Cell type and regulatory analysis in amphioxus illuminates evolutionary origin of the vertebrate head.

To shed light on the enigmatic origin of the vertebrate head, our study employs an integrated approach that combines single-cell transcriptomics, perturbations in signaling pathways, and cis-regulatory analysis in amphioxus. As a representative of a basal lineage within the chordate phylum, amphioxus retains many characteristics thought to have been present in the common chordate ancestor. Through cell type characterization, we identify the presence of prechordal plate-like, pre-migratory, and migratory neural crest-like cell populations in the developing amphioxus embryo. Functional analysis establishes conserved roles of the Nodal and Hedgehog signaling pathways in prechordal plate-like populations, and of the Wnt signaling pathway in neural crest-like populations' development. Furthermore, our trans-species transgenic experiments highlight similarities in the regulatory environments that drive neural crest-like and prechordal plate-like developmental programs in both vertebrates and amphioxus. Our findings provide evidence that the key features of vertebrate head development can be traced back to the common ancestor of all chordates.

Widespread Adaptive Introgression of Major Histocompatibility Complex Genes across Vertebrate Hybrid Zones.

Interspecific introgression is a potentially important source of novel variation of adaptive significance. Although multiple cases of adaptive introgression are well documented, broader generalizations about its targets and mechanisms are lacking. Multiallelic balancing selection, particularly when acting through rare allele advantage, is an evolutionary mechanism expected to favor adaptive introgression. This is because introgressed alleles are likely to confer an immediate selective advantage, facilitating their establishment in the recipient species even in the face of strong genomic barriers to introgression. Vertebrate major histocompatibility complex genes are well-established targets of long-term multiallelic balancing selection, so widespread adaptive major histocompatibility complex introgression is expected. Here, we evaluate this hypothesis using data from 29 hybrid zones formed by fish, amphibians, squamates, turtles, birds, and mammals at advanced stages of speciation. The key prediction of more extensive major histocompatibility complex introgression compared to genome-wide introgression was tested with three complementary statistical approaches. We found evidence for widespread adaptive introgression of major histocompatibility complex genes, providing a link between the process of adaptive introgression and an underlying mechanism. Our work identifies major histocompatibility complex introgression as a general mechanism by which species can acquire novel, and possibly regain previously lost, variation that may enhance defense against pathogens and increase adaptive potential.

The evolution of tenascins.

BACKGROUND: The evolution of extracellular matrix is tightly linked to the evolution of organogenesis in metazoans. Tenascins are extracellular matrix glycoproteins of chordates that participate in integrin-signaling and morphogenetic events. Single tenascins are encoded by invertebrate chordates, and multiple tenascin paralogs are found in vertebrates (designated tenascin-C, tenascin-R, tenascin-W and tenascin-X) yet, overall, the evolution of this family has remained unclear. RESULTS: This study examines the genomes of hemichordates, cephalochordates, tunicates, agnathans, cartilaginous fishes, lobe-finned fishes, ray-finned fishes and representative tetrapods to identify predicted tenascin proteins. We comprehensively assess their evolutionary relationships by sequence conservation, molecular phylogeny and examination of conservation of synteny of the encoding genes. The resulting new evolutionary model posits the origin of tenascin in an ancestral chordate, with tenascin-C-like and tenascin-R-like paralogs emerging after a whole genome duplication event in an ancestral vertebrate. Tenascin-X appeared following a second round of whole genome duplication in an ancestral gnathostome, most likely from duplication of the gene encoding the tenascin-R homolog. The fourth gene, encoding tenascin-W (also known as tenascin-N), apparently arose from a local duplication of tenascin-R. CONCLUSIONS: The diversity of tenascin paralogs observed in agnathans and gnathostomes has evolved through selective retention of novel genes that arose from a combination of whole genome and local duplication events. The evolutionary appearance of specific tenascin paralogs coincides with the appearance of vertebrate-specific cell and tissue types where the paralogs are abundantly expressed, such as the endocranium and facial skeleton (tenascin-C), an expanded central nervous system (tenascin-R), and bone (tenascin-W).

Genomic transfers help to decipher the ancient evolution of filoviruses and interactions with vertebrate hosts.

