Cnidaria, the sister group to Bilateria, is a highly diverse group of animals in terms of morphology, lifecycles, ecology, and development. How this diversity originated and evolved is not well understood because phylogenetic relationships among major cnidarian lineages are unclear, and recent studies present contrasting phylogenetic hypotheses. Here, we use transcriptome data from 15 newly-sequenced species in combination with 26 publicly available genomes and transcriptomes to assess phylogenetic relationships among major cnidarian lineages. Phylogenetic analyses using different partition schemes and models of molecular evolution, as well as topology tests for alternative phylogenetic relationships, support the monophyly of Medusozoa, Anthozoa, Octocorallia, Hydrozoa, and a clade consisting of Staurozoa, Cubozoa, and Scyphozoa. Support for the monophyly of Hexacorallia is weak due to the equivocal position of Ceriantharia. Taken together, these results further resolve deep cnidarian relationships, largely support traditional phylogenetic views on relationships, and provide a historical framework for studying the evolutionary processes involved in one of the most ancient animal radiations.

BackgroundA colony of the hydrozoan Hydractinia symbiolongicarpus comprises genetically identical yet morphologically distinct and functionally specialized polyp types. The main labor divisions are between feeding, reproduction and defense. In H. symbiolongicarpus, the feeding polyp (called a gastrozooid) has elongated tentacles and a mouth, which are absent in the reproductive polyp (gonozooid) and defensive polyp (dactylozooid). Instead, the dactylozooid has an extended body column with an abundance of stinging cells (nematocysts) and the gonozooid bears gonophores on its body column. Morphological differences between polyp types can be attributed to simple changes in their axial patterning during development, and it has long been hypothesized that these specialized polyps arose through evolutionary alterations in oral-aboral patterning of the ancestral gastrozooid.ResultsAn assembly of 66,508 transcripts (>200 bp) were generated using short-read Illumina RNA-Seq libraries constructed from feeding, reproductive, and defensive polyps of H. symbiolongicarpus. Using several different annotation methods, approximately 54% of the transcripts were annotated. Differential expression analyses were conducted between these three polyp types to isolate genes that may be involved in functional, histological, and pattering differences between polyp types. Nearly 7 K transcripts were differentially expressed in a polyp-specific manner, including members of the homeodomain, myosin, toxin and BMP gene families. We report the spatial expression of a subset of these polyp-specific transcripts to validate our differential expression analyses.ConclusionsWhile potentially originating through simple changes in patterning, polymorphic polyps in Hydractinia are the result of differentially expressed functional, structural, and patterning genes.The differentially expressed genes identified in our study provide a starting point for future investigations of the developmental patterning and functional differences that are displayed in the different polyp types that confer a division of labor within a colony of H. symbiolongicarpus.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-406) contains supplementary material, which is available to authorized users.

BackgroundHydractinia symbiolongicarpus, a colonial cnidarian, is a tractable model system for many cnidarian-specific and general biological questions. Until recently, tests of gene function in Hydractinia have relied on laborious forward genetic approaches, randomly integrated transgenes, or transient knockdown of mRNAs.ResultsHere, we report the use of CRISPR/Cas9 genome editing to generate targeted genomic insertions in H. symbiolonigcarpus. We used CRISPR/Cas9 to promote homologous recombination of two fluorescent reporters, eGFP and tdTomato, into the Eukaryotic elongation factor 1 alpha (Eef1a) locus. We demonstrate that the transgenes are expressed ubiquitously and are stable over two generations of breeding. We further demonstrate that CRISPR/Cas9 genome editing can be used to mark endogenous proteins with FLAG or StrepII-FLAG affinity tags to enable in vivo and ex vivo protein studies.ConclusionsThis is the first account of CRISPR/Cas9 mediated knockins in Hydractinia and the first example of the germline transmission of a CRISPR/Cas9 inserted transgene in a cnidarian. The ability to precisely insert exogenous DNA into the Hydractinia genome will enable sophisticated genetic studies and further development of functional genomics tools in this understudied cnidarian model.Electronic supplementary materialThe online version of this article (10.1186/s12864-018-5032-z) contains supplementary material, which is available to authorized users.

Hydrozoans are known for their complex life cycles, which can alternate between an asexually reproducing polyp stage and a sexually reproducing medusa stage. Most hydrozoan species, however, lack a free-living medusa stage and instead display a developmentally truncated form, called a medusoid or sporosac, which generally remains attached to the polyp. Although evolutionary transitions in medusa truncation and loss have been investigated phylogenetically, little is known about the genes involved in the development and loss of this life cycle stage. Here, we present a new workflow for evaluating differential expression (DE) between two species using short read Illumina RNA-seq data. Through interspecific DE analyses between two hydractiniid hydrozoans, Hydractinia symbiolongicarpus and Podocoryna carnea, we identified genes potentially involved in the developmental, functional, and morphological differences between the fully developed medusa of P. carnea and reduced sporosac of H. symbiolongicarpus. A total of 10,909 putative orthologs of H. symbiolongicarpus and P. carnea were identified from de novo assemblies of short read Illumina data. DE analysis revealed 938 of these are differentially expressed between P. carnea developing and adult medusa, when compared with H. symbiolongicarpus sporosacs, the majority of which have not been previously characterized in cnidarians. In addition, several genes with no corresponding ortholog in H. symbiolongicarpus were expressed in developing medusa of P. carnea. Results presented here show interspecific DE analyses of RNA-seq data to be a sensitive and reliable method for identifying genes and gene pathways potentially involved in morphological and life cycle differences between species.

