Revisiting genomes of non-model species with long reads yields new insights into their biology and evolution.

High-quality genomes obtained using long-read data allow not only for a better understanding of heterozygosity levels, repeat content, and more accurate gene annotation and prediction when compared to those obtained with short-read technologies, but also allow to understand haplotype divergence. Advances in long-read sequencing technologies in the last years have made it possible to produce such high-quality assemblies for non-model organisms. This allows us to revisit genomes, which have been problematic to scaffold to chromosome-scale with previous generations of data and assembly software. Nematoda, one of the most diverse and speciose animal phyla within metazoans, remains poorly studied, and many previously assembled genomes are fragmented. Using long reads obtained with Nanopore R10.4.1 and PacBio HiFi, we generated highly contiguous assemblies of a diploid nematode of the Mermithidae family, for which no closely related genomes are available to date, as well as a collapsed assembly and a phased assembly for a triploid nematode from the Panagrolaimidae family. Both genomes had been analysed before, but the fragmented assemblies had scaffold sizes comparable to the length of long reads prior to assembly. Our new assemblies illustrate how long-read technologies allow for a much better representation of species genomes. We are now able to conduct more accurate downstream assays based on more complete gene and transposable element predictions.

Parthenogenomics: Insights on mutation rates and nucleotide diversity in parthenogenetic Panagrolaimus nematodes.

Asexual reproduction is assumed to lead to the accumulation of deleterious mutations, and reduced heterozygosity due to the absence of recombination. Panagrolaimid nematode species display different modes of reproduction. Sexual reproduction with distinct males and females, asexual reproduction through parthenogenesis in the genus Panagrolaimus, and hermaphroditism in Propanagrolaimus. Here, we compared genomic features of free-living nematodes in populations and species isolated from geographically distant regions to study diversity, and genome-wide differentiation under different modes of reproduction. We firstly estimated genome-wide spontaneous mutation rates in a triploid parthenogenetic Panagrolaimus, and a diploid hermaphroditic Propanagrolaimus via long-term mutation accumulation lines. Secondly, we calculated population genetic parameters including nucleotide diversity, and fixation index (F ST) between populations of asexually and sexually reproducing nematodes. Thirdly, we used phylogenetic network methods on sexually and asexually reproducing Panagrolaimus populations to understand evolutionary relationships between them. The estimated mutation rate was slightly lower for the asexual population, as expected for taxa with this reproductive mode. Natural polyploid asexual populations revealed higher nucleotide diversity. Despite their common ancestor, a gene network revealed a high level of genetic differentiation among asexual populations. The elevated heterozygosity found in the triploid parthenogens could be explained by the third genome copy. Given their tendentially lower mutation rates it can be hypothesized that this is part of the mechanism to evade Muller's ratchet. Our findings in parthenogenetic triploid nematode populations seem to challenge common expectations of evolution under asexuality.

A novel nematode species from the Siberian permafrost shares adaptive mechanisms for cryptobiotic survival with C. elegans dauer larva.

Some organisms in nature have developed the ability to enter a state of suspended metabolism called cryptobiosis when environmental conditions are unfavorable. This state-transition requires execution of a combination of genetic and biochemical pathways that enable the organism to survive for prolonged periods. Recently, nematode individuals have been reanimated from Siberian permafrost after remaining in cryptobiosis. Preliminary analysis indicates that these nematodes belong to the genera Panagrolaimus and Plectus. Here, we present precise radiocarbon dating indicating that the Panagrolaimus individuals have remained in cryptobiosis since the late Pleistocene (~46,000 years). Phylogenetic inference based on our genome assembly and a detailed morphological analysis demonstrate that they belong to an undescribed species, which we named Panagrolaimus kolymaensis. Comparative genome analysis revealed that the molecular toolkit for cryptobiosis in P. kolymaensis and in C. elegans is partly orthologous. We show that biochemical mechanisms employed by these two species to survive desiccation and freezing under laboratory conditions are similar. Our experimental evidence also reveals that C. elegans dauer larvae can remain viable for longer periods in suspended animation than previously reported. Altogether, our findings demonstrate that nematodes evolved mechanisms potentially allowing them to suspend life over geological time scales.

CRISPR/Cas9 mediated gene editing in non-model nematode Panagrolaimus sp. PS1159.

