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The emergence of new structures can often be linked to the evolution of novel cell types that follows the rewiring of developmental gene regulatory subnetworks. Vertebrates are characterized by a complex body plan compared to the other chordate clades and the question remains of whether and how the emergence of vertebrate morphological innovations can be related to the appearance of new embryonic cell populations. We previously proposed, by studying mesoderm development in the cephalochordate amphioxus, a scenario for the evolution of the vertebrate head mesoderm. To further test this scenario at the cell population level, we used scRNA-seq to construct a cell atlas of the amphioxus neurula, stage at which the main mesodermal compartments are specified. Our data allowed us to validate the presence of a prechordal-plate like territory in amphioxus. Additionally, the transcriptomic profile of somite cell populations supports the homology between specific territories of amphioxus somites and vertebrate cranial/pharyngeal and lateral plate mesoderm. Finally, our work provides evidence that the appearance of the specific mesodermal structures of the vertebrate head was associated to both segregation of pre-existing cell populations, and co-option of new genes for the control of myogenesis., (© 2024. The Author(s).)

Phylogenetic analyses over the last two decades have united a few small, and previously orphan clades, the nematodermatids, acoels and xenoturbelids, into the phylum Xenacoelomorpha. Some phylogenetic analyses support a sister relationship between Xenacoelomorpha and Ambulacraria (Xenambulacraria), while others suggest that Xenacoelomorpha may be sister to the rest of the Bilateria (Nephrozoa). An understanding of the cell type complements of Xenacoelomorphs is essential to assessing these alternatives as well as to our broader understanding of bilaterian cell type evolution. Employing whole organism single-cell RNA-seq in the marine xenacoelomorph worm Xenoturbella bocki, we show that Xenambulacrarian nerve nets share regulatory features and a peptidergic identity with those found in cnidarians and protostomes and more broadly share muscle and gland cell similarities with other metazoans. Taken together, these data are consistent with broad homologies of animal gland, muscle, and neurons as well as more specific affinities between Xenoturbella and acoel gut and epidermal tissues, consistent with the monophyly of Xenacoelomorpha., (© 2024. The Author(s).)

The assembly of the neuronal and other major cell type programs occurred early in animal evolution. We can reconstruct this process by studying non-bilaterians like placozoans. These small disc-shaped animals not only have nine morphologically described cell types and no neurons but also show coordinated behaviors triggered by peptide-secreting cells. We investigated possible neuronal affinities of these peptidergic cells using phylogenetics, chromatin profiling, and comparative single-cell genomics in four placozoans. We found conserved cell type expression programs across placozoans, including populations of transdifferentiating and cycling cells, suggestive of active cell type homeostasis. We also uncovered fourteen peptidergic cell types expressing neuronal-associated components like the pre-synaptic scaffold that derive from progenitor cells with neurogenesis signatures. In contrast, earlier-branching animals like sponges and ctenophores lacked this conserved expression. Our findings indicate that key neuronal developmental and effector gene modules evolved before the advent of cnidarian/bilaterian neurons in the context of paracrine cell signaling., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Histones and associated chromatin proteins have essential functions in eukaryotic genome organization and regulation. Despite this fundamental role in eukaryotic cell biology, we lack a phylogenetically comprehensive understanding of chromatin evolution. Here, we combine comparative proteomics and genomics analysis of chromatin in eukaryotes and archaea. Proteomics uncovers the existence of histone post-translational modifications in archaea. However, archaeal histone modifications are scarce, in contrast with the highly conserved and abundant marks we identify across eukaryotes. Phylogenetic analysis reveals that chromatin-associated catalytic functions (for example, methyltransferases) have pre-eukaryotic origins, whereas histone mark readers and chaperones are eukaryotic innovations. We show that further chromatin evolution is characterized by expansion of readers, including capture by transposable elements and viruses. Overall, our study infers detailed evolutionary history of eukaryotic chromatin: from its archaeal roots, through the emergence of nucleosome-based regulation in the eukaryotic ancestor, to the diversification of chromatin regulators and their hijacking by genomic parasites., (© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)