Hydrozoans are known for their complex life cycles, alternating between benthic, asexually reproducing polyps and pelagic, sexually reproducing medusae. Although patterning in hydrozoan polyps has been well studied, little is known about the signaling mechanisms governing medusa development. In order to investigate the role of Wnt signaling in medusa development, we use RNA-Seq data collected from three discrete life cycle stages of Podocoryna carnea to assemble, annotate, and assess enrichment and differential expression (DE) of Wnt pathway elements in P. carnea's transcriptome. Enrichment analyses revealed a statistically significant enrichment of DE Wnt signaling transcripts in the transcriptome of P. carnea, of which, the vast majority of these were significantly up-regulated in developing and adult medusae stages. Whole mount in situ hybridization (ISH) reveals co-expression of the Wnt ligand, Wnt3, and a membrane bound Wnt receptor, frizzled3, at the distal and oral ends of the developmental axes of medusae and polyps in P. carnea. DE and ISH results presented here reveal expression of Wnt signaling components consistent with it playing a role in medusa development. Specifically, Wnt ligand expression in the oral region suggests that the Wnt pathway may play a role in medusa patterning, similar to that of polyps. Previous Wnt expression studies in hydrozoan taxa with reduced medusa have failed to detect co-expression of Wnt3 and a frizzled receptor at their truncated developmental axes, suggesting that down regulation of Wnt pathway elements may play a key role in the loss of the medusa life cycle stage in hydrozoan evolution.

We have studied the evolution of Wnt genes in cnidarians and the expression pattern of all Wnt ligands in the hydrozoan Hydractinia echinata. Current views favor a scenario in which 12 Wnt sub-families were jointly inherited by cnidarians and bilaterians from their last common ancestor. Our phylogenetic analyses clustered all medusozoan genes in distinct, well-supported clades, but many orthologous relationships between medusozoan Wnts and anthozoan and bilaterian Wnt genes were poorly supported. Only seven anthozoan genes, Wnt2, Wnt4, Wnt5, Wnt6, Wnt 10, Wnt11, and Wnt16 were recovered with strong support with bilaterian genes and of those, only the Wnt2, Wnt5, Wnt11, and Wnt16 clades also included medusozoan genes. Although medusozoan Wnt8 genes clustered with anthozoan and bilaterian genes, this was not well supported. In situ hybridization studies revealed poor conservation of expression patterns of putative Wnt orthologs within Cnidaria. In polyps, only Wnt1, Wnt3, and Wnt7 were expressed at the same position in the studied cnidarian models Hydra, Hydractinia, and Nematostella. Different expression patterns are consistent with divergent functions. Our data do not fully support previous assertions regarding Wnt gene homology, and suggest a more complex history of Wnt family genes than previously suggested. This includes high rates of sequence divergence and lineage-specific duplications of Wnt genes within medusozoans, followed by functional divergence over evolutionary time scales.

Hydrozoan colonies display a variety of shapes and sizes including encrusting, upright and pelagic forms. Phylogenetic patterns reveal a complex evolutionary history of these distinct colony forms, as well as colony loss. Within a species, phenotypic variation in colonies as a response to changing environmental cues and resources has been documented. The patterns of branching of colony specific tissue, called stolons in encrusting colonies and stalks in upright colonies, are likely under the control of signaling mechanisms whose changing expression in evolution and development are responsible for the diversity of hydrozoan colony forms. Although mechanisms of polyp development have been well studied, little research has focused on colony development and patterning. In the few studies that investigated mechanisms governing colony patterning, the Wnt signaling pathway has been implicated. The diversity of colony form, evolutionary patterns and mechanisms of colony variation in Hydrozoa are reviewed here.

Sex determination occurs across animal species, but most of our knowledge about the mechanisms of sex determination comes from only a handful of bilaterian taxa. This limits our ability to infer the evolutionary history of sex determination within animals. In this study, we generated a linkage map of the genome of the colonial cnidarian Hydractinia symbiolongicarpus and used this map to determine that this species has an XX/XY sex determination system. We delineate the pseudoautosomal and non-recombining regions of the Y chromosome and show that the latter encodes a number of genes with male gonad-specific expression. These findings establish Hydractinia as a tractable non-bilaterian model system for the study of sex determination.