Reconstruction of protein domain evolution using single-cell amplified royalsocietypublishing.org/journal/rstb genomes of uncultured choanoflagellates sheds light on the origin of animals Research David López-Escardó 1,2 , Xavier Grau-Bové 1,3,4 , Amy Guillaumet-Adkins 5,6 , Marta Gut 5,6 , Michael E. Sieracki 7 and Iñaki Ruiz-Trillo 1,3,8 Cite this article: López-Escardó D, Grau-Bové X, Guillaumet-Adkins A, Gut M, Sieracki ME, 1Institut de Biologia Evolutiva (CSIC-Universitat Pompeu Fabra), Passeig Marítim de la Barceloneta 37-49, Ruiz-Trillo I. 2019 Reconstruction of protein 08003 Barcelona, Catalonia, Spain 2Institut de Ciències del Mar (ICM-CSIC), Passeig Marítim de la Barceloneta 37-49, 08003 Barcelona, Catalonia, Spain domain evolution using single- cell amplified 3Departament de Genètica, Microbiologia i Estadística, Universitat de Barcelona, 08028 Barcelona, Catalonia, Spain genomes of uncultured choanoflagellates sheds 4Department of Vector Biology, Liverpool School of Tropical Medicine, Pembroke Place, Liverpool, L3 5QA, UK light on the origin of animals. Phil. 5CNAG-CRG, Centre for Genomic Regulation (CRG), Barcelona Institute of Science and Technology (BIST), 08028 Trans. R. Soc. B 374 : 20190088. Barcelona, Spain 6Universitat Pompeu Fabra (UPF), 08003 Barcelona, Spain http://dx.doi.org/10.1098/rstb.2019.0088 7National Science Foundation, Arlington, VA 22314, USA 8ICREA, Pg. Lluís Companys 23, 08010 Barcelona, Spain Accepted: 15 June 2019 DL-E, 0000-0002-9122-6771; XG-B, 0000-0003-1978-5824; IR-T, 0000-0001-6547-5304 One contribution of 18 to a discussion meeting Understanding the origins of animal multicellularity is a fundamental biologi- cal question. Recent genome data have unravelled the role that co-option of issue ‘Single cell ecology ’. pre-existing genes played in the origin of animals. However, there were also some important genetic novelties at the onset of Metazoa. To have a clear Subject Areas: understanding of the specific genetic innovations and how they appeared, genomics, evolution, molecular biology, we need the broadest taxon sampling possible, especially among early- microbiology, genetics branching animals and their unicellular relatives. Here, we take advantage of single-cell genomics to expand our understanding of the genomic diversity of choanoflagellates, the sister-group to animals. With these genomes, we have Keywords: performed an updated and taxon-rich reconstruction of protein evolution protein domain evolution, choanoflagellates, from the Last Eukaryotic Common Ancestor (LECA) to animals. Our novel animal multicellularity, single-cell genomics data re-defines the origin of some genes previously thought to be metazoan- specific, like the POU transcription factor, which we show appeared earlier in evolution. Moreover, our data indicate that the acquisition of new genes at Authors for correspondence: the stem of Metazoa was mainly driven by duplications and protein domain David López-Escardó rearrangement processes at the stem of Metazoa. Furthermore, our analysis e-mail: [email protected] allowed us to reveal protein domains that are essential to the maintenance Iñaki Ruiz-Trillo of animal multicellularity. Our analyses also demonstrate the utility of e-mail: [email protected] sin gle-cell genomics from uncultured taxa to address evolutionary questions. This article is part of a discussion meeting issue ‘Single cell ecology ’. 1. Introduction Metazoa is the eukaryotic kingdom with most described species so far, around 1.3 million [1], and it is the multicellular group of eukaryotes for which the most differential cell types have been described [2]. Animals ’ success might be tightly linked to their multicellular complexity, which has been considered unique in the eukaryotic world [3], compared to other multicellular eukaryotic transitions [3,4]. Thus, the uniqueness of animal multicellularity raises the question of which mechanisms shaped the emergence of such special types of organisms from a unicellular ancestor more than 600 million years ago [4,5]. To address this question, efforts have been made, over the past decade, to Electronic supplementary material is available reconstruct the Urmetazoan genomic content by comparing the genomic online at https://doi.org/10.6084/m9.figshare. c.4643798. © 2019 The Authors. Published by the Royal Society under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/4.0/, which permits unrestricted use, provided the original author and source are credited. sequences of a broad and diverse spectrum of animals [6 –8] species marine and with a tectiform lorica (around 150 species) 2 with the genomes of their closest unicellular relatives: the [24], although there are 5-6 species described with nudiform