Bitterness is perceived in humans by 25 subtypes of bitter taste receptors (hTAS2R) that range from broadly tuned to more narrowly tuned receptors. hTAS2R5 is one of the most narrowly tuned bitter taste receptors in humans. In this study, we review the literature on this receptor and show there is no consensus about its role. We then compare the possible role of hTAS2R5 with that of the proteins of the TAS2R family in rat, mouse, and pig. A phylogenetic tree of all mammalian TAS2R domain-containing proteins showed that human hTAS2R5 has no ortholog in pig, mouse, or rat genomes. By comparing the agonists that are common to hTAS2R5 and other members of the family, we observed that hTAS2R39 is the receptor that shares most agonists with hTAS2R5. In mouse, some of these agonists activate mTas2r105 and mTas2r144, which are distant paralogs of hTAS2R5. mTas2r144 seems to be the receptor that is most similar to hTAS2R5 because they are both activated by the same agonists and have affinities in the same range of values. Then, we can conclude that hTAS2R5 has a unique functional specificity in humans as it is activated by selective agonists and that its closest functional homolog in mouse is the phylogenetically distant mTas2r144., (© 2022 The Authors. The FASEB Journal published by Wiley Periodicals LLC on behalf of Federation of American Societies for Experimental Biology.)

Possvm (Phylogenetic Ortholog Sorting with Species oVerlap and MCL [Markov clustering algorithm]) is a tool that automates the process of identifying clusters of orthologous genes from precomputed phylogenetic trees and classifying gene families. It identifies orthology relationships between genes using the species overlap algorithm to infer taxonomic information from the gene tree topology, and then uses the MCL to identify orthology clusters and provide annotated gene families. Our benchmarking shows that this approach, when provided with accurate phylogenies, is able to identify manually curated orthogroups with very high precision and recall. Overall, Possvm automates the routine process of gene tree inspection and annotation in a highly interpretable manner, and provides reusable outputs and phylogeny-aware gene annotations that can be used to inform comparative genomics and gene family evolution analyses., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.)

Stony corals are colonial cnidarians that sustain the most biodiverse marine ecosystems on Earth: coral reefs. Despite their ecological importance, little is known about the cell types and molecular pathways that underpin the biology of reef-building corals. Using single-cell RNA sequencing, we define over 40 cell types across the life cycle of Stylophora pistillata. We discover specialized immune cells, and we uncover the developmental gene expression dynamics of calcium-carbonate skeleton formation. By simultaneously measuring the transcriptomes of coral cells and the algae within them, we characterize the metabolic programs involved in symbiosis in both partners. We also trace the evolution of these coral cell specializations by phylogenetic integration of multiple cnidarian cell type atlases. Overall, this study reveals the molecular and cellular basis of stony coral biology., Competing Interests: Declation of interests The authors declare no competing interests., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

The causes and consequences of genome reduction in animals are unclear because our understanding of this process mostly relies on lineages with often exceptionally high rates of evolution. Here, we decode the compact 73.8-megabase genome of Dimorphilus gyrociliatus, a meiobenthic segmented worm. The D. gyrociliatus genome retains traits classically associated with larger and slower-evolving genomes, such as an ordered, intact Hox cluster, a generally conserved developmental toolkit and traces of ancestral bilaterian linkage. Unlike some other animals with small genomes, the analysis of the D. gyrociliatus epigenome revealed canonical features of genome regulation, excluding the presence of operons and trans-splicing. Instead, the gene-dense D. gyrociliatus genome presents a divergent Myc pathway, a key physiological regulator of growth, proliferation and genome stability in animals. Altogether, our results uncover a conservative route to genome compaction in annelids, reminiscent of that observed in the vertebrate Takifugu rubripes.