The phylum Nematoda harbors a huge diversity of species in a broad range of ecosystems and habitats. Nematodes share a largely conserved Bauplan but major differences have been found in early developmental processes. The development of the nematode model organism Caenorhabditis elegans has been studied in great detail for decades. These efforts have provided the community with a large number of protocols and methods. Unfortunately, many of these tools are not easily applicable in non-Caenorhabditis nematodes. In recent years it has become clear that many crucial genes in the C. elegans developmental toolkit are absent in other nematode species. It is thus necessary to study the developmental program of other nematode species in detail to understand evolutionary conservation and novelty in the phylum. Panagrolaimus sp. PS1159 is a non-parasitic nematode exhibiting parthenogenetic reproduction and we are establishing the species to comparatively study evolution, biodiversity, and alternative reproduction and survival strategies. Here, we demonstrate the first successful application of the CRISPR/Cas9 system for genome editing in Panagrolaimus sp. PS1159 and the closely related hermaphroditic species Propanagrolaimus sp. JU765 applying the non-homologous end joining and the homology-directed repair (HDR) mechanisms. Using microinjections and modifying published protocols from C. elegans and P. pacificus we induced mutations in the orthologue of unc-22. This resulted in a visible uncoordinated twitching phenotype. We also compared the HDR efficiency following the delivery of different single-stranded oligodeoxynucleotides (ssODNs). Our work will expand the applicability for a wide range of non-model nematodes from across the tree and facilitate functional analysis into the evolution of parthenogenesis, changes in the developmental program of Nematoda, and cryptobiosis.

FGF signalling is involved in cumulus migration in the common house spider Parasteatoda tepidariorum.

Cell migration is a fundamental component during the development of most multicellular organisms. In the early spider embryo, the collective migration of signalling cells, known as the cumulus, is required to set the dorsoventral body axis. Here, we show that FGF signalling plays an important role during cumulus migration in the spider Parasteatoda tepidariorum. Spider embryos with reduced FGF signalling show reduced or absent cumulus migration and display dorsoventral patterning defects. Our study reveals that the transcription factor Ets4 regulates the expression of several FGF signalling components in the cumulus. In conjunction with a previous study, we show that the expression of fgf8 in the germ-disc is regulated via the Hedgehog signalling pathway. We also demonstrate that FGF signalling influences the BMP signalling pathway activity in the region around cumulus cells. Finally, we show that FGFR signalling might also influence cumulus migration in basally branching spiders and we propose that fgf8 might act as a chemo-attractant to guide cumulus cells towards the future dorsal pole of the spider embryo.

Systematic errors in orthology inference and their effects on evolutionary analyses.

The availability of complete sets of genes from many organisms makes it possible to identify genes unique to (or lost from) certain clades. This information is used to reconstruct phylogenetic trees; identify genes involved in the evolution of clade specific novelties; and for phylostratigraphy-identifying ages of genes in a given species. These investigations rely on accurately predicted orthologs. Here we use simulation to produce sets of orthologs that experience no gains or losses. We show that errors in identifying orthologs increase with higher rates of evolution. We use the predicted sets of orthologs, with errors, to reconstruct phylogenetic trees; to count gains and losses; and for phylostratigraphy. Our simulated data, containing information only from errors in orthology prediction, closely recapitulate findings from empirical data. We suggest published downstream analyses must be informed to a large extent by errors in orthology prediction that mimic expected patterns of gene evolution.

Evolutionary dynamics of transposable elements in bdelloid rotifers.

Transposable elements (TEs) are selfish genomic parasites whose ability to spread autonomously is facilitated by sexual reproduction in their hosts. If hosts become obligately asexual, TE frequencies and dynamics are predicted to change dramatically, but the long-term outcome is unclear. Here, we test current theory using whole-genome sequence data from eight species of bdelloid rotifers, a class of invertebrates in which males are thus far unknown. Contrary to expectations, we find a variety of active TEs in bdelloid genomes, at an overall frequency within the range seen in sexual species. We find no evidence that TEs are spread by cryptic recombination or restrained by unusual DNA repair mechanisms. Instead, we find that that TE content evolves relatively slowly in bdelloids and that gene families involved in RNAi-mediated TE suppression have undergone significant expansion, which might mitigate the deleterious effects of active TEs and compensate for the consequences of long-term asexuality.

Conserved Patterns in Developmental Processes and Phases, Rather than Genes, Unite the Highly Divergent Bilateria.

