HOST SWITCHING IN DICYEMIDS (PHYLUM DICYEMIDA).

Dicyemids (phylum Dicyemida) are the most common and most characteristic endosymbionts in the renal sacs of benthic cephalopod molluscs: octopuses and cuttlefishes. Typically, 2 or 3 dicyemid species are found in a single specimen of the host, and most dicyemids have high host specificity. Host-specific parasites are restricted to a limited range of host species by ecological barriers that impede dispersal and successful establishment; therefore, phylogenies of interacting groups are often congruent due to repeated co-speciation. Most frequently, however, host and parasite phylogenies are not congruent, which can be explained by processes such as host switching and other macro-evolutionary events. Here, the history of dicyemids and their host cephalopod associations were studied by comparing their phylogenies. Dicyemid species were collected from 8 decapodiform species and 12 octopodiform species in Japanese waters. Using whole mitochondrial cytochrome c oxidase subunit 1 (COI) sequences, a phylogeny of 37 dicyemid species, including 4 genera representing the family Dicyemidae, was reconstructed. Phylogenetic trees derived from analyses of COI genes consistently suggested that dicyemid species should be separated into 3 major clades and that the most common genera, Dicyema and Dicyemennea, are not monophyletic. Thus, morphological classification does not reflect the phylogenetic relationships of these 2 genera. Divergence (speciation) of dicyemid species seems to have occurred within a single host species. Possible host-switching events may have occurred between the Octopodiformes and Decapodiformes or within the Octopodiformes or the Decapodiformes. Therefore, the mechanism of dicyemid speciation may be a mixture of host switching and intra-host speciation. This is the first study in which the process of dicyemid diversification involving cephalopod hosts has been evaluated with a large number of dicyemid species and genera.

A practical guide for the husbandry of cave and surface invertebrates as the first step in establishing new model organisms.

While extensive research on traditional model species has significantly advanced the biological sciences, the ongoing search for new model organisms is essential to tackle contemporary challenges such as human diseases or climate change, and fundamental phenomena including adaptation or speciation. Recent methodological advances such as next-generation sequencing, gene editing, and imaging are widely applicable and have simplified the selection of species with specific traits from the wild. However, a critical milestone in this endeavor remains the successful cultivation of selected species. A historically overlooked but increasingly recognized group of non-model organisms are cave dwellers. These unique animals offer invaluable insights into the genetic basis of human diseases like eye degeneration, metabolic and neurological disorders, and basic evolutionary principles and the origin of adaptive phenotypes. However, to take advantage of the beneficial traits of cave-dwelling animals, laboratory cultures must be established-a practice that remains extremely rare except for the cavefish Astyanax mexicanus. For most cave-dwelling organisms, there are no published culturing protocols. In this study, we present the results of our multi-year effort to establish laboratory cultures for a variety of invertebrate groups. We have developed comprehensive protocols for housing, feeding, and husbandry of cave dwellers and their surface relatives. Our recommendations are versatile and can be applied to a wide range of species. Hopefully our efforts will facilitate the establishment of new laboratory animal facilities for cave-dwelling organisms and encourage their greater use in experimental biology.

From DNA to diagnostics: A case study using macroinvertebrate metabarcoding to assess the effectiveness of restoration measures in a Dutch stream.

Stream ecosystems are under pressure due to multiple stressors. Restoration measures can halt further degradation and improve their ecological status. However, assessment of the effectiveness of the implemented measures is often insufficient because of logistic and financial constraints. DNA-metabarcoding has been proposed to scale up sample processing, although its application as a diagnostic tool has received less attention. The aim of our study was to evaluate if DNA-metabarcoding of stream macroinvertebrates can be used to compute a stressor-specific index to assess the effectiveness of a stream restoration project. For this purpose, we sampled the upstream, restored, and downstream section of a recently restored lowland stream in the Netherlands. At each site, we applied three different methods of macroinvertebrate identification: morphological identification of bulk samples (morphology), DNA-metabarcoding of the same bulk samples (DNA) and metabarcoding of eDNA extracted from the water (eDNA). First, we compared the community composition identified by each method. The communities identified by morphology and DNA were highly similar, whereas the communities generated by the eDNA differed. Second, we analysed whether the identification methods could be used to assess the effectiveness of the restoration project, focussing on a stressor-specific index for flow as the restoration measures aimed at improving flow conditions. Both the morphology and bulk DNA samples indicated improved flow conditions in the restored section of the stream (i.e., less stress from the reduction or absence of flow than in the unrestored sections). Contrary, the eDNA-water samples did not differentiate the amount of stress throughout the catchment, although applying recent developments in eDNA sampling could lead to more robust results. In conclusion, this study forms proof of concept that DNA from bulk samples can be utilized to assess the effectiveness of restoration measures, showing the added value of this approach for water managers.