Vector population control using insecticides is a key element of current strategies to prevent malaria transmission in Africa. The introduction of effective insecticides, such as the organophosphate pirimiphos-methyl, is essential to overcome the recurrent emergence of resistance driven by the highly diverse Anopheles genomes. Here, we use a population genomic approach to investigate the basis of pirimiphos-methyl resistance in the major malaria vectors Anopheles gambiae and A. coluzzii. A combination of copy number variation and a single non-synonymous substitution in the acetylcholinesterase gene, Ace1, provides the key resistance diagnostic in an A. coluzzii population from Côte d'Ivoire that we used for sequence-based association mapping, with replication in other West African populations. The Ace1 substitution and duplications occur on a unique resistance haplotype that evolved in A. gambiae and introgressed into A. coluzzii, and is now common in West Africa primarily due to selection imposed by other organophosphate or carbamate insecticides. Our findings highlight the predictive value of this complex resistance haplotype for phenotypic resistance and clarify its evolutionary history, providing tools to for molecular surveillance of the current and future effectiveness of pirimiphos-methyl based interventions., Competing Interests: The authors have declared that no competing interests exist.

The evolution of insecticide resistance mechanisms in natural populations of Anopheles malaria vectors is a major public health concern across Africa. Using genome sequence data, we study the evolution of resistance mutations in the resistance to dieldrin locus (Rdl), a GABA receptor targeted by several insecticides, but most notably by the long-discontinued cyclodiene, dieldrin. The two Rdl resistance mutations (296G and 296S) spread across West and Central African Anopheles via two independent hard selective sweeps that included likely compensatory nearby mutations, and were followed by a rare combination of introgression across species (from A. gambiae and A. arabiensis to A. coluzzii) and across nonconcordant karyotypes of the 2La chromosomal inversion. Rdl resistance evolved in the 1950s as the first known adaptation to a large-scale insecticide-based intervention, but the evolutionary lessons from this system highlight contemporary and future dangers for management strategies designed to combat development of resistance in malaria vectors., (© The Author(s) 2020. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.)

RNA editing is a source of molecular diversity that regulates the functional repertoire of animal transcriptomes. Multiple studies in Drosophila have revealed that conserved editing events can be a source of evolutionary adaptations, and there is a solid body of evidence linking editing and the fine-tuning of neural genes, which are often targeted by insecticides used in vector control. Yet, despite these suggestive connections, genome-wide analyses of editing in insect vectors are conspicuously lacking. Future advances will require complementing the growing wealth of vector genomes with targeted transcriptome analyses. Here, we review recent investigations of the genetic footprints of adaptive RNA editing in insects and provide an overview of new methodologies applicable to studies of RNA editing in insect vectors., (Copyright © 2020 Elsevier Inc. All rights reserved.)

The surface of insects is coated in cuticular hydrocarbons (CHCs); variations in the composition of this layer affect a range of traits including adaptation to arid environments and defence against pathogens and toxins. In the African malaria vector, Anopheles gambiae quantitative and qualitative variance in CHC composition have been associated with speciation, ecological habitat and insecticide resistance. Understanding how these modifications arise will inform us of how mosquitoes are responding to climate change and vector control interventions. CHCs are synthesised in sub-epidermal cells called oenocytes that are very difficult to isolate from surrounding tissues. Here we utilise a transgenic line with fluorescent oenocytes to purify these cells for the first time. Comparative transcriptomics revealed the enrichment of biological processes related to long chain fatty acyl-CoA biosynthesis and elongation of mono-, poly-unsaturated and saturated fatty acids and enabled us to delineate, and partially validate, the hydrocarbon biosynthetic pathway in An. gambiae ., Competing Interests: LG, XG, HC, GL, HR No competing interests declared, (© 2020, Grigoraki et al.)

Understanding the origins of animal multicellularity is a fundamental biological question. Recent genome data have unravelled the role that co-option of 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 understanding of the specific genetic innovations and how they appeared, we need the broadest taxon sampling possible, especially among early-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 performed an updated and taxon-rich reconstruction of protein evolution from the Last Eukaryotic Common Ancestor (LECA) to animals. Our novel 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 the stem of Metazoa was mainly driven by duplications and protein domain rearrangement processes at the stem of Metazoa. Furthermore, our analysis allowed us to reveal protein domains that are essential to the maintenance of animal multicellularity. Our analyses also demonstrate the utility of single-cell genomics from uncultured taxa to address evolutionary questions. This article is part of a discussion meeting issue 'Single cell ecology'.