Bilateria are the predominant clade of animals on Earth. Despite having evolved a wide variety of body plans and developmental modes, they are characterized by common morphological traits. By default, researchers have tried to link clade-specific genes to these traits, thus distinguishing bilaterians from non-bilaterians, by their gene content. Here we argue that it is rather biological processes that unite Bilateria and set them apart from their non-bilaterian sisters, with a less complex body morphology. To test this hypothesis, we compared proteomes of bilaterian and non-bilaterian species in an elaborate computational pipeline, aiming to search for a set of bilaterian-specific genes. Despite the limited confidence in their bilaterian specificity, we nevertheless detected Bilateria-specific functional and developmental patterns in the sub-set of genes conserved in distantly related Bilateria. Using a novel multi-species GO-enrichment method, we determined the functional repertoire of genes that are widely conserved among Bilateria. Analyzing expression profiles in three very distantly related model species-D. melanogaster, D. rerio and C. elegans-we find characteristic peaks at comparable stages of development and a delayed onset of expression in embryos. In particular, the expression of the conserved genes appears to peak at the phylotypic stage of different bilaterian phyla. In summary, our study illustrate how development connects distantly related Bilateria after millions of years of divergence, pointing to processes potentially separating them from non-bilaterians. We argue that evolutionary biologists should return from a purely gene-centric view of evolution and place more focus on analyzing and defining conserved developmental processes and periods.

Computational discovery of hidden breaks in 28S ribosomal RNAs across eukaryotes and consequences for RNA Integrity Numbers.

In some eukaryotes, a 'hidden break' has been described in which the 28S ribosomal RNA molecule is cleaved into two subparts. The break is common in protostome animals (arthropods, molluscs, annelids etc.), but a break has also been reported in some vertebrates and non-metazoan eukaryotes. We present a new computational approach to determine the presence of the hidden break in 28S rRNAs using mapping of RNA-Seq data. We find a homologous break is present across protostomes although it has been lost in a small number of taxa. We show that rare breaks in vertebrate 28S rRNAs are not homologous to the protostome break. A break is found in just 4 out of 331 species of non-animal eukaryotes studied and, in three of these, the break is located in the same position as the protostome break suggesting a striking instance of convergent evolution. RNA Integrity Numbers (RIN) rely on intact 28S rRNA and will be consistently underestimated in the great majority of animal species with a break.

Signatures of the Evolution of Parthenogenesis and Cryptobiosis in the Genomes of Panagrolaimid Nematodes.

Most animal species reproduce sexually and fully parthenogenetic lineages are usually short lived in evolution. Still, parthenogenesis may be advantageous as it avoids the cost of sex and permits colonization by single individuals. Panagrolaimid nematodes have colonized environments ranging from arid deserts to Arctic and Antarctic biomes. Many are obligatory meiotic parthenogens, and most have cryptobiotic abilities, being able to survive repeated cycles of complete desiccation and freezing. To identify systems that may contribute to these striking abilities, we sequenced and compared the genomes and transcriptomes of parthenogenetic and outcrossing panagrolaimid species, including cryptobionts and non-cryptobionts. The parthenogens are triploids, most likely originating through hybridization. Adaptation to cryptobiosis shaped the genomes of panagrolaimid nematodes and is associated with the expansion of gene families and signatures of selection on genes involved in cryptobiosis. All panagrolaimids have acquired genes through horizontal gene transfer, some of which are likely to contribute to cryptobiosis.

Mitigating Anticipated Effects of Systematic Errors Supports Sister-Group Relationship between Xenacoelomorpha and Ambulacraria.

Xenoturbella and the acoelomorph worms (Xenacoelomorpha) are simple marine animals with controversial affinities. They have been placed as the sister group of all other bilaterian animals (Nephrozoa hypothesis), implying their simplicity is an ancient characteristic [1, 2]; alternatively, they have been linked to the complex Ambulacraria (echinoderms and hemichordates) in a clade called the Xenambulacraria [3-5], suggesting their simplicity evolved by reduction from a complex ancestor. The difficulty resolving this problem implies the phylogenetic signal supporting the correct solution is weak and affected by inadequate modeling, creating a misleading non-phylogenetic signal. The idea that the Nephrozoa hypothesis might be an artifact is prompted by the faster molecular evolutionary rate observed within the Acoelomorpha. Unequal rates of evolution are known to result in the systematic artifact of long branch attraction, which would be predicted to result in an attraction between long-branch acoelomorphs and the outgroup, pulling them toward the root [6]. Other biases inadequately accommodated by the models used can also have strong effects, exacerbated in the context of short internal branches and long terminal branches [7]. We have assembled a large and informative dataset to address this problem. Analyses designed to reduce or to emphasize misleading signals show the Nephrozoa hypothesis is supported under conditions expected to exacerbate errors, and the Xenambulacraria hypothesis is preferred in conditions designed to reduce errors. Our reanalyses of two other recently published datasets [1, 2] produce the same result. We conclude that the Xenacoelomorpha are simplified relatives of the Ambulacraria.