royalsocietypublishing.org/journal/rstb Choanoflagellatea [9,10], Filasterea [11,12] and Teretosporea, lorica [25]. Furthermore, there are other clades of choanoflagel- which include ichth yosporeans and Corallochytium limacisporum lates, such as Clade L [29], FRESCHOs and MACHOs [30], that [13,14]. These unicellular lineages and metazoans conform the have been defined only by environmental sequences (18S Holozoa clade. Holozoa, together with Fungi and their unicel- rDNA gene). Thus, there is a vast hidden choanoflagellate lular relatives compose the eukaryotic supergroup known as diversity, which is uncultured and may be relevant to address Opisthokonta [15]. Thus, understanding the evolution of animal origins. opisthokonts is critical to address the origin of animals. In this work, we aim to improve this view by sequencing The first genomic comparisons between animals and their four single-cell amplified genomes (SAGs) of uncultured unicellular relatives at a genomic level revealed a complex choanoflagellates belonging to distinct taxa and collected pre-metazoan genetic toolkit, already equipped with a rich during the TARA Oceans expedition [31]. With the novel repertoire of genes involved in multicellular functions. These genomic information, we perform a new, taxon-rich phyloge- included developmental transcription factors (like Brachyury , nomic analysis of the opisthokonts. Moreover, we reconstruct Phil. Trans. R. Soc. B MYC, Runx, or P53), cell adhesion proteins (ECM elements, the evolutionary history of protein domains from the last integrins, cadherins, and C-type lectins) and cell signalling eukaryotic common ancestor (LECA) to animals. Protein receptors andtransducers [9,10,16 –20]. These findings suggested domains are the basic building blocks that determine the that co-option of ancestral genes into new functions was an structure and the function of the proteins [32]. Thus, under- important mechanism that occurred in the transition from the standing the gains/losses of protein domains is crucial to unicellular ancestor of animals to the Urmetazoa [20]. However, better understand the genomic changes that mediated the 374 it was also found that not all the components of manyanimal sig- transition towards animal multicellularity [33]. Also, recon- : 20190088 nalling pathways, like the Hippo pathway, had a pre-metazoan struction of protein domains is the most reliable method to origin. In some cases, only some ligands or receptors were pre- get informative evolutionary insights when using single-cell sent before the emergence of multicellularity. Those ancestral genomics data, in which genomes appear fragmented and ligands and receptors were later on putatively co-opted in with low completeness values [34]. functioning within these animal signalling pathways [20,21]. Our protein domain reconstruction analysis shows that, Thus, the acquisition of new genes might have also played an contrary to previous genetic reconstructions, the Metazoa important role in the emergence of animal multicellularity. ancestor did not suffer an important acquisition of new In two recent studies aiming at better reconstructing the protein domains. This suggests that the genome innovation Urmetazoan genome, bursts of new genic innovation were at the stem of Metazoa was mainly driven by duplications shown at the stem of Metazoa. One of those studies focused and protein domain shuffling processes. In addition, our on the search of animal genetic innovations using a new analysis reveals key essential protein domains for animal method to infer homology [22], while the other expanded the functions and redefines the origin of some genes thought to genomic information of choanoflagellates by sequencing the be metazoan-specific, which appeared earlier in the unicellular transcriptomes of 19 choanoflagellate species [23]. Both studies ancestor of animals like the POU transcription factor. claimed that approximately 1500 –1700 genes were acquired during the transition towards animal multicellularity (three times more gene acquisition than has been reported in their uni- 2. Methods cellular ancestors). In particular, the most conserved animal- specific genes in extant metazoans were genes related to major (a) Cell collection and whole genome amplification Cells for single-cell genomics were collected from the signalling pathways such as components of TGF-beta or Wnt Mediterranean sea and different places in the Indian Ocean signalling pathways, and transcription factors like ETS or POU during the Tara Oceans expedition [35] and cryopreserved as [22]. However, the results also showed genes that had probably described before [36]. Flow cytometry cell sorting, single cell been overlooked for their potential role