In animals, cellularization of a coenocyte is a specialized form of cytokinesis that results in the formation of a polarized epithelium during early embryonic development. It is characterized by coordinated assembly of an actomyosin network, which drives inward membrane invaginations. However, whether coordinated cellularization driven by membrane invagination exists outside animals is not known. To that end, we investigate cellularization in the ichthyosporean Sphaeroforma arctica , a close unicellular relative of animals. We show that the process of cellularization involves coordinated inward plasma membrane invaginations dependent on an actomyosin network and reveal the temporal order of its assembly. This leads to the formation of a polarized layer of cells resembling an epithelium. We show that this stage is associated with tightly regulated transcriptional activation of genes involved in cell adhesion. Hereby we demonstrate the presence of a self-organized, clonally-generated, polarized layer of cells in a unicellular relative of animals., Competing Interests: OD, AO, XG, AH, AT, HS, JB, IR No competing interests declared, (© 2019, Dudin et al.)

Aphelids are little-known phagotrophic parasites of algae whose life cycle and morphology resemble those of the parasitic rozellids (Cryptomycota, Rozellomycota). In previous phylogenetic analyses of RNA polymerase and rRNA genes, aphelids, rozellids and Microsporidia (parasites of animals) formed a clade, named Opisthosporidia, which appeared as the sister group to Fungi. However, the statistical support for the Opisthosporidia was always moderate. Here, we generated full life-cycle transcriptome data for the aphelid species Paraphelidium tribonemae . In-depth multi-gene phylogenomic analyses using several protein datasets place this aphelid as the closest relative of fungi to the exclusion of rozellids and Microsporidia. In contrast with the comparatively reduced Rozella allomycis genome, we infer a rich, free-living-like aphelid proteome, with a metabolism similar to fungi, including cellulases likely involved in algal cell-wall penetration and enzymes involved in chitin biosynthesis. Our results suggest that fungi evolved from complex aphelid-like ancestors that lost phagotrophy and became osmotrophic., Competing Interests: The authors declare no competing interests.

Background: Alternative splicing, particularly through intron retention and exon skipping, is a major layer of pre-translational regulation in eukaryotes. While intron retention is believed to be the most prevalent mode across non-animal eukaryotes, animals have unusually high rates of exon skipping. However, when and how this high prevalence of exon skipping evolved is unknown. Since exon skipping can greatly expand proteomes, answering these questions sheds light on the evolution of higher organismal complexity in metazoans., Results: We used RNA-seq data to quantify exon skipping and intron retention frequencies across 65 eukaryotic species, with particular focus on early branching animals and unicellular holozoans. We found that only bilaterians have significantly increased their exon skipping frequencies compared to all other eukaryotic groups. Unlike in other eukaryotes, however, exon skipping in nearly all animals, including non-bilaterians, is strongly enriched for frame-preserving sequences, suggesting that exon skipping involvement in proteome expansion predated the increase in frequency. We also identified architectural features consistently associated with higher exon skipping rates within all studied eukaryotic genomes. Remarkably, these architectures became more prevalent during animal evolution, indicating co-evolution between genome architectures and exon skipping frequencies., Conclusion: We suggest that the increase of exon skipping rates in animals followed a two-step process. First, exon skipping in early animals became enriched for frame-preserving events. Second, bilaterian ancestors dramatically increased their exon skipping frequencies, likely driven by the interplay between a shift in their genome architectures towards more exon definition and recruitment of frame-preserving exon skipping events to functionally diversify their cell-specific proteomes.