Fitness Landscape of the Fission Yeast Genome.

The relationship between DNA sequence, biochemical function, and molecular evolution is relatively well-described for protein-coding regions of genomes, but far less clear in noncoding regions, particularly, in eukaryote genomes. In part, this is because we lack a complete description of the essential noncoding elements in a eukaryote genome. To contribute to this challenge, we used saturating transposon mutagenesis to interrogate the Schizosaccharomyces pombe genome. We generated 31 million transposon insertions, a theoretical coverage of 2.4 insertions per genomic site. We applied a five-state hidden Markov model (HMM) to distinguish insertion-depleted regions from insertion biases. Both raw insertion-density and HMM-defined fitness estimates showed significant quantitative relationships to gene knockout fitness, genetic diversity, divergence, and expected functional regions based on transcription and gene annotations. Through several analyses, we conclude that transposon insertions produced fitness effects in 66-90% of the genome, including substantial portions of the noncoding regions. Based on the HMM, we estimate that 10% of the insertion depleted sites in the genome showed no signal of conservation between species and were weakly transcribed, demonstrating limitations of comparative genomics and transcriptomics to detect functional units. In this species, 3'- and 5'-untranslated regions were the most prominent insertion-depleted regions that were not represented in measures of constraint from comparative genomics. We conclude that the combination of transposon mutagenesis, evolutionary, and biochemical data can provide new insights into the relationship between genome function and molecular evolution.

The mitochondrial genomes of the mesozoans Intoshia linei, Dicyema sp. and Dicyema japonicum.

The Dicyemida and Orthonectida are two groups of tiny, simple, vermiform parasites that have historically been united in a group named the Mesozoa. Both Dicyemida and Orthonectida have just two cell layers and appear to lack any defined tissues. They were initially thought to be evolutionary intermediates between protozoans and metazoans but more recent analyses indicate that they are protostomian metazoans that have undergone secondary simplification from a complex ancestor. Here we describe the first almost complete mitochondrial genome sequence from an orthonectid, Intoshia linei, and describe nine and eight mitochondrial protein-coding genes from Dicyema sp. and Dicyema japonicum, respectively. The 14,247 base pair long I. linei sequence has typical metazoan gene content, but is exceptionally AT-rich, and has a unique gene order. The data we have analysed from the Dicyemida provide very limited support for the suggestion that dicyemid mitochondrial genes are found on discrete mini-circles, as opposed to the large circular mitochondrial genomes that are typical of the Metazoa. The cox1 gene from dicyemid species has a series of conserved, in-frame deletions that is unique to this lineage. Using cox1 genes from across the genus Dicyema, we report the first internal phylogeny of this group.

Orthonectids Are Highly Degenerate Annelid Worms.

The animal groups of Orthonectida and Dicyemida are tiny, extremely simple, vermiform endoparasites of various marine animals and have been linked in the Mesozoa (Figure 1). The Orthonectida (Figures 1A and 1B) have a few hundred cells, including a nervous system of just ten cells [2], and the Dicyemida (Figure 1C) are even simpler, with ∼40 cells [3]. They are classic "Problematica" [4]-the name Mesozoa suggests an evolutionary position intermediate between Protozoa and Metazoa (animals) [5] and implies that their simplicity is a primitive state, but molecular data have shown they are members of Lophotrochozoa within Bilateria [6-9], which means that they derive from a more complex ancestor. Their precise affinities remain uncertain, however, and it is disputed whether they even constitute a clade. Ascertaining their affinities is complicated by the very fast evolution observed in their genes, potentially leading to the common systematic error of long-branch attraction (LBA) [10]. Here, we use mitochondrial and nuclear gene sequence data and show that both dicyemids and orthonectids are members of the Lophotrochozoa. Carefully addressing the effects of unequal rates of evolution, we show that the Mesozoa is polyphyletic. While the precise position of dicyemids remains unresolved within Lophotrochozoa, we identify orthonectids as members of the phylum Annelida. This result reveals one of the most extreme cases of body-plan simplification in the animal kingdom; our finding makes sense of an annelid-like cuticle in orthonectids [2] and suggests that the circular muscle cells repeated along their body [11] may be segmental in origin.

The gene regulatory program of Acrobeloides nanus reveals conservation of phylum-specific expression.

