AbstractsBiology & Animal Science

DNA barcoding and eDNA barcoding in diatoms

by Jonas Zimmermann

Institution: Universität Giessen
Department: FB 08 - Biologie und Chemie
Degree: PhD
Year: 2015
Record ID: 1098091
Full text PDF: http://geb.uni-giessen.de/geb/volltexte/2015/11281


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. This dissertation presents environmental DNA (eDNA) barcoding of diatom communities as an alternative methodology to the routine use of light microscopy. The V4 locus of the 18S rRNA gene is introduced in this work as an adequate DNA barcoding marker for diatoms: the locus is flanked by regions that are conserved throughout the diatoms, thus serving as universal primer binding sites. The 18S V4 region also contains sufficient sequence variation for diatom discrimination on a stretch of only approximately 390 bp; therefore it is also suitable for next generation sequencing. A standard laboratory protocol for the amplification of this marker from diatoms in culture as well as from environmental samples is specified in the present dissertation. Furthermore, this work includes the agreement of the CBOL Protist Working Group (ProWG) on the 18S V4 locus as pre]barcode for the assessment of general protist diversity. In addition to that each protist group could apply a second marker region to further examine taxon delimitation and diversity, if necessary. 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. The present study investigates the taxonomic discriminatory power of eDNA barcoding in comparison to light microscopy. It is demonstrated that the DNA based approach provides a more refined taxon detection than the microscopical approach. As the simple BLAST algorithm proved to be insufficient for the assignment of sequences to taxonomic entities, the phylogenetic]based coalescent model approach (PCMA) is 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. It is also shown that the quality of sequence assignment to taxonomic entities is directly related to the quality of the taxonomic treatment of the reference sequences; and the information in the most commonly used reference database INSDC (International Nucleotide Sequence Database Collaboration; incl. Genbank, EMBL/ENA and DDBJ) is of questionable quality. This work therefore includes 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…