Eukaryotic diversity through the lens of metabarcoding and metagenomics

Resumen

*Abstract* Eukaryotes encompass an unprecedented diversity of forms, sizes and lifestyles. However, tackling the real breadth of that diversity is a challenging task. In the last decades, the assessment of biodiversity has seen substantial progress due to the incorporation of culture-independent techniques based on Next Generation DNA Sequencing. This thesis is a composition of three independent projects that implement these techniques aiming to expand our understanding of the extant eukaryotic biodiversity. In the first project, we obtain the nuclear and mitochondrial genomes of Txikispora philomaios, an uncultured unicellular parasite, using a metagenomic approach. We define the phylogenetic position of T.philomaios that branches within Filasterea, a group of unicellular eukaryotes that is closely related to animals and is key to elucidate the transition to animal multicellularity. Despite T.philomaios possessing a reduced genome in comparison to other Filasterea, it has a complete flagellar toolkit and its genome encodes many proteins that are related to multicellularity in animals. In the second project, we seek undescribed molecular diversity inside the phylum Platyhelminthes, one of the most diverse and biomedically important animal phyla. To this end, we analyze global metabarcoding data of the 18S rDNA gene from marine and freshwater habitats. Our results show that a large part of the molecular diversity of Platyhelminthes remains undocumented and identify freshwater environments as potential reservoirs for novel species of flatworms. Finally, in the third project, we investigate the molecular novelty, the taxonomic composition and the structure of the eukaryotic community in Sanabria Lake by sequencing the V4 hypervariable region of the 18S rDNA gene. Here, we show to which extent the choice of the analytical methods (ASVs or OTUs) affects the final results and conclusions in a biodiversity survey. Altogether, our results broaden our perspective of eukaryotic diversity and enhance our understanding of the distribution, the ecology, the molecular novelty and the genomic traits of eukaryotes. *Keywords* metabarcoding, metagenomics, protists, eukaryotic diversity, multicellularity, biodiversity, platyhelminthes *Motivation and content presentation* The world that we see, hear, smell, touch, and perceive with our finite sensorial systems is reduced compared to the world that exists. However, technology and science have permitted us to surpass our human limits and understand the world beyond the point of our biological limitations. One of the most fundamental questions that we have tried to address since the beginning of humankind is with whom we share the planet. First, we have observed life with our naked eyes, and later we have used magnifying lenses to study and document it. We have reached a point where we thought we know almost everything about our planet's inhabitants. And then, we have been given "molecular eyes"... and the known living world expanded one more time! This work is an attempt to push the known living world's limits a little further. I observe the eukaryotic diversity through the lenses of metabarcoding and metagenomics, trying to discover hidden molecular diversity. In the first part of the introduction (Section 1.1), I present the main eukaryotic taxonomic groups as the latest classification system defines them. After introducing eukaryotes, I raise the issue of the hidden diversity among them, and I enumerate the reasons that, in my opinion, have obscured our perception of diversity to date (Section 1.2). Finally, I present the latest technological advances and tools that permit us to overcome the limitations in biodiversity assessment and get a more realistic insight into eukaryotic diversity (Section 1.3). The main results are structured as three independent projects that altogether aim to expand our understanding of extant eukaryotic biodiversity. In the first project (Section 3.1), I present the genome of an uncultured parasite acquired using a metagenomic approach. The genome is analyzed under a phylogenomic and comparative genomics prism to gain insights into the novel organism's biology. In the second project (Section 3.2), I explore a global dataset of metabarcodes from freshwater and marine habitats, searching for unknown molecular diversity inside the phylum Platyhelminthes, a diverse and biomedically important animal phylum with many microscopic representatives. The third project (Section 3.3) is a metabarcoding study of a natural freshwater reservoir that aims to explore the taxonomic composition and the structure of its protist community and search for molecular novelty among microbial eukaryotes. The discussion is structured in five blocks. First, I briefly explain how the genome of Txikispora philomaios has broadened our understanding of the biology and evolution of Filasterea, a group of unicellular eukaryotes that has a key phylogenetic position to understand one of the major evolutionary transitions, the transition to animal multicellularity (Section 4.1). Then, I report state of the art in eukaryotic metagenomics and discuss points that should be considered for successful future applications (Section 4.2). In Section 4.3, I compare two commonly used taxonomic units of metabarcoding pipelines, ASVs and OTUs, focusing on how each unit's choice affects the final results in a biodiversity study. Section 4.4 presents the types of molecular diversity and discusses its importance for conservation. Finally, I go through the current limitations of biodiversity assessment based on metabarcoding and future perspectives that will permit overcoming these limitations (Section 4.5).