Microsatellite abundance across the Anthozoa and Hydrozoa in the phylum Cnidaria

Background

Microsatellite loci have high mutation rates and thus are indicative of mutational processes within the genome. By concentrating on the symbiotic and aposymbiotic cnidarians, we investigated if microsatellite abundances follow a phylogenetic or ecological pattern. Individuals from eight species were shotgun sequenced using 454 GS-FLX Titanium technology. Sequences from the three available cnidarian genomes (Nematostella vectensis, Hydra magnipapillata and Acropora digitifera) were added to the analysis for a total of eleven species representing two classes, three subclasses and eight orders within the phylum Cnidaria.

Results

Trinucleotide and tetranucleotide repeats were the most abundant motifs, followed by hexa- and dinucleotides. Pentanucleotides were the least abundant motif in the data set. Hierarchical clustering and log likelihood ratio tests revealed a weak relationship between phylogeny and microsatellite content. Further, comparisons between cnidaria harboring intracellular dinoflagellates and those that do not, show microsatellite coverage is higher in the latter group.

Conclusions

Our results support previous studies that found tri- and tetranucleotides to be the most abundant motifs in invertebrates. Differences in microsatellite coverage and composition between symbiotic and non-symbiotic cnidaria suggest the presence/absence of dinoflagellates might place restrictions on the host genome.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-939) contains supplementary material, which is available to authorized users.     

Character evolution in Hydrozoa (phylum Cnidaria)

The diversity of hydrozoan life cycles, as manifested in the wide range of polyp, colony, and medusa morphologies, has been appreciated for centuries. Unraveling the complex history of characters involved in this diversity is critical for understanding the processes driving hydrozoan evolution. In this study, we use a phylogenetic approach to investigate the evolution of morphological characters in Hydrozoa. A molecular phylogeny is reconstructed using ribosomal DNA sequence data. Several characters involving polyp, colony, and medusa morphology are coded in the terminal taxa. These characters are mapped onto the phylogeny and then the ancestral character states are reconstructed. This study confirms the complex evolutionary history of hydrozoan morphological characters. Many of the characters involving polyp, colony, and medusa morphology appear as synapomorphies for major hydrozoan clades, yet homoplasy is commonplace.     

Early development, pattern, and reorganization of the planula nervous system in Aurelia (Cnidaria, Scyphozoa)

We examined the development of the nervous system in Aurelia (Cnidaria, Scyphozoa) from the early planula to the polyp stage using confocal and transmission electron microscopy. Fluorescently labeled anti-FMRFamide, antitaurine, and antityrosinated tubulin antibodies were used to visualize the nervous system. The first detectable FMRFamide-like immunoreactivity occurs in a narrow circumferential belt toward the anterior/aboral end of the ectoderm in the early planula. As the planula matures, the FMRFamide-immunoreactive cells send horizontal processes (i.e., neurites) basally along the longitudinal axis. Neurites extend both anteriorly/aborally and posteriorly/orally, but the preference is for anterior neurite extension, and neurites converge to form a plexus at the aboral/anterior end at the base of the ectoderm. In the mature planula, a subset of cells in the apical organ at the anterior/aboral pole begins to show FMRFamide-like and taurine-like immunoreactivity, suggesting a sensory function of the apical organ. During metamorphosis, FMRFamide-like immunoreactivity diminishes in the ectoderm but begins to occur in the degenerating primary endoderm, indicating that degenerating FMRFamide-immunoreactive neurons are taken up by the primary endoderm. FMRFamide-like expression reappears in the ectoderm of the oral disc and the tentacle anlagen of the growing polyp, indicating metamorphosis-associated restructuring of the nervous system. These observations are discussed in the context of metazoan nervous system evolution.     

Lipoprotein secretion during the formation of the axial skeleton in Leptogorgia virgulata (Cnidaria: Gorgonacea)

Summary A lipoprotein-secreting role is associated with a population of skeletogenic cells in the gorgonian coral, Leptogorgia virgulata (Lam.). Electron-microscopical and cytochemical studies show that these cells, the medullocytes, occur almost exclusively at the growing skeletal tip and are morphologically and functionally distinct from those cells that thicken the skeletal cortex. It is postulated that the lipoprotein serves as a hydrophobic substratum onto which cortical collagen is secreted.     

