Exploring the potential of small RNA subunit and ITS sequences for resolving phylogenetic relationships within the phylum Ctenophora

Ctenophores are a phylum of non-bilaterian marine (mostly planktonic) animals, characterised by several unique synapomorphies (e.g., comb rows, apical organ). Relationships between and within the nine recognised ctenophore orders are far from understood, notably due to a paucity of phylogenetically informative anatomical characters. Previous attempts to address ctenophore phylogeny using molecular data (18S rRNA) led to poorly resolved trees but demonstrated the paraphyly of the order Cydippida. Here we compiled an updated 18S rRNA data set, notably including a few newly sequenced species representing previously unsampled families (Lampeidae, Euryhamphaeidae), and we constructed an additional more rapidly evolving ITS1 + 5.8S rRNA + ITS2 alignment. These data sets were analysed separately and in combination under a probabilistic framework, using different methods (maximum likelihood, Bayesian inference) and models (e.g., doublet model to accommodate secondary structure; data partitioning). An important lesson from our exploration of these datasets is that the fast-evolving internal transcribed spacer (ITS) regions are useful markers for reconstructing high-level relationships within ctenophores. Our results confirm the paraphyly of the order Cydippida (and thus a “cydippid-like” ctenophore common ancestor) and suggest that the family Mertensiidae could be the sister group of all other ctenophores. The family Lampeidae (also part of the former “Cydippida”) is probably the sister group of the order Platyctenida (benthic ctenophores). The order Beroida might not be monophyletic, due to the position of Beroe abyssicola outside of a clade grouping the other Beroe species and members of the “Cydippida” family Haeckeliidae. Many relationships (e.g. between Pleurobrachiidae, Beroida, Cestida, Lobata, Thalassocalycida) remain unresolved. Future progress in understanding ctenophore phylogeny will come from the use of additional rapidly evolving markers and improvement of taxonomic sampling.     

Bacterial communities associated with the ctenophores Mnemiopsis leidyi and Beroe ovata

Residing in a phylum of their own, ctenophores are gelatinous zooplankton that drift through the ocean's water column. Although ctenophores are known to be parasitized by a variety of eukaryotes, no studies have examined their bacterial associates. This study describes the bacterial communities associated with the lobate ctenophore Mnemiopsis leidyi and its natural predator Beroe ovata in Tampa Bay, Florida, USA. Investigations using terminal restriction fragment length polymorphism (T-RFLP) and cloning and sequencing of 16S rRNA genes demonstrated that ctenophore bacterial communities were distinct from the surrounding water. In addition, each ctenophore genus contained a unique microbiota. Ctenophore samples contained fewer bacterial operational taxonomic units (OTUs) by T-RFLP and lower diversity communities by 16S rRNA gene sequencing than the water column. Both ctenophore genera contained sequences related to bacteria previously described in marine invertebrates, and sequences similar to a sea anemone pathogen were abundant in B.?ovata. Temporal sampling revealed that the ctenophore-associated bacterial communities varied over time, with no single OTU detected at all time points. This is the first report of distinct and dynamic bacterial communities associated with ctenophores, suggesting that these microbial consortia may play important roles in ctenophore ecology. Future work needs to elucidate the functional roles and mode of acquisition of these bacteria.     

Expansion of the SOX gene family predated the emergence of the Bilateria

Members of the SOX gene family are involved in regulating many developmental processes including neuronal determination and differentiation, and in carcinogenesis. So far they have only been identified in species from the Bilateria (deuterostomes and protostomes). To understand the origins of the SOX family, we used a PCR-based strategy to obtain 28 new sequences of SOX gene HMG domains from four non-bilaterian Metazoa: two sponge species, one ctenophore and one cnidarian. One additional SOX sequence was retrieved from EST sequences of the cnidarian species Clytia hemisphaerica. Unexpected SOX gene diversity was found in these species, especially in the cnidarian and the ctenophore. The topology of gene