Anciently duplicated genes continuously recruited to heart expression in vertebrate evolution are associated with heart chamber increase.

Although gene/genome duplications in the early stage of vertebrates have been thought to provide major resources of raw genetic materials for evolutionary innovations, it is unclear whether they continuously contribute to the evolution of morphological complexity during the course of vertebrate evolution, such as the evolution from two heart chambers (fishes) to four heart chambers (mammals and birds). We addressed this issue by our heart RNA-Seq experiments combined with published data, using 13 vertebrates and one invertebrate (sea squirt, as an outgroup). Our evolutionary transcriptome analysis showed that number of ancient paralogous genes expressed in heart tends to increase with the increase of heart chamber number along the vertebrate phylogeny, in spite that most of them were duplicated at the time near to the origin of vertebrates or even more ancient. Moreover, those paralogs expressed in heart exert considerably different functions from heart-expressed singletons: the former are functionally enriched in cardiac muscle and muscle contraction-related categories, whereas the latter play more basic functions of energy generation like aerobic respiration. These findings together support the notion that recruiting anciently paralogous genes that are expressed in heart is associated with the increase of chamber number in vertebrate evolution.

Revisiting the evolution of Family B1 GPCRs and ligands: Insights from mollusca.

Family B1 G protein-coupled receptors (GPCRs) are one of the most well studied neuropeptide receptor families since they play a central role in many biological processes including endocrine, gastrointestinal, cardiovascular and reproduction in animals. The genes for these receptors emerged from a common ancestral gene in bilaterian genomes and evolved via gene/genome duplications and deletions in vertebrate and invertebrate genomes. Their existence and function have mostly been characterized in vertebrates and few studies exist in invertebrate species. Recently, an increased interest in molluscs, means a series of genomes have become available, and since they are less modified than insect and nematode genomes, they are ideal to explore the origin and evolution of neuropeptide gene families. This review provides an overview of Family B1 GPCRs and their peptide ligands and incorporates new data obtained from Mollusca genomes and taking a comparative approach challenges existing models on their origin and evolution.

Comparative mitogenomics of Brachiopods reveals conservatism in articulate species and unusualness in inarticulate species.

BACKGROUND: Brachiopods are a phylum of marine invertebrates with over 10,000 fossil species. Today, there are fewer than 500 extant species assigned to the class Articulata or Inarticulata and for which knowledge of evolutionary genetics and genomics is still poor. Until now, complete mitogenome sequences of two inarticulate species and four articulate species were available. METHODS AND RESULTS: The complete mitogenome of the inarticulate brachiopod species Lingula reevii (20,778 bp) was obtained by using next generation sequencing. It contains 12 protein-coding genes (the annotation of atp8 is unsure), two ribosomal RNA genes, 26 transfer RNA genes, and one supernumerary ORF that is also conserved in the inarticulate species Lingula anatina. It is hypothesized that this ORF could represent a Lingula-specific mtORFan gene (without obvious homology to other genes). Comparative mitogenomics indicate the mitochondrial gene order of L. reevii is unique among brachiopods, and that compared to articulate species, inarticulate species exhibit massive mitogenome rearrangements, deviant ATP8 protein sequences and supernumerary ORFs, possibly representing species- or lineage-specific mtORFan genes. CONCLUSION: The results of this study enrich genetics knowledge of extant brachiopods, which may eventually help to test hypotheses about their decline.

Genome-Wide Comparative Analysis of SRCR Gene Superfamily in Invertebrates Reveals Massive and Independent Gene Expansions in the Sponge and Sea Urchin.

