European phylogeography and genetic structure of wood and yellow-necked mice Apodemus sylvaticus and Apodemus flavicollis based on whole-genome, high-density genotyping by restriction-site-associated DNA sequencing (RAD-seq)

Rodents of the genus Apodemus are one the most common mammals in the Palaearctic region. They play an important role in ecosystems by participating in seed dispersal and being a part of the diet of many carnivores. They contribute to the spread of human diseases such as Lyme disease and tick–borne encephalitis, are a reservoir of hantaviruses that cause hemorrhagic fever with renal syndrome (HFRS) and exhibit interesting karyotype feature – B chromosomes. They are, however, very underdeveloped in terms of genetic and genomic resources available for their study.

Apodemus flavicollis and Apodemus sylvaticus live in sympatry in the European Plain. They are phylogenetically related and exhibit similar behaviour and morphology. They have long been studied for elucidation of post-glaciation migration patterns where previous studies using microsatellite and mtDNA markers revealed glacial refugia in southern Europe and suggested the possibility of the existence of a northern refugium. Here, we employ double digestion restriction site-associated DNA sequencing (ddRAD-seq) to study Apodemus phylogeography in Europe.

I first established the feasibility of this approach in a pilot study with 82 samples from both species (72 Apodemus flavicollis and 10 Apodemus sylvaticus) from four locations spanning 500 km in north-eastern Poland. My results shown that despite presumed relatively low mobility of the species, A. flavicollis in the north-eastern Poland effectively constitutes a single population with neligible structure and moderate heterozygosity. Based on 21377 common loci, I was able to estimate the average genetic divergence between the two species at 1.51% and an evolutionary rate of 0.0019 substitutions per site per million of years. I also generated a catalogue of 632063 loci to enable clear genetic differentiation of the two species, and successfully verified its performance on 20 unrelated samples from Europe and Tunisia.

Based on the pilot project experience, I developed a new library preparation protocol that incorporated longer barcodes and degenerate base regions to allow detection of both PCR duplicates and chimeric sequences. After testing the efficiency of the new protocol on a set of samples with variable DNA quality, I applied it to a large scale pan-European study of Apodemus in one of the first application of the RAD-seq in mammals. My results show, for the first time, the existance of post-glacial northern groups, not only on
A. sylvaticus but also on A. flavicollis, as well as long distance movements on A. sylvaticus but not A. flavicollis.

This thesis constitutes the first application of a whole-genome approach to study these organisms. It has allowed us to generate sequences from thousands of loci for both species, identify tens of thousands of SNPs markers and perform continental-scale analysis of the relationships between multiple populations, contributing to the development of Apodemus as a model organism.