Development of molecular tools to study Daphnia - parasite dynamics

Evolutionary dynamics of hosts and their parasites are complex processes. In order to study these processes on genotype level, reliable molecular tools have to be developed. The goal of this thesis was to develop such tools for freshwater crustaceans - Daphnia longispina species complex and its parasites. On one hand, from the host side - an interspecific hybridization plays an important role. For tracing community dynamics and reticulate evolution in such a hybrid species complex, long-term comparative studies of natural populations are necessary. In order to conduct such a study, it is essential to access historical samples. These samples are usually suffering from low DNA quality due to the preservation chemical such as formaldehyde or denaturated ethanol, therefore traditional genotyping through length-based markers (such as microsatellites or allozymes) proved to be insufficient. For circumventing these issues, SNP- based markers were developed. Based on transcriptome data of one species belonging to the complex, it was possible to amplify and sequence several unlinked loci, which were then scanned for species-specific SNPs. Altogether 11 loci distinguishing all three species of the complex and their hybrids were developed and incorporated into PCR-RFLP assay. By comparing the taxon assignment from microsatellite and SNP data, there was found nearly perfect concordance. Finally, the genotyping method was successfully tested on samples dating back to the year 1960. On the other hand, parasite genetic studies are much more limited, in terms of availability of molecular markers. Only handful of parasites allows their cultivation under laboratory conditions inside the hosts. Even then, their disproportional amount of DNA compared to hosts and additional presence of other organisms present in media is forcing to rely on the traditional markers such as internal transcribed spacer (ITS). The traditional method of obtaining sequences to access the diversity (Sanger sequencing preceded by cloning – due to high intragenomic variation of this region) is becoming inefficient due to its high costs in terms of funds and time. Therefore, there was developed a molecular pipeline able to produce and process larger amount of sequence data with more accurate processing, specifically using a next-generation-sequencing platform (454). Afterwards a new bioinformatic pipeline QRS (quantification of representative sequences) was developed, inferring the representative sequences from the next generation sequencing (NGS) data sets (based on neighbor joining or statistical parsimony) and calculating their frequencies. Verification of the method was done by comparing the dataset with the previous study of population structure of Daphnia parasite Caullerya mesnili based on cloning and Sanger sequencing. Pipelines were then used for accessing genetic diversity of the two parasite microsporidian species (Berwaldia and MIC1) commonly infecting Daphnia longispina complex in Central Europe. Specifically, the patterns of geographic population structure, intraspecific genetic variation, and the recombination events were examined, which are necessary for better characterization of the biology of these parasites. The limited geographical variation that was observed in Berwaldia and the different lake origin of recombinant and parental sequences supports usage of a mobile secondary host hypothesis during the life cycle of this species. Similarly MIC1 seems to have a secondary host, however the secondary hosts of both parasites likely differ and the Berwaldia`s one is assumed to have a higher mobility than the one transmitting MIC1.