The evolution of development has been studied through the lens of gene regulation by examining either closely related species or extremely distant animals of different phyla. In nematodes, detailed cell- and stage-specific expression analyses are focused on the model Caenorhabditis elegans, in part leading to the view that the developmental expression of gene cascades in this species is archetypic for the phylum. Here, we compared two species of an intermediate evolutionary distance: the nematodes C. elegans (clade V) and Acrobeloides nanus (clade IV). To examine A. nanus molecularly, we sequenced its genome and identified the expression profiles of all genes throughout embryogenesis. In comparison with C. elegans, A. nanus exhibits a much slower embryonic development and has a capacity for regulative compensation of missing early cells. We detected conserved stages between these species at the transcriptome level, as well as a prominent middevelopmental transition, at which point the two species converge in terms of their gene expression. Interestingly, we found that genes originating at the dawn of the Ecdysozoa supergroup show the least expression divergence between these two species. This led us to detect a correlation between the time of expression of a gene and its phylogenetic age: evolutionarily ancient and young genes are enriched for expression in early and late embryogenesis, respectively, whereas Ecdysozoa-specific genes are enriched for expression during the middevelopmental transition. Our results characterize the developmental constraints operating on each individual embryo in terms of developmental stages and genetic evolutionary history.

Evolutionary analysis indicates that DNA alkylation damage is a byproduct of cytosine DNA methyltransferase activity.

Methylation at the 5 position of cytosine in DNA (5meC) is a key epigenetic mark in eukaryotes. Once introduced, 5meC can be maintained through DNA replication by the activity of 'maintenance' DNA methyltransferases (DNMTs). Despite their ancient origin, DNA methylation pathways differ widely across animals, such that 5meC is either confined to transcribed genes or lost altogether in several lineages. We used comparative epigenomics to investigate the evolution of DNA methylation. Although the model nematode Caenorhabditis elegans lacks DNA methylation, more basal nematodes retain cytosine DNA methylation, which is targeted to repeat loci. We found that DNA methylation coevolved with the DNA alkylation repair enzyme ALKB2 across eukaryotes. In addition, we found that DNMTs introduced the toxic lesion 3-methylcytosine into DNA both in vitro and in vivo. Alkylation damage is therefore intrinsically associated with DNMT activity, and this may promote the loss of DNA methylation in many species.

Differences in the genetic control of early egg development and reproduction between C. elegans and its parthenogenetic relative D. coronatus.

BACKGROUND: The free-living nematode Diploscapter coronatus is the closest known relative of Caenorhabditis elegans with parthenogenetic reproduction. It shows several developmental idiosyncracies, for example concerning the mode of reproduction, embryonic axis formation and early cleavage pattern (Lahl et al. in Int J Dev Biol 50:393-397, 2006). Our recent genome analysis (Hiraki et al. in BMC Genomics 18:478, 2017) provides a solid foundation to better understand the molecular basis of developmental idiosyncrasies in this species in an evolutionary context by comparison with selected other nematodes. Our genomic data also yielded indications for the view that D. coronatus is a product of interspecies hybridization. RESULTS: In a genomic comparison between D. coronatus, C. elegans, other representatives of the genus Caenorhabditis and the more distantly related Pristionchus pacificus and Panagrellus redivivus, certain genes required for central developmental processes in C. elegans like control of meiosis and establishment of embryonic polarity were found to be restricted to the genus Caenorhabditis. The mRNA content of early D. coronatus embryos was sequenced and compared with similar stages in C. elegans and Ascaris suum. We identified 350 gene families transcribed in the early embryo of D. coronatus but not in the other two nematodes. Looking at individual genes transcribed early in D. coronatus but not in C. elegans and A. suum, we found that orthologs of most of these are present in the genomes of the latter species as well, suggesting heterochronic shifts with respect to expression behavior. Considerable genomic heterozygosity and allelic divergence lend further support to the view that D. coronatus may be the result of an interspecies hybridization. Expression analysis of early acting single-copy genes yields no indication for silencing of one parental genome. CONCLUSIONS: Our comparative cellular and molecular studies support the view that the genus Caenorhabditis differs considerably from the other studied nematodes in its control of development and reproduction. The easy-to-culture parthenogenetic D. coronatus, with its high-quality draft genome and only a single chromosome when haploid, offers many new starting points on the cellular, molecular and genomic level to explore alternative routes of nematode development and reproduction.

Genome analysis of Diploscapter coronatus: insights into molecular peculiarities of a nematode with parthenogenetic reproduction.