Although several filoviruses are dangerous human pathogens, there is conflicting evidence regarding their origins and interactions with animal hosts. Here we attempt to improve this understanding using the paleoviral record over a geological time scale, protein structure predictions, tests for evolutionary maintenance, and phylogenetic methods that alleviate sources of bias and error. We found evidence for long branch attraction bias in the L gene tree for filoviruses, and that using codon-specific models and protein structural comparisons of paleoviruses ameliorated conflict and bias. We found evidence for four ancient filoviral groups, each with extant viruses and paleoviruses with open reading frames. Furthermore, we found evidence of repeated transfers of filovirus-like elements to mouse-like rodents. A filovirus-like nucleoprotein ortholog with an open reading frame was detected in three subfamilies of spalacid rodents (present since the Miocene). We provide evidence that purifying selection is acting to maintain amino acids, protein structure and open reading frames in these elements. Our finding of extant viruses nested within phylogenetic clades of paleoviruses informs virus discovery methods and reveals the existence of Lazarus taxa among RNA viruses. Our results resolve a deep conflict in the evolutionary framework for filoviruses and reveal that genomic transfers to vertebrate hosts with potentially functional co-options have been more widespread than previously appreciated.

The protein domains of vertebrate species in which selection is more effective have greater intrinsic structural disorder.

The nearly neutral theory of molecular evolution posits variation among species in the effectiveness of selection. In an idealized model, the census population size determines both this minimum magnitude of the selection coefficient required for deleterious variants to be reliably purged, and the amount of neutral diversity. Empirically, an 'effective population size' is often estimated from the amount of putatively neutral genetic diversity and is assumed to also capture a species' effectiveness of selection. A potentially more direct measure of the effectiveness of selection is the degree to which selection maintains preferred codons. However, past metrics that compare codon bias across species are confounded by among-species variation in %GC content and/or amino acid composition. Here, we propose a new Codon Adaptation Index of Species (CAIS), based on Kullback-Leibler divergence, that corrects for both confounders. We demonstrate the use of CAIS correlations, as well as the Effective Number of Codons, to show that the protein domains of more highly adapted vertebrate species evolve higher intrinsic structural disorder.

Evolution is the process through which populations change over time, starting with mutations in the genetic sequence of an organism. Many of these mutations harm the survival and reproduction of an organism, but only by a very small amount. Some species, especially those with large populations, can purge these slightly harmful mutations more effectively than other species. This fact has been used by the 'drift barrier theory' to explain various profound differences amongst species, including differences in biological complexity. In this theory, the effectiveness of eliminating slightly harmful mutations is specified by an 'effective' population size, which depends on factors beyond just the number of individuals in the population. Effective population size is normally calculated from the amount of time a 'neutral' mutation (one with no effect at all) stays in the population before becoming lost or taking over. Estimating this time requires both representative data for genetic diversity and knowledge of the mutation rate. A major limitation is that these data are unavailable for most species. A second limitation is that a brief, temporary reduction in the number of individuals has an oversized impact on the metric, relative to its impact on the number of slighly harmful mutations accumulated. Weibel, Wheeler et al. developed a new metric to more directly determine how effectively a species purges slightly harmful mutations. Their approach is based on the fact that the genetic code has 'synonymous' sequences. These sequences code for the same amino acid building block, with one of these sequences being only slightly preferred over others. The metric by Weibel, Wheeler et al. quantifies the proportion of the genome from which less preferred synonymous sequences have been effectively purged. It judges a population to have a higher effective population size when the usage of synonymous sequences departs further from the usage predicted from mutational processes. The researchers expected that natural selection would favour 'ordered' proteins with robust three-dimensional structures, i.e., that species with a higher effective population size would tend to have more ordered versions of a protein. Instead, they found the opposite: species with a higher effective population size tend to have more disordered versions of the same protein. This changes our view of how natural selection acts on proteins. Why species are so different remains a fundamental question in biology. Weibel, Wheeler et al. provide a useful tool for future applications of drift barrier theory to a broad range of ways that species differ.

Single-cell analysis of the amphioxus hepatic caecum and vertebrate liver reveals genetic mechanisms of vertebrate liver evolution.

