella comensis and closely related taxa. Diatom Research 29 (4): 423-440
Kistenich, S., Dressler, M., Zimmermann, J., Hübener, T., Bastrop, R. & Jahn, R.
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(2014). Does the Cosmopolitan Diatom Gomphonema parvulum (Kützing) Kützing have a Biogeography? PLoS ONE 9(1): e86885
Abarca, N., Jahn, R., Zimmermann, J. & Enke, N.
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(2014). Metabarcoding vs. morphological identification to assess diatom diversity in environmental studies. Molecular Ecology Resources
Zimmermann, J., Glöckner, G., Jahn, R., Enke, N. & Gemeinholzer, B.
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(2014). Taxonomic Reference Libraries for Environmental Barcoding: a Best Practice Example from Diatom Research. PLoS ONE 9(9): e108793
Zimmermann, J., Abarca, N., Skibbe, O., Kusber, W.-H., Enke, N. & Jahn, R.