Eukaryotic microbes have three primary mechanisms for obtaining nutrients and energy: phagotrophy, photosynthesis and osmotrophy. Traits associated with the latter two functions arose independently multiple times in the eukaryotes. The Fungi successfully coupled osmotrophy with filamentous growth, and similar traits are also manifested in the Pseudofungi (oomycetes and hyphochytriomycetes). Both the Fungi and the Pseudofungi encompass a diversity of plant and animal parasites. Genome-sequencing efforts have focused on host-associated microbes (mutualistic symbionts or parasites), providing limited comparisons with free-living relatives. Here we report the first draft genome sequence of a hyphochytriomycete 'pseudofungus'; Hyphochytrium catenoides Using phylogenomic approaches, we identify genes of recent viral ancestry, with related viral derived genes also present on the genomes of oomycetes, suggesting a complex history of viral coevolution and integration across the Pseudofungi. H. catenoides has a complex life cycle involving diverse filamentous structures and a flagellated zoospore with a single anterior tinselate flagellum. We use genome comparisons, drug sensitivity analysis and high-throughput culture arrays to investigate the ancestry of oomycete/pseudofungal characteristics, demonstrating that many of the genetic features associated with parasitic traits evolved specifically within the oomycete radiation. Comparative genomics also identified differences in the repertoire of genes associated with filamentous growth between the Fungi and the Pseudofungi, including differences in vesicle trafficking systems, cell-wall synthesis pathways and motor protein repertoire, demonstrating that unique cellular systems underpinned the convergent evolution of filamentous osmotrophic growth in these two eukaryotic groups., (© 2018 The Authors.)

Single-cell genomics (SCG) appeared as a powerful technique to get genomic information from uncultured organisms. However, SCG techniques suffer from biases at the whole genome amplification step that can lead to extremely variable numbers of genome recovery (5-100%). Thus, it is unclear how useful can SCG be to address evolutionary questions on uncultured microbial eukaryotes. To provide some insights into this, we here analysed 3 single-cell amplified genomes (SAGs) of the choanoflagellate Monosiga brevicollis, whose genome is known. Our results show that each SAG has a different, independent bias, yielding different levels of genome recovery for each cell (6-36%). Genes often appear fragmented and are split into more genes during annotation. Thus, analyses of gene gain and losses, gene architectures, synteny and other genomic features can not be addressed with a single SAG. However, the recovery of phylogenetically-informative protein domains can be up to 55%. This means SAG data can be used to perform accurate phylogenomic analyses. Finally, we also confirm that the co-assembly of several SAGs improves the general genomic recovery. Overall, our data show that, besides important current limitations, SAGs can still provide interesting and novel insights from poorly-known, uncultured organisms.

Which genomic innovations underpinned the origin of multicellular animals is still an open debate. Here, we investigate this question by reconstructing the genome architecture and gene family diversity of ancestral premetazoans, aiming to date the emergence of animal-like traits. Our comparative analysis involves genomes from animals and their closest unicellular relatives (the Holozoa), including four new genomes: three Ichthyosporea and Corallochytrium limacisporum . Here, we show that the earliest animals were shaped by dynamic changes in genome architecture before the emergence of multicellularity: an early burst of gene diversity in the ancestor of Holozoa, enriched in transcription factors and cell adhesion machinery, was followed by multiple and differently-timed episodes of synteny disruption, intron gain and genome expansions. Thus, the foundations of animal genome architecture were laid before the origin of complex multicellularity - highlighting the necessity of a unicellular perspective to understand early animal evolution.

Ubiquitination is a relevant cell regulatory mechanism to determine protein fate and function. Most data has focused on the role of ubiquitin as a tag molecule to target substrates to proteasome degradation, and on its impact in the control of cell cycle, protein homeostasis and cancer. Only recently, systematic assays have pointed to the relevance of the ubiquitin pathway in the development and differentiation of tissues and organs, and its implication in hereditary diseases. Moreover, although the activity and composition of ubiquitin ligases has been largely addressed, the role of the deubiquitinating enzymes (DUBs) in specific tissues, such as the retina, remains mainly unknown. In this work, we undertook a systematic analysis of the transcriptional levels of DUB genes in the adult mouse retina by RT-qPCR and analyzed the expression pattern by in situ hybridization and fluorescent immunohistochemistry, thus providing a unique spatial reference map of retinal DUB expression. We also performed a systematic phylogenetic analysis to understand the origin and the presence/absence of DUB genes in the genomes of diverse animal taxa that represent most of the known animal diversity. The expression landscape obtained supports the potential subfunctionalization of paralogs in those families that expanded in vertebrates. Overall, our results constitute a reference framework for further characterization of the DUB roles in the retina and suggest new candidates for inherited retinal disorders.