The evolution of the vertebrate liver is a prime example of the evolution of complex organs, yet the driving genetic factors behind it remain unknown. Here we study the evolutionary genetics of liver by comparing the amphioxus hepatic caecum and the vertebrate liver, as well as examining the functional transition within vertebrates. Using in vivo and in vitro experiments, single-cell/nucleus RNA-seq data and gene knockout experiments, we confirm that the amphioxus hepatic caecum and vertebrate liver are homologous organs and show that the emergence of ohnologues from two rounds of whole-genome duplications greatly contributed to the functional complexity of the vertebrate liver. Two ohnologues, kdr and flt4, play an important role in the development of liver sinusoidal endothelial cells. In addition, we found that liver-related functions such as coagulation and bile production evolved in a step-by-step manner, with gene duplicates playing a crucial role. We reconstructed the genetic footprint of the transfer of haem detoxification from the liver to the spleen during vertebrate evolution. Together, these findings challenge the previous hypothesis that organ evolution is primarily driven by regulatory elements, underscoring the importance of gene duplicates in the emergence and diversification of a complex organ.

40†years of the homeobox: mechanisms of Hox spatial-temporal collinearity in vertebrates.

Animal body plans are established during embryonic development by the Hox genes. This patterning process relies on the differential expression of Hox genes along the head-to-tail axis. Hox spatial collinearity refers to the relationship between the organization of Hox genes in clusters and the differential Hox expression, whereby the relative order of the Hox genes within a cluster mirrors the spatial sequence of expression in the developing embryo. In vertebrates, the cluster organization is also associated with the timing of Hox activation, which harmonizes Hox expression with the progressive emergence of axial tissues. Thereby, in vertebrates, Hox temporal collinearity is intimately linked to Hox spatial collinearity. Understanding the mechanisms contributing to Hox temporal and spatial collinearity is thus key to the comprehension of vertebrate patterning. Here, we provide an overview of the main discoveries pertaining to the mechanisms of Hox spatial-temporal collinearity.

The making of a nucleic acid sensor at the dawn of jawed vertebrate evolution.

Self and nonself discrimination is fundamental to immunity. However, it remains largely enigmatic how the mechanisms of distinguishing nonself from self originated. As an intracellular nucleic acid sensor, protein kinase R (PKR) recognizes double-stranded RNA (dsRNA) and represents a crucial component of antiviral innate immunity. Here, we combine phylogenomic and functional analyses to show that PKR proteins probably originated from a preexisting kinase protein through acquiring dsRNA binding domains at least before the last common ancestor of jawed vertebrates during or before the Silurian period. The function of PKR appears to be conserved across jawed vertebrates. Moreover, we repurpose a protein closely related to PKR proteins into a putative dsRNA sensor, recapturing the making of PKR. Our study illustrates how a nucleic acid sensor might have originated via molecular tinkering with preexisting proteins and provides insights into the origins of innate immunity.

Varying Selection Pressure for a Na+ Sensing Site in Epithelial Na+ Channel Subunits Reflect Divergent Roles in Na+ Homeostasis.

The epithelial Na+ channel (ENaC) emerged early in vertebrates and has played a role in Na+ and fluid homeostasis throughout vertebrate evolution. We previously showed that proteolytic activation of the channel evolved at the water-to-land transition of vertebrates. Sensitivity to extracellular Na+, known as Na+ self-inhibition, reduces ENaC function when Na+ concentrations are high and is a distinctive feature of the channel. A fourth ENaC subunit, δ, emerged in jawed fishes from an α subunit gene duplication. Here, we analyzed 849 α and δ subunit sequences and found that a key Asp in a postulated Na+ binding site was nearly always present in the α subunit, but frequently lost in the δ subunit (e.g. human). Analysis of site evolution and codon substitution rates provide evidence that the ancestral α subunit had the site and that purifying selection for the site relaxed in the δ subunit after its divergence from the α subunit, coinciding with a loss of δ subunit expression in renal tissues. We also show that the proposed Na+ binding site in the α subunit is a bona fide site by conferring novel function to channels comprising human δ subunits. Together, our findings provide evidence that ENaC Na+ self-inhibition improves fitness through its role in Na+ homeostasis in vertebrates.

Study of the TEAD-binding domain of the VGLL1, VGLL2 and VGLL3 proteins from vertebrates.