First record of sympagic hydroids (Hydrozoa,Cnidaria) in Arctic coastal fast ice

We report on the first record of interstitial cnidarians in sea ice. Ice core samples were collected during eight field periods between February 2003 and June 2006 in the coastal fast ice off Barrow, Alaska (71°N, 156°W) at four locations. A total of 194 solitary, small (0.2–1.1 mm) elongated specimens of a previously unknown interstitial hydroid taxon were found. By cnidome composition and the occurrence of a highly retractable pedal disc formed by epidermal tissue only, the specimens are tentatively assigned to representatives of the family Protohydridae, subclass Anthomedusae. The hydroids were found almost exclusively in the bottom 10 cm-layer (at the ice–water interface) of 118 ice cores, with abundances ranging from 0 to 27 individuals per core section (0–4,244 ind m⁻²) and a grand mean of 269 ind m⁻² in bottom 10 cm-layer sections. Abundances were lower in December and late May than in months in between with considerable site variability. A factor analysis using 12 variables showed that hydroid abundance correlated highest with abundances of copepod nauplii and polychaete juveniles suggesting a trophic relationship.     

Phylogenetic analysis of the Cnidaria

The recent members of the phylum Cnidaria were analyzed with phylogenetic methodology and the help of the PAUP Computer program. The Cnidaria are established as a monophylum by their cnidocysts, planula larva, and a polyp stage. The Ctenophora were seen as the most probable sister group of the Cnidaria. Arguments for the monophyly of the cnidarian classes Anthozoa, Scyphozoa, Cubozoa, and Hydrozoa were providea. For the ground plan of the Cnidaria the following characters were postulated: triphasic life cycle consisting of a planula larva, a benthic polyp stage, and a sexually propagating medusa like stage. For the polyp a radial symmetry, lack of septae, and hollow tentacles were assumed. The original medusa probably was tetraradial and developed from the polyp stage by a total metamorphosis. Twelve polarized characters were used to generate cladograms. The most parsimonious one showed the Anthozoa as the first offshoot of the tree with the united Scyphozoa, Cubozoa and Hydrozoa forming its sister group. Within this sister group the Scyphozoa and Cubozoa were seen as sistergroups to each other. Both groups united are then the sistergroup of the Hydrozoa. A bootstrap analysis yielded the same tree with high probabilities for the internal nodes. Despite assuming a planktonic origin of the Cnidaria in this investigation, the resulting cladogram is also compatible with an evolution of the medusa stage within the Cnidaria after the splitting-off of the Anthozoa. The possible loss of the medusa stage in the Anthozoa is discussed.     

Responses of solitary and colonial coronate polyps (Cnidaria, Scyphozoa, Coronatae) to sedimentation and burial