The Opisthokonta are a eukaryotic supergroup divided in two main lineages: animals and related protistan taxa, and fungi and their allies [1, 2]. There is a great diversity of lifestyles and morphologies among unicellular opisthokonts, from free-living phagotrophic flagellated bacterivores and filopodiated amoebas to cell-walled osmotrophic parasites and saprotrophs. However, these characteristics do not group into monophyletic assemblages, suggesting rampant convergent evolution within Opisthokonta. To test this hypothesis, we assembled a new phylogenomic dataset via sequencing 12 new strains of protists. Phylogenetic relationships among opisthokonts revealed independent origins of filopodiated amoebas in two lineages, one related to fungi and the other to animals. Moreover, we observed that specialized osmotrophic lifestyles evolved independently in fungi and protistan relatives of animals, indicating convergent evolution. We therefore analyzed the evolution of two key fungal characters in Opisthokonta, the flagellum and chitin synthases. Comparative analyses of the flagellar toolkit showed a previously unnoticed flagellar apparatus in two close relatives of animals, the filasterean Ministeria vibrans and Corallochytrium limacisporum. This implies that at least four different opisthokont lineages secondarily underwent flagellar simplification. Analysis of the evolutionary history of chitin synthases revealed significant expansions in both animals and fungi, and also in the Ichthyosporea and C. limacisporum, a group of cell-walled animal relatives. This indicates that the last opisthokont common ancestor had a complex toolkit of chitin synthases that was differentially retained in extant lineages. Thus, our data provide evidence for convergent evolution of specialized lifestyles in close relatives of animals and fungi from a generalist ancestor., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Lysyl oxidases (LOX) are copper-dependent enzymes that oxidize primary amine substrates to reactive aldehydes. The best-studied role of LOX enzymes is the remodeling of the extracellular matrix (ECM) in animals by cross-linking collagens and elastin, although intracellular functions have been reported as well. Five different LOX enzymes have been identified in mammals, LOX and LOX-like (LOXL) 1 to 4, showing a highly conserved catalytic carboxy terminal domain and more divergence in the rest of the sequence. Here we have surveyed a wide selection of genomes in order to infer the evolutionary history of LOX. We identified LOX proteins not only in animals, but also in many other eukaryotes, as well as in bacteria and archaea - which reveals a pre-metazoan origin for this gene family. LOX genes expanded during metazoan evolution resulting in two superfamilies, LOXL2/L3/L4 and LOX/L1/L5. Considering the current knowledge on the function of mammalian LOX isoforms in ECM remodeling, we propose that LOXL2/L3/L4 members might have preferentially been involved in making cross-linked collagen IV-based basement membrane, whereas the diversification of LOX/L1/L5 forms contributed to chordate/vertebrate-specific ECM innovations, such as elastin and fibronectin. Our work provides a novel view on the evolution of this family of enzymes.