Without general adaptative immunity, invertebrates evolved a vast number of heterogeneous non-self recognition strategies. One of those well-known adaptations is the expansion of the immune receptor gene superfamily coding for scavenger receptor cysteine-rich domain containing proteins (SRCR) in a few invertebrates. Here, we investigated the evolutionary history of the SRCR gene superfamily (SRCR-SF) across 29 metazoan species with an emphasis on invertebrates. We analyzed their domain architectures, genome locations and phylogenetic distribution. Our analysis shows extensive genome-wide duplications of the SRCR-SFs in Amphimedon queenslandica and Strongylocentrotus purpuratus. Further molecular evolution study reveals various patterns of conserved cysteines in the sponge and sea urchin SRCR-SFs, indicating independent and convergent evolution of SRCR-SF expansion during invertebrate evolution. In the case of the sponge SRCR-SFs, a novel motif with seven conserved cysteines was identified. Exon-intron structure analysis suggests the rapid evolution of SRCR-SFs during gene duplications in both the sponge and the sea urchin. Our findings across nine representative metazoans also underscore a heightened expression of SRCR-SFs in immune-related tissues, notably the digestive glands. This observation indicates the potential role of SRCR-SFs in reinforcing distinct immune functions in these invertebrates. Collectively, our results reveal that gene duplication, motif structure variation, and exon-intron divergence might lead to the convergent evolution of SRCR-SF expansions in the genomes of the sponge and sea urchin. Our study also suggests that the utilization of SRCR-SF receptor duplication may be a general and basal strategy to increase immune diversity and tissue specificity for the invertebrates.

Analysis of codon usage bias of exonuclease genes in invertebrate iridescent viruses.

Invertebrate iridescent viruses (IIVs) are double-stranded DNA viruses that belong to the Iridoviridae family. IIVs result diseases that vary in severity from subclinical to lethal in invertebrate hosts. Codon usage bias (CUB) analysis is a versatile method for comprehending the genetic and evolutionary aspects of species. In this study, we analyzed the CUB in 10 invertebrate iridescent viruses exonuclease genes by calculating and comparing the nucleotide contents, effective number of codons (ENC), codon adaptation index (CAI), relative synonymous codon usage (RSCU), and others. The results revealed that IIVs exonuclease genes are rich in A/T. The ENC analysis displayed a low codon usage bias in IIVs exonuclease genes. ENC-plot, neutrality plot, and parity rule 2 plot demonstrated that besides mutational pressure, other factors like natural selection, dinucleotide content, and aromaticity also contributed to CUB. The findings could enhance our understanding of the evolution of IIVs exonuclease genes.

Functional Characterization of Gonadotropin-Releasing Hormone and Corazonin Signaling Systems in Pacific Abalone: Toward Reclassification of Invertebrate Neuropeptides.

INTRODUCTION: The proposed evolutionary origins and corresponding nomenclature of bilaterian gonadotropin-releasing hormone (GnRH)-related neuropeptides have changed tremendously with the aid of receptor deorphanization. However, the reclassification of the GnRH and corazonin (CRZ) signaling systems in Lophotrochozoa remains unclear. METHODS: We characterized GnRH and CRZ receptors in the mollusk Pacific abalone, Haliotis discus hannai (Hdh), by phylogenetic and gene expression analyses, bioluminescence-based reporter, Western blotting, substitution of peptide amino acids, in vivo neuropeptide injection, and RNA interference assays. RESULTS: Two Hdh CRZ-like receptors (Hdh-CRZR-A and Hdh-CRZR-B) and three Hdh GnRH-like receptors (Hdh-GnRHR1-A, Hdh-GnRHR1-B, and Hdh-GnRHR2) were identified. In phylogenetic analysis, Hdh-CRZR-A and -B grouped within the CRZ-type receptors, whereas Hdh-GnRHR1-A/-B and Hdh-GnRHR2 clustered within the GnRH/adipokinetic hormone (AKH)/CRZ-related peptide-type receptors. Hdh-CRZR-A/-B and Hdh-GnRHR1-A were activated by Hdh-CRZ (pQNYHFSNGWHA-NH2) and Hdh-GnRH (pQISFSPNWGT-NH2), respectively. Hdh-CRZR-A/-B dually coupled with the Gαq and Gαs signaling pathways, whereas Hdh-GnRHR1-A was linked only with Gαq signaling. Analysis of substituted peptides, [I2S3]Hdh-CRZ and [N2Y3H4]Hdh-GnRH, and in silico docking models revealed that the N-terminal amino acids of the peptides are critical for the selectivity of Hdh-CRZR and Hdh-GnRHR. Two precursor transcripts for Hdh-CRZ and Hdh-GnRH peptides and their receptors were mainly expressed in the neural ganglia, and their levels increased in starved abalones. Injection of Hdh-CRZ peptide into abalones decreased food consumption, whereas Hdh-CRZR knockdown increased food consumption. Moreover, Hdh-CRZ induced germinal vesicle breakdown in mature oocytes. CONCLUSION: Characterization of Hdh-CRZRs and Hdh-GnRHRs and their cognate peptides provides new insight into the evolutionary route of GnRH-related signaling systems in bilaterians.