Coronate polyps retract their soft bodies into a protective peridermal tube after mechanical irritation. Sediment may enter the tube of contracted polyps, however, and block the opening [Jarms, G., 1990. Neubeschreibung dreier Arten der Gattung Nausithoe (Coronatae, Scyphozoa) sowie Wiederbeschreibung der Art Nausithoe marginata Kölliker, 1853. Mitt. Hamb. Zool. Mus. Inst. 87, 7-39; Silveira, F.L. da, Jarms, G., Morandini, A.C., 2003. Experiments in nature and laboratory observations with Nausithoe aurea (Scyphozoa: Coronatae) support the concept of perennation by tissue saving and confirm dormancy. Biota Neotropica 2 (2), 1-25]. In the present study, the ability of different coronate species [Nausithoe aurea Silveira and Morandini, 1997, Nausithoe planulophora (Werner, 1971), Thecoscyphus zibrowii Werner, 1984, Linuche unguiculata (Swarts, 1788)] to expel sediment particles from the tube was investigated. In laboratory experiments, sand grains and mussel shell fragments were inserted into the tubes and the responses of polyps were observed. Particles were ingested as polyps extended themselves, and after extension, they were defecated. Ingestion was effected by an aborally directed flagellar beat of the flagellated gastrodermal epithelia that was reversed for defecation. Particles only slightly smaller than the tube opening could be expelled, and extension of the polyp was possible even if grains blocked 2/3 of the tube. However, if particles became stuck in the tube, ingestion was impossible and polyp extension failed. Comparisons among the tested species showed that expulsion success depended on tube shape and polyp morphology. In N. aurea and N. planulophora, less than 5% of tubes were permanently blocked. The cave-dwelling species T. zibrowii was not able to ingest particles due to its particular morphology, and in 25% of experiments with shell fragments tubes were permanently blocked. Thin, elongate tubes of the colonial polyps L. unguiculata were also often permanently blocked by shell fragments (50%), but new polyps were developed from the scyphorhiza to ensure survival of the colony. Solitary polyps were able to survive more than 5 months retracted beneath any blocking particles. After tubes were cut off beneath such particles normal polyps developed. From our observations, we suggest that coronate polyps can exist in habitats with moderate sedimentation, and that they can survive being temporarily buried.     

Ultrastructure of nematocyst discharge in catch tentacles of the sea anemone Haliplanella luciae (Cnidaria: Anthozoa)

The mature nematocyst lies just beneath the cnidodyte plasma membrane. A microtubule array surrounds the nematocyst capsule just beneath the capsule tip. We propose that the array helps to hold the capsule at the cnidocyte cell surface until discharge. The undischarged capsule tip is sealed by three apical flaps, joined together along complex radial seams. The seams are filled with subunits that appear to bind the flaps together. Upon discharge, the flaps separate along the radial seams to permit thread eversion. The everted thread is lined on both sides by subunits that are stained by antimonate, indicating that they bind calcium. We suggest that, together, the subunits hold the uneverted thread in its folded and coiled configuration. Thread eversion would follow subunit uncoupling. The capsule and thread interiors of partially discharged nematocysts are stained by antimonate. In contrast, the capsule and thread interiors of fully discharged nematocysts are not stained by antimonate. Thus, nematocyst calcium might be injected into the target tissue where it is presumed to act in conjunction with nematocyst venom to promote cell death.     

Induction of metamorphosis from the larval to the polyp stage is similar in Hydrozoa and a subgroup of Scyphozoa (Cnidaria, Semaeostomeae)

Larvae of cnidarians need an external cue for metamorphosis to start. The larvae of various hydrozoa, in particular of Hydractinia echinata, respond to Cs⁺, Li⁺, NH₄ ⁺ and seawater in which the concentration of Mg²⁺ ions is reduced. They further respond to the phorbolester, tetradecanoyl-phorbol-13-acetate (TPA) and the diacylglycerol (DAG) diC8, which both are argued to stimulate a protein kinase C. The only well-studied scyphozoa, Cassiopea spp., respond differently, i.e. to TPA and diC8 only. We found that larvae of the scyphozoa Aurelia aurita, Chrysaora hysoscella and Cyanea lamarckii respond to all the compounds mentioned. Trigonelline (N-methylnicotinic acid), a metamorphosis inhibitor found in Hydractinia larvae, is assumed to act by delivering a methyl group for transmethylation processes antagonising metamorphosis induction in Chrysaora hysoscella and Cyanea lamarckii. The three species tested are scyphozoa belonging to the subgroup of semaeostomeae, while Cassiopea spp. belong to the rhizostomeae. The results obtained may contribute to the discussion concerning the evolution of cnidarians and may help to clarify whether the way metamorphosis can be induced in rhizostomeae as a whole is different from that in hydrozoa and those scyphozoa belonging to the subgroup semaeostomeae. Electronic Publication     

An ultrastructural and cytochemical analysis of the cellular basis for tyrosine-derived collagen crosslinks in Leptogorgia virgulata (Cnidaria: Gorgonacea)