The origin of the eukaryotic cell is one of the most important transitions in the history of life. However, the emergence and early evolution of eukaryotes remains poorly understood. Recent data have shown that the last eukaryotic common ancestor (LECA) was much more complex than previously thought. The LECA already had the genetic machinery encoding the endomembrane apparatus, spliceosome, nuclear pore, and myosin and kinesin cytoskeletal motors. It is unclear, however, when the functional regulation of these cellular components evolved. Here, we address this question by analyzing the origin and evolution of the ubiquitin (Ub) signaling system, one of the most important regulatory layers in eukaryotes. We delineated the evolution of the whole Ub, Small-Ub-related MOdifier (SUMO), and Ub-fold modifier 1 (Ufm1) signaling networks by analyzing representatives from all major eukaryotic, bacterial, and archaeal lineages. We found that the Ub toolkit had a pre-eukaryotic origin and is present in three extant archaeal groups. The pre-eukaryotic Ub toolkit greatly expanded during eukaryogenesis, through massive gene innovation and diversification of protein domain architectures. This resulted in a LECA with essentially all of the Ub-related genes, including the SUMO and Ufm1 Ub-like systems. Ub and SUMO signaling further expanded during eukaryotic evolution, especially labeling and delabeling enzymes responsible for substrate selection. Additionally, we analyzed protein domain architecture evolution and found that multicellular lineages have the most complex Ub systems in terms of domain architectures. Together, we demonstrate that the Ub system predates the origin of eukaryotes and that a burst of innovation during eukaryogenesis led to a LECA with complex posttranslational regulation., (© The Author 2014. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.)

Myosins are key components of the eukaryotic cytoskeleton, providing motility for a broad diversity of cargoes. Therefore, understanding the origin and evolutionary history of myosin classes is crucial to address the evolution of eukaryote cell biology. Here, we revise the classification of myosins using an updated taxon sampling that includes newly or recently sequenced genomes and transcriptomes from key taxa. We performed a survey of eukaryotic genomes and phylogenetic analyses of the myosin gene family, reconstructing the myosin toolkit at different key nodes in the eukaryotic tree of life. We also identified the phylogenetic distribution of myosin diversity in terms of number of genes, associated protein domains and number of classes in each taxa. Our analyses show that new classes (i.e., paralogs) and domain architectures were continuously generated throughout eukaryote evolution, with a significant expansion of myosin abundance and domain architectural diversity at the stem of Holozoa, predating the origin of animal multicellularity. Indeed, single-celled holozoans have the most complex myosin complement among eukaryotes, with paralogs of most myosins previously considered animal specific. We recover a dynamic evolutionary history, with several lineage-specific expansions (e.g., the myosin III-like gene family diversification in choanoflagellates), convergence in protein domain architectures (e.g., fungal and animal chitin synthase myosins), and important secondary losses. Overall, our evolutionary scheme demonstrates that the ancestral eukaryote likely had a complex myosin repertoire that included six genes with different protein domain architectures. Finally, we provide an integrative and robust classification, useful for future genomic and functional studies on this crucial eukaryotic gene family.

The posttranslational modification of proteins by the ubiquitination pathway is an important regulatory mechanism in eukaryotes. To date, however, studies on the evolutionary history of the proteins involved in this pathway have been restricted to E1 and E2 enzymes, whereas E3 studies have been focused mainly in metazoans and plants. To have a wider perspective, here we perform a genomic survey of the HECT family of E3 ubiquitin-protein ligases, an important part of this posttranslational pathway, in genomes from representatives of all major eukaryotic lineages. We classify eukaryotic HECTs and reconstruct, by phylogenetic analysis, the putative repertoire of these proteins in the last eukaryotic common ancestor (LECA). Furthermore, we analyze the diversity and complexity of protein domain architectures of HECTs along the different extant eukaryotic lineages. Our data show that LECA had six different HECTs and that protein expansion and N-terminal domain diversification shaped HECT evolution. Our data reveal that the genomes of animals and unicellular holozoans considerably increased the molecular and functional diversity of their HECT system compared with other eukaryotes. Other eukaryotes, such as the Apusozoa Thecanomas trahens or the Heterokonta Phytophthora infestans, independently expanded their HECT repertoire. In contrast, plant, excavate, rhodophyte, chlorophyte, and fungal genomes have a more limited enzymatic repertoire. Our genomic survey and phylogenetic analysis clarifies the origin and evolution of different HECT families among eukaryotes and provides a useful phylogenetic framework for future evolutionary studies of this regulatory pathway.