The TEAD transcription factors are the final effectors of the Hippo pathway, and to exert their transcriptional activity they need to interact with other proteins. The three paralogous vestigial-like proteins VGLL1, VGLL2 and VGLL3 bind to TEAD via a conserved short linear sequence, the Tondu motif. The TEAD-binding domain of human VGLL2 contains in addition an Ω-loop, which is also present in Vg (vestigial) from arthropods and the YAP proteins, another family of TEAD interactors. In this report, using the available structural data, we study the amino acid sequence of the TEAD-binding domain of more than 2400 putative VGLL proteins from vertebrates. This analysis shows a strong link between sequence conservation and functional role for the residues from the Tondu motif. It also reveals that one protein sequence containing both a Tondu motif and an Ω-loop is present in most (if not all) vertebrate species. This suggests that there is a selective pressure to keep a VGLL paralog with a functional Ω-loop in vertebrates. Finally, this study identifies, particularly in mammals, variants of VGLL2 and VGLL3 with an altered TEAD-binding domain suggesting that they may have a different biological function than their homologs.

Functional and regulatory diversification of Period genes responsible for circadian rhythm in vertebrates.

The Period genes (Per) play essential roles in modulating the molecular circadian clock timing in a broad range of species, which regulates the physiological and cellular rhythms through the transcription-translation feedback loop. While the Period gene paralogs are widely observed among vertebrates, the evolutionary history and the functional diversification of Per genes across vertebrates are not well known. In this study, we comprehensively investigated the evolution of Per genes at the copy number and sequence levels, including de novo binding motif discovery by comparative genomics. We also determined the lineage-specific transcriptome landscape across tissues and developmental stages and phenotypic effects in public RNA-seq data sets of model species. We observed multiple lineage-specific gain and loss events Per genes, though no simple association was observed between ecological factors and Per gene numbers in each species. Among salmonid fish species, the per3 gene has been lost in the majority, whereas those retaining the per3 gene exhibit not a signature of relaxed selective constraint but rather a signature of intensified selection. We also determined the signature of adaptive diversification of the CRY-binding region in Per1 and Per3, which modulates the circadian rhythm. We also discovered putative regulatory sequences, which are lineage-specific, suggesting that these cis-regulatory elements may have evolved rapidly and divergently across different lineages. Collectively, our findings revealed the evolution of Per genes and their fine-tuned contribution to the plastic and precise regulation of circadian rhythms in various vertebrate taxa.

Sulfotransferase 4A1 Coding Sequence and Protein Structure Are Highly Conserved in Vertebrates.

Cytosolic sulfotransferases (SULTs) are Phase 2 drug-metabolizing enzymes that catalyze the conjugation of sulfonate to endogenous and xenobiotic compounds, increasing their hydrophilicity and excretion from cells. To date, 13 human SULTs have been identified and classified into five families. SULT4A1 mRNA encodes two variants: (1) the wild type, encoding a 284 amino acid, ~33 kDa protein, and (2) an alternative spliced variant resulting from a 126 bp insert between exon 6 and 7, which introduces a premature stop codon that enhances nonsense-mediated decay. SULT4A1 is classified as an SULT based on sequence and structural similarities, including PAPS-domains, active-site His, and the dimerization domain; however, the catalytic pocket lid 'Loop 3' size is not conserved. SULT4A1 is uniquely expressed in the brain and localized in the cytosol and mitochondria. SULT4A1 is highly conserved, with rare intronic polymorphisms that have no outward manifestations. However, the SULT4A1 haplotype is correlated with Phelan-McDermid syndrome and schizophrenia. SULT4A1 knockdown revealed potential SULT4A1 functions in photoreceptor signaling and knockout mice display hampered neuronal development and behavior. Mouse and yeast models revealed that SULT4A1 protects the mitochondria from endogenously and exogenously induced oxidative stress and stimulates cell division, promoting dendritic spines' formation and synaptic transmission. To date, no physiological enzymatic activity has been associated with SULT4A1.

Progressive modifications during evolution involving epigenetic changes have determined loss of regeneration mainly in terrestrial animals: A hypothesis.