Molecular evolution of the Thrombospondin superfamily.

Thrombospondins (TSPs) are multidomain, calcium-binding glycoproteins that have wide-ranging roles in vertebrates in cell interactions, extracellular matrix (ECM) organisation, angiogenesis, tissue remodelling, synaptogenesis, and also in musculoskeletal and cardiovascular functions. Land animals encode five TSPs, which assembly co-translationally either as trimers (subgroup A) or pentamers (subgroup B). The vast majority of research has focused on this canonical TSP family, which evolved through the whole-genome duplications that took place early in the vertebrate lineage. With benefit of the growth in genome- and transcriptome-predicted proteomes of a much wider range of animal species, examination of TSPs throughout metazoan phyla has revealed extensive conservation of subgroup B-type TSPs in invertebrates. In addition, these searches established that canonical TSPs are, in fact, one branch within a TSP superfamily that includes other clades designated mega-TSPs, sushi-TSPs and poriferan-TSPs. Despite the apparent simplicity of poriferans and cnidarians as organisms, these phyla encode a greater diversity of TSP superfamily members than vertebrates. We discuss here the molecular characteristics of the TSP superfamily members, current knowledge of their expression profiles and functions in invertebrates, and models for the evolution of this complex ECM superfamily.

FicD genes in invertebrates: A tale of transposons, pathogenic and integrated viruses.

Many gene families are shared across the tree of life between distantly related species because of horizontal gene transfers (HGTs). However, the frequency of HGTs varies strongly between gene families and biotic realms suggesting differential selection pressures and functional bias. One gene family with a wide distribution are FIC-domain containing enzymes (FicDs). FicDs catalyze AMPylation, a post-translational protein modification consisting in the addition of adenosine monophosphate to accessible residues of target proteins. Beside the well-known conservation of FicDs in deuterostomes, we report the presence of a conserved FicD gene ortholog in a large number of protostomes and microbial eukaryotes. We also reported additional FicD gene copies in the genomes of some rotifers, parasitic worms and bivalves. A few dsDNA viruses of these invertebrates, including White spot syndrome virus, Cherax quadricarinatus iridovirus, Ostreid herpesvirus-1 and the beetle nudivirus, carry copies of FicDs, with phylogenetic analysis suggesting a common origin of these FicD copies and the duplicated FicDs of their invertebrate hosts. HGTs and gene duplications possibly mediated by endogenous viruses or genetic mobile elements seem to have contributed to the transfer of AMPylation ability from bacteria and eukaryotes to pathogenic viruses, where this pathway could have been hijacked to promote viral infection.

The MetaInvert soil invertebrate genome resource provides insights into below-ground biodiversity and evolution.

Soil invertebrates are among the least understood metazoans on Earth. Thus far, the lack of taxonomically broad and dense genomic resources has made it hard to thoroughly investigate their evolution and ecology. With MetaInvert we provide draft genome assemblies for 232 soil invertebrate species, representing 14 common groups and 94 families. We show that this data substantially extends the taxonomic scope of DNA- or RNA-based taxonomic identification. Moreover, we confirm that theories of genome evolution cannot be generalised across evolutionarily distinct invertebrate groups. The soil invertebrate genomes presented here will support the management of soil biodiversity through molecular monitoring of community composition and function, and the discovery of evolutionary adaptations to the challenges of soil conditions.

APGW/AKH Precursor from Rotifer Brachionus plicatilis and the DNA Loss Model Explain Evolutionary Trends of the Neuropeptide LWamide, APGWamide, RPCH, AKH, ACP, CRZ, and GnRH Families.