BACKGROUND: Sexual reproduction involving the fusion of egg and sperm is prevailing among eukaryotes. In contrast, the nematode Diploscapter coronatus, a close relative of the model Caenorhabditis elegans, reproduces parthenogenetically. Neither males nor sperm have been observed and some steps of meiosis are apparently skipped in this species. To uncover the genomic changes associated with the evolution of parthenogenesis in this nematode, we carried out a genome analysis. RESULTS: We obtained a 170 Mbp draft genome in only 511 scaffolds with a N50 length of 1 Mbp. Nearly 90% of these scaffolds constitute homologous pairs with a 5.7% heterozygosity on average and inversions and translocations, meaning that the 170 Mbp sequences correspond to the diploid genome. Fluorescent staining shows that the D. coronatus genome consists of two chromosomes (2n = 2). In our genome annotation, we found orthologs of 59% of the C. elegans genes. However, a number of genes were missing or very divergent. These include genes involved in sex determination (e.g. xol-1, tra-2) and meiosis (e.g. the kleisins rec-8 and coh-3/4) giving a possible explanation for the absence of males and the second meiotic division. The high degree of heterozygosity allowed us to analyze the expression level of individual alleles. Most of the homologous pairs show very similar expression levels but others exhibit a 2-5-fold difference. CONCLUSIONS: Our high-quality draft genome of D. coronatus reveals the peculiarities of the genome of parthenogenesis and provides some clues to the genetic basis for parthenogenetic reproduction. This draft genome should be the basis to elucidate fundamental questions related to parthenogenesis such as its origin and mechanisms through comparative analyses with other nematodes. Furthermore, being the closest outgroup to the genus Caenorhabditis, the draft genome will help to disclose many idiosyncrasies of the model C. elegans and its congeners in future studies.

The mitochondrial genomes of the acoelomorph worms Paratomella rubra, Isodiametra pulchra and Archaphanostoma ylvae.

Acoels are small, ubiquitous - but understudied - marine worms with a very simple body plan. Their internal phylogeny is still not fully resolved, and the position of their proposed phylum Xenacoelomorpha remains debated. Here we describe mitochondrial genome sequences from the acoels Paratomella rubra and Isodiametra pulchra, and the complete mitochondrial genome of the acoel Archaphanostoma ylvae. The P. rubra and A. ylvae sequences are typical for metazoans in size and gene content. The larger I. pulchra  mitochondrial genome contains both ribosomal genes, 21 tRNAs, but only 11 protein-coding genes. We find evidence suggesting a duplicated sequence in the I. pulchra mitochondrial genome. The P. rubra, I. pulchra and A. ylvae mitochondria have a unique genome organisation in comparison to other metazoan mitochondrial genomes. We found a large degree of protein-coding gene and tRNA overlap with little non-coding sequence in the compact P. rubra genome. Conversely, the A. ylvae and I. pulchra genomes have many long non-coding sequences between genes, likely driving genome size expansion in the latter. Phylogenetic trees inferred from mitochondrial genes retrieve Xenacoelomorpha as an early branching taxon in the deuterostomes. Sequence divergence analysis between P. rubra sampled in England and Spain indicates cryptic diversity.

The cnidarian Hydractinia echinata employs canonical and highly adapted histones to pack its DNA.

BACKGROUND: Cnidarians are a group of early branching animals including corals, jellyfish and hydroids that are renowned for their high regenerative ability, growth plasticity and longevity. Because cnidarian genomes are conventional in terms of protein-coding genes, their remarkable features are likely a consequence of epigenetic regulation. To facilitate epigenetics research in cnidarians, we analysed the histone complement of the cnidarian model organism Hydractinia echinata using phylogenomics, proteomics, transcriptomics and mRNA in situ hybridisations. RESULTS: We find that the Hydractinia genome encodes 19 histones and analyse their spatial expression patterns, genomic loci and replication-dependency. Alongside core and other replication-independent histone variants, we find several histone replication-dependent variants, including a rare replication-dependent H3.3, a female germ cell-specific H2A.X and an unusual set of five H2B variants, four of which are male germ cell-specific. We further confirm the absence of protamines in Hydractinia. CONCLUSIONS: Since no protamines are found in hydroids, we suggest that the novel H2B variants are pivotal for sperm DNA packaging in this class of Cnidaria. This study adds to the limited number of full histone gene complements available in animals and sets a comprehensive framework for future studies on the role of histones and their post-translational modifications in cnidarian epigenetics. Finally, it provides insight into the evolution of spermatogenesis.