In order to address a biological explanation for the different regenerative abilities present among animals, a new evolutionary speculation is presented. It is hypothesized that epigenetic mechanisms have lowered or erased regeneration during the evolution of terrestrial invertebrates and vertebrates. The hypothesis indicates that a broad regeneration can only occur in marine or freshwater conditions, and that life on land does not allow for high regeneration. This is due to the physical, chemical and microbial conditions present in the terrestrial environment with respect to those of the aquatic environment. The present speculation provides examples of hypothetic evolutionary animal lineages that colonized the land, such as parasitic annelids, terrestrial mollusks, arthropods and amniotes. These are the animals where regeneration is limited or absent and their injuries are only repaired through limited healing or scarring. It is submitted that this loss derived from changes in the developmental gene pathways sustaining regeneration in the aquatic environment but that cannot be expressed on land. Once regeneration was erased in terrestrial species, re-adaptation to freshwater niches could not reactivate the previously altered gene pathways that determined regeneration. Therefore a broad regeneration was no longer possible or became limited and heteromorphic in the derived, extant animals. Only in few cases extensive healing abilities or regengrow, a healing process where regeneration overlaps with somatic growth, have evolved among arthropods and amniotes. The present paper is an extension of previous speculations trying to explain in biological terms the different regenerative abilities present among metazoans.

Century-old chromatin architecture revealed in formalin-fixed vertebrates.

Gene expression is regulated by changes in chromatin architecture intrinsic to cellular differentiation and as an active response to environmental stimuli. Chromatin dynamics are a major driver of phenotypic diversity, regulation of development, and manifestation of disease. Remarkably, we know little about the evolutionary dynamics of chromatin reorganisation through time, data essential to characterise the impact of environmental stress during the ongoing biodiversity extinction crisis (20th-21st century). Linking the disparate fields of chromatin biology and museum science through their common use of the preservative formaldehyde (a constituent of formalin), we have generated historical chromatin profiles in museum specimens up to 117 years old. Historical chromatin profiles are reproducible, tissue-specific, sex-specific, and environmental condition-dependent in vertebrate specimens. Additionally, we show that over-fixation modulates differential chromatin accessibility to enable semi-quantitative estimates of relative gene expression in vertebrates and a yeast model. Our approach transforms formalin-fixed biological collections into an accurate, comprehensive, and global record of environmental impact on gene expression and phenotype.

The Evolution of Ultraconserved Elements in Vertebrates.

Ultraconserved elements were discovered two decades ago, arbitrarily defined as sequences that are identical over a length ≥ 200 bp in the human, mouse, and rat genomes. The definition was subsequently extended to sequences ≥ 100 bp identical in at least three of five mammalian genomes (including dog and cow), and shown to have undergone rapid expansion from ancestors in fish and strong negative selection in birds and mammals. Since then, many more genomes have become available, allowing better definition and more thorough examination of ultraconserved element distribution and evolutionary history. We developed a fast and flexible analytical pipeline for identifying ultraconserved elements in multiple genomes, dedUCE, which allows manipulation of minimum length, sequence identity, and number of species with a detectable ultraconserved element according to specified parameters. We suggest an updated definition of ultraconserved elements as sequences ≥ 100 bp and ≥97% sequence identity in ≥50% of placental mammal orders (12,813 ultraconserved elements). By mapping ultraconserved elements to ∼200 species, we find that placental ultraconserved elements appeared early in vertebrate evolution, well before land colonization, suggesting that the evolutionary pressures driving ultraconserved element selection were present in aquatic environments in the Cambrian-Devonian periods. Most (>90%) ultraconserved elements likely appeared after the divergence of gnathostomes from jawless predecessors, were largely established in sequence identity by early Sarcopterygii evolution-before the divergence of lobe-finned fishes from tetrapods-and became near fixed in the amniotes. Ultraconserved elements are mainly located in the introns of protein-coding and noncoding genes involved in neurological and skeletomuscular development, enriched in regulatory elements, and dynamically expressed throughout embryonic development.

Sperm-Specific CatSper is Not Conserved in All Vertebrates and May Not be the Only Progesterone-Responsive Ion Channel Present in Sperm.