In the year 2002, DNA loss model (DNA-LM) postulated that neuropeptide genes to emerged through codons loss via the repair of damaged DNA from ancestral gene namely Neuropeptide Precursor Predictive (NPP), which organization correspond two or more neuropeptides precursors evolutive related. The DNA-LM was elaborated according to amino acids homology among LWamide, APGWamide, red pigment-concentrating hormone (RPCH), adipokinetic hormones (AKHs) and in silico APGW/RPCH NPPAPGW/AKH NPP were proposed. With the above principle, it was proposed the evolution of corazonin (CRZ), gonadotropin-releasing hormone (GnRH), AKH, and AKH/CRZ (ACP), but any NPP never was considered. However, the evolutive relation via DNA-LM among these neuropeptides precursors not has been established yet. Therefore, the transcriptomes from crabs Callinectes toxotes and Callinectes arcuatus were used to characterized ACP and partial CRZ precursors, respectively. BLAST alignment with APGW/RPCH NPP and APGW/AKH NPP allow identified similar NPP in the rotifer Brachionus plicatilis and other invertebrates. Moreover, three bioinformatics algorithms and manual verification were used to purify 13,778 sequences, generating a database with 719 neuropeptide precursors. Phylogenetic trees with the DNA-LM parameters showed that some ACP, CRZ, AKH2 and two NPP share nodes with GnRH from vertebrates and some of this neuropeptide had nodes in invertebrates. Whereas the phylogenetic tree with standard parameters do not showed previous node pattern. Robinson-Foulds metric corroborates the differences among phylogenetic trees. Homology relationship showed four putative orthogroups; AKH4, CRZ, and protostomes GnRH had individual group. This is the first demonstration of NPP in species and would explain the evolution neuropeptide families by the DNA-LM.

Genomic and transcriptomic survey of bryozoan Hox and ParaHox genes with emphasis on phylactolaemate bryozoans.

BACKGROUND: Bryozoans are mostly sessile aquatic colonial invertebrates belonging to the clade Lophotrochozoa, which unites many protostome bilaterian phyla such as molluscs, annelids and brachiopods. While Hox and ParaHox genes have been extensively studied in various lophotrochozoan lineages, investigations on Hox and ParaHox gene complements in bryozoans are scarce. RESULTS: Herein, we present the most comprehensive survey of Hox and ParaHox gene complements in bryozoans using four genomes and 35 transcriptomes representing all bryozoan clades: Cheilostomata, Ctenostomata, Cyclostomata and Phylactolaemata. Using similarity searches, phylogenetic analyses and detailed manual curation, we have identified five Hox genes in bryozoans (pb, Dfd, Lox5, Lox4 and Post2) and one ParaHox gene (Cdx). Interestingly, we observed lineage-specific duplication of certain Hox and ParaHox genes (Dfd, Lox5 and Cdx) in some bryozoan lineages. CONCLUSIONS: The bryozoan Hox cluster does not retain the ancestral lophotrochozoan condition but appears relatively simple (includes only five genes) and broken into two genomic regions, characterized by the loss and duplication of serval genes. Importantly, bryozoans share the lack of two Hox genes (Post1 and Scr) with their proposed sister-taxon, Phoronida, which suggests that those genes were missing in the most common ancestor of bryozoans and phoronids.

Evidence for an ancient aquatic origin of the RNA viral order Articulavirales.

The emergence of previously unknown disease-causing viruses in mammals is in part the result of a long-term evolutionary process. Reconstructing the deep phylogenetic histories of viruses helps identify major evolutionary transitions and contextualizes the emergence of viruses in new hosts. We used a combination of total RNA sequencing and transcriptome data mining to extend the diversity and evolutionary history of the RNA virus order Articulavirales, which includes the influenza viruses. We identified instances of Articulavirales in the invertebrate phylum Cnidaria (including corals), constituting a novel and divergent family that we provisionally named the "Cnidenomoviridae." We further extended the evolutionary history of the influenza virus lineage by identifying four divergent, fish-associated influenza-like viruses, thereby supporting the hypothesis that fish were among the first hosts of influenza viruses. In addition, we substantially expanded the phylogenetic diversity of quaranjaviruses and proposed that this genus be reclassified as a family-the "Quaranjaviridae." Within this putative family, we identified a novel arachnid-infecting genus, provisionally named "Cheliceravirus." Notably, we observed a close phylogenetic relationship between the Crustacea- and Chelicerata-infecting "Quaranjaviridae" that is inconsistent with virus-host codivergence. Together, these data suggest that the Articulavirales has evolved over at least 600 million years, first emerging in aquatic animals. Importantly, the evolution of the Articulavirales was likely shaped by multiple aquatic-terrestrial transitions and substantial host jumps, some of which are still observable today.

A trait-based approach to assess niche overlap and functional distinctiveness between non-indigenous and native species.

Our understanding of the community assembly processes acting on non-indigenous species (NIS), as well as the relationship with native species is limited, especially in marine ecosystems. To overcome this knowledge gap we here develop a trait-based approach based on the functional distinctiveness metric to assess niche overlap between NIS and native species, using high-resolution data on benthic invertebrate communities in the Baltic Sea. Our results show that NIS retain a certain degree of similarity with native species, but display one or a few singular unique traits (e.g., bioturbation ability). Furthermore, we demonstrate that community assembly processes, including both environmental filtering and limiting similarity affect NIS establishment, but that their effects may be highly context dependent, as illustrated by pronounced spatial patterns in distinctiveness. Finally, our trait-based approach provides a generic framework applicable to other areas and organisms, to better understand and address biological invasions.

Dinucleotide biases in RNA viruses that infect vertebrates or invertebrates.

IMPORTANCE: Akin to a molecular signature, dinucleotide composition can be exploited by the zinc-finger antiviral protein (ZAP) to restrict CpG-rich (and UpA-rich) RNA viruses. ZAP evolved in tetrapods, and it is not encoded by invertebrates and fish. Because a systematic analysis is missing, we analyzed the genomes of RNA viruses that infect vertebrates or invertebrates. We show that vertebrate single-stranded (ss) RNA(+) viruses and, to a lesser extent, double-stranded RNA viruses tend to have stronger CpG bias than invertebrate viruses. Conversely, ssRNA(-) viruses have similar dinucleotide composition whether they infect vertebrates or invertebrates. Analysis of ssRNA(+) viruses that infect mammals, reptiles, and fish indicated that ZAP is unlikely to be a major driver of CpG depletion. We also show that, compared to other coronaviruses, the genome of SARS-CoV-2 is not homogeneously CpG-depleted. Our study provides new insights into virus evolution and strategies for recoding RNA virus genomes.

Stepwise emergence of the neuronal gene expression program in early animal evolution.

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.

Discovery of Paralogous GnRH and Corazonin Signaling Systems in an Invertebrate Chordate.

Gonadotropin-releasing hormone (GnRH) is a key regulator of reproductive function in vertebrates. GnRH is related to the corazonin (CRZ) neuropeptide which influences metabolism and stress responses in insects. Recent evidence suggests that GnRH and CRZ are paralogous and arose by a gene duplication in a common ancestor of bilaterians. Here, we report the identification and complete characterization of the GnRH and CRZ signaling systems in the amphioxus Branchiostoma floridae. We have identified a novel GnRH peptide (YSYSYGFAP-NH2) that specifically activates two GnRH receptors and a CRZ peptide (FTYTHTW-NH2) that activates three CRZ receptors in B. floridae. The latter appear to be promiscuous, as two CRZ receptors can also be activated by GnRH in the physiological range. Hence, there is a potential for cross-talk between these closely related signaling systems. Discovery of both the GnRH and CRZ signaling systems in one of the closest living relatives of vertebrates provides a framework to discover their roles at the transition from invertebrates to vertebrates.

Direct observation of the evolution of cell-type-specific microRNA expression signatures supports the hematopoietic origin model of endothelial cells.

The evolution of specialized cell-types is a long-standing interest of biologists, but given the deep time-scales very difficult to reconstruct or observe. microRNAs have been linked to the evolution of cellular complexity and may inform on specialization. The endothelium is a vertebrate-specific specialization of the circulatory system that enabled a critical new level of vasoregulation. The evolutionary origin of these endothelial cells is unclear. We hypothesized that Mir-126, an endothelial cell-specific microRNA may be informative. We here reconstruct the evolutionary history of Mir-126. Mir-126 likely appeared in the last common ancestor of vertebrates and tunicates, which was a species without an endothelium, within an intron of the evolutionary much older EGF Like Domain Multiple (Egfl) locus. Mir-126 has a complex evolutionary history due to duplications and losses of both the host gene and the microRNA. Taking advantage of the strong evolutionary conservation of the microRNA among Olfactores, and using RNA in situ hybridization, we localized Mir-126 in the tunicate Ciona robusta. We found exclusive expression of the mature Mir-126 in granular amebocytes, supporting a long-proposed scenario that endothelial cells arose from hemoblasts, a type of proto-endothelial amoebocyte found throughout invertebrates. This observed change of expression of Mir-126 from proto-endothelial amoebocytes in the tunicate to endothelial cells in vertebrates is the first direct observation of the evolution of a cell-type in relation to microRNA expression indicating that microRNAs can be a prerequisite of cell-type evolution.