Environmental sequencing to infer patterns of eukaryotic evolution : Combining long-read and short-read metabarcoding

Sammanfattning: Our view of eukaryotes is biased towards plants, animals, and fungi. But the vast majority of eukaryotic diversity is microbial in nature. These microbial eukaryotes are key players in all ecosystems on earth and are collectively known as protists. Over the past decade we have gathered a better understanding of environmental protist diversity and ecology through metabarcoding studies, which routinely generate millions of reads corresponding to short fragments (< 500 bp) of the 18S gene. However, the limited phylogenetic signal of these short reads hinders their use in investigating questions of an evolutionary nature. To overcome this limitation, we introduced a method for long-read metabarcoding in Paper I of this thesis. We validated this method by amplifying DNA from three soil samples and sequencing with PacBio to obtain a ca. 4500 bp region of the ribosomal DNA operon spanning the 18S and 28S genes. The long-reads were taxonomically annotated using a phylogeny-aware approach, and were used to infer robust 18S-28S phylogenies of the environmental diversity. In Paper II, we investigated habitat evolution across the eukaryotic tree of life, using a unique combination of long-read and short-read metabarcoding data in a phylogenetic framework. We showed that transitions across the marine-terrestrial habitat boundary are more frequent than previously assumed, and that eukaryotic groups vary in their ability to cross this habitat boundary. We inferred that the last eukaryotic common ancestor inhabited non-marine environments, and that subsequent transitions across the marine-terrestrial boundary likely played a key role in eukaryotic evolution by opening new niches to fill.Paper III focused on determining the effects of habitat and latitude on the rates of molecular evolution of protists. Analyses on phylogenies inferred from long-read metabarcoding data found no systematic differences in the evolutionary rates of marine and terrestrial species. Additionally, contrary to expectations, not all eukaryotic groups showed an increase in evolutionary rates towards the equator, with some groups displaying the opposite trend. Finally Paper IV isolates the parasite of the endangered freshwater pearl mussel in Sweden, and phylogenetic analyses including long-read metabarcoding data identifies it as a gregarine belonging to the genus Nematopsis. In summary, this thesis introduces a new method for environmental sequencing of protists, and urges future studies to use both long-read and short-read metabarcoding data to study outstanding questions in eukaryotic evolution and ecology.