Progesterone (P4) acts as a key conserved signalling molecule in vertebrate reproduction. P4 is especially important for mature sperm physiology and subsequent reproductive success. "CatSpermasome", a multi-unit molecular complex, has been suggested to be the main if not the only P4-responsive atypical Ca2+-ion channel present in mature sperm. Altogether, here we analyse the protein sequences of CatSper1-4 from more than 500 vertebrates ranging from early fishes to humans. CatSper1 becomes longer in mammals due to sequence gain mainly at the N-terminus. Overall the conservation of full-length CatSper1-4 as well as the individual TM regions remain low. The lipid-water-interface residues (i.e. a 5 amino acid stretch sequence present on both sides of each TM region) also remain highly diverged. No specific patterns of amino acid distributions were observed. The total frequency of positively charged, negatively charged or their ratios do not follow in any specific pattern. Similarly, the frequency of total hydrophobic, total hydrophilic residues or even their ratios remain random and do not follow any specific pattern. We noted that the CatSper1-4 genes are missing in amphibians and the CatSper1 gene is missing in birds. The high variability of CatSper1-4 and gene-loss in certain clades indicate that the "CatSpermasome" is not the only P4-responsive ion channel. Data indicate that the molecular evolution of CatSper is mostly guided by diverse hydrophobic ligands rather than only P4. The comparative data also suggest possibilities of other Ca2+-channel/s in vertebrate sperm that can also respond to P4.

Macroecological correlates of Darwinian shortfalls across terrestrial vertebrates.

Most described species have not been explicitly included in phylogenetic trees-a problem named the Darwinian shortfall-owing to a lack of molecular and/or morphological data, thus hampering the explicit incorporation of evolution into large-scale biodiversity analyses. We investigate potential drivers of the Darwinian shortfall in tetrapods, a group in which at least one-third of described species still lack phylogenetic data, thus necessitating the imputation of their evolutionary relationships in fully sampled phylogenies. We show that the number of preserved specimens in scientific collections is the main driver of phylogenetic knowledge accumulation, highlighting the major role of biological collections in unveiling novel biodiversity data and the importance of continued sampling efforts to reduce knowledge gaps. Additionally, large-bodied and wide-ranged species, as well as terrestrial and aquatic amphibians and reptiles, are phylogenetically better known. Future efforts should prioritize phylogenetic research on organisms that are narrow-ranged, small-bodied and underrepresented in scientific collections, such as fossorial species. Addressing the Darwinian shortfall will be imperative for advancing our understanding of evolutionary drivers shaping biodiversity patterns and implementing comprehensive conservation strategies.

Neuropeptides and receptors in the cephalochordate: A crucial model for understanding the origin and evolution of vertebrate neuropeptide systems.

Genomes and transcriptomes from diverse organisms are providing a wealth of data to explore the evolution and origin of neuropeptides and their receptors in metazoans. While most neuropeptide-receptor systems have been extensively studied in vertebrates, there is still a considerable lack of understanding regarding their functions in invertebrates, an extraordinarily diverse group that account for the majority of animal species on Earth. Cephalochordates, commonly known as amphioxus or lancelets, serve as the evolutionary proxy of the chordate ancestor. Their key evolutionary position, bridging the invertebrate to vertebrate transition, has been explored to uncover the origin, evolution, and function of vertebrate neuropeptide systems. Amphioxus genomes exhibit a high degree of sequence and structural conservation with vertebrates, and sequence and functional homologues of several vertebrate neuropeptide families are present in cephalochordates. This review aims to provide a comprehensively overview of the recent findings on neuropeptides and their receptors in cephalochordates, highlighting their significance as a model for understanding the complex evolution of neuropeptide signaling in vertebrates.

Vertebrate centromere architecture: from chromatin threads to functional structures.

Centromeres are chromatin structures specialized in sister chromatid cohesion, kinetochore assembly, and microtubule attachment during chromosome segregation. The regional centromere of vertebrates consists of long regions of highly repetitive sequences occupied by the Histone H3 variant CENP-A, and which are flanked by pericentromeres. The three-dimensional organization of centromeric chromatin is paramount for its functionality and its ability to withstand spindle forces. Alongside CENP-A, key contributors to the folding of this structure include components of the Constitutive Centromere-Associated Network (CCAN), the protein CENP-B, and condensin and cohesin complexes. Despite its importance, the intricate architecture of the regional centromere of vertebrates remains largely unknown. Recent advancements in long-read sequencing, super-resolution and cryo-electron microscopy, and chromosome conformation capture techniques have significantly improved our understanding of this structure at various levels, from the linear arrangement of centromeric sequences and their epigenetic landscape to their higher-order compaction. In this review, we discuss the latest insights on centromere organization and place them in the context of recent findings describing a bipartite higher-order organization of the centromere.