Summary Electron-microscopical autoradiography and cytochemical techniques have been used to identify the distinct and separate subcellular structures involved in the secretion of 1) procollagen, 2) dihydroxyphenylalanine (DOPA), which is a precursor of a collagen-crosslinking compound, and 3) DOPA oxidase, which converts DOPA to a putative crosslinking compound of collagen in the axial skeleton of the gorgonian coral Leptogorgia virgulata. Some skeletal-protein hydrolysates contain material that co-elutes with DOPA. The data indicate that these skeletogenic cells, corticocytes, are capable of modifying the number of non-reducible, tyrosine-derived crosslinkages of collagen by the secretion of a crosslinking compound that acts extracellularly on collagen. A mechanism for a cell-mediated control of the mechanical properties of collagen is thereby presented.     

Immunohistochemical localization of a retinoic acid-like receptor in nerve cells of two colonial anthozoans (Cnidaria)

Retinoic acid is known to induce vertebrate stem cells to differentiate into a variety of cell types, including neurons. Although retinoic acid was reported to affect morphogenetic pattern specification in the hydrozoan Hydractinia (Müller, W.A., 1984. Retinoids and pattern formation in a hydroid. J. Embryol. Exp. Morph. 81, 253-271) and a retinoid RXR receptor was cloned in the jellyfish Tripedalia (Kostrouch, Z., Kostrouchova, M., Love, W., Jannini, E., Piatigorsky, J., Rall, J.E., 1998. Retinoic acid X receptor in the diploblast, Tripedalia cystophora. Proc. Natl. Acad. Sci. U.S.A. 95, 13442-13447), the cellular targets of retinoids were not investigated. We used Western immunoblotting and immunohistochemistry to investigate the presence and cellular distribution of a RXR-like receptor in the sea pansy Renilla koellikeri and in the staghorn coral Acropora millepora (Cnidaria, Anthozoa). Western blots revealed a 64 kDa protein from a sea pansy extract in a band that co-migrated with a RXR protein from the rat brain. Using antibodies raised against an epitope of human alpha RXR, we visualized putative ectodermal sensory cells in the polyp column of the adult sea pansy. Immunoreactivity was absent in staghorn coral larvae but present in the polyp column of adult colonies in the form of clusters of neuron-like cells in the basiectoderm near the ectoderm-mesoglea interface. These observations suggest that a RXR-like receptor is involved in epithelial nerve cell specification in adult anthozoans and that this role is conserved throughout evolution.     

A phylogenetic study of the Anthozoa (phylum Cnidaria) based on morphological and molecular characters

The phylogenetic relationships within the Anthozoa were re-evaluated based on 41 morphological characters and nuclear sequences of 18S ribosomal DNA (29 anthozoans as ingroups and 3 hydrozoans as outgroups). The parsimony trees derived from the morphological data did not coincide closely with the molecular data, and the presence of several polytomies at some nodes of the trees resulted in ambiguities among the systematic relationships. On the other hand, the combined analysis using total evidence presents a more resolved and highly supported topology, as is indicated by higher bootstrap values and decay indices than either analysis alone. However, strict and semi-strict consensus trees derived from taxonomic congruence show a poorer resolution for the phylogeny of Anthozoa. The trees constructed from the molecular data, using neighbor-joining and maximum-likelihood methods, are nearly congruent with the result from the total evidence. Based on these results, Anthozoa is divided into three subclasses: Alcyonaria, Zoantharia, and Ceriantipatharia. The Ceriantipatharia now includes only one order, Ceriantharia, since the order Antipatharia is more closely related to orders within the Zoantharia. The Alcyonaria is a monophyletic group, in which the order Pennatulacea is basal, and orders Alcyonacea and Telestacea branch later. The order Gorgonacea is divided into two suborders, Holaxonia and Scleraxonia. Bellonela is more related to order Stolonifera, forming a monophyletic group. In Zoantharia, the order Zoanthinaria is basal, and the remaining taxa are divided into two clades: one includes the order Actiniaria and the other includes orders Antipatharia, Corallimorpharia, and Scleractinia. The latter two orders form a monophyletic group. This study presents a different phylogeny of actiniarians from the earlier hypothesis of scleractinian ancestry.     

Gametogenic cycles and reproduction in the beadlet sea anemone Actinia equina (Cnidaria: Anthozoa)

Sexual Actinia equina have been maintained in the laboratory. Monthly biopsies show that these animals have annual gametogenic cycles. Regular samples from several natural populations suggest that animals in the wild show similar gametogenic periodicity. In the laboratory, males release sperm, but females brood young, at least for a few weeks. Members of these broods are released sporadically, usually after a water change. Males and non-sexual adults kept in isolation for over a year release a few juveniles during and at the end of that time. These young are likely to be asexually reproduced. There appears to be geographical variation of anemone size and in the frequency of sexual individuals in populations round the British Coast, so that modes of reproduction may vary.     

Boundary cells of endodermal origin define the mouth of Hydra vulgaris (Cnidaria)

We investigated morphology, dynamics and origin of cells surrounding the mouth of Hydra vulgaris using the monoclonal antibody L96. This antibody recognises a one cell-thick ring of endodermal epithelial cells exactly at the boundary between endoderm (gastrodermis) and ectoderm (epidermis). L96⁺ cells can stretch considerably without any cell rupture during mouth opening. Thus, our data prove the existence of a distinct cell population defining hydra's mouth. A model for mouth opening is proposed and the significance of L96⁺ cells for boundary formation between ectoderm and endoderm is discussed.     

The Embryonic Origin of Neurosensory Cells and the Role of Nerve Cells in Metamorphosis in Phialidium gregarium (Cnidaria,Hydrozoa)

Summary

The embryonic origin of the nervous system in Phialidium gregarium was investigated. Entoderm-free planulae, surgically produced by bisection at mid-gastrulation, and normal planulae were examined by light and electron microscopy to determine their cellular composition. The cell types that occur in the epidermis of the normal planula were described. The entoderm-free planulae were found to be devoid of interstitial cells and their derivatives, the nematocytes and ganglion cells. Neurosensory cells were present, however, indicating that they are derivatives of the ectodermal epithelium.

The role of nerve elements in the initiation of metamorphosis was also examined. Normal and entoderm-free planulae treated for four hours with 0.4% colchicine at two, three, or four days of development fail to undergo cesium-induced metamorphosis. Since such treatment in other hydrozoans eliminates interstitial cells and their derivatives [1-3], it might be argued that ganglion cells are necessary to initiate metamorphosis. The observation that entoderm-free planulae, devoid of interstitial cell derivatives, are capable of responding to induction by bacteria or cesium, however, indicates that in Phialidium the colchicine effect is on other cell types. The results are compared with findings for other Cnidaria.     

Evolutionary origins of germline segregation in Metazoa: evidence for a germ stem cell lineage in the coral Orbicella faveolata (Cnidaria,Anthozoa)

The ability to segregate a committed germ stem cell (GSC) lineage distinct from somatic cell lineages is a characteristic of bilaterian Metazoans. However, the occurrence of GSC lineage specification in basally branching Metazoan phyla, such as Cnidaria, is uncertain. Without an independently segregated GSC lineage, germ cells and their precursors must be specified throughout adulthood from continuously dividing somatic stem cells, generating the risk of propagating somatic mutations within the individual and its gametes. To address the potential for existence of a GSC lineage in Anthozoa, the sister-group to all remaining Cnidaria, we identified moderate- to high-frequency somatic mutations and their potential for gametic transfer in the long-lived coral Orbicella faveolata (Anthozoa, Cnidaria) using a 2b-RAD sequencing approach. Our results demonstrate that somatic mutations can drift to high frequencies (up to 50%) and can also generate substantial intracolonial genetic diversity. However, these somatic mutations are not transferable to gametes, signifying the potential for an independently segregated GSC lineage in O. faveolata. In conjunction with previous research on germ cell development in other basally branching Metazoan species, our results suggest that the GSC system may be a Eumetazoan characteristic that evolved in association with the emergence of greater complexity in animal body plan organization and greater specificity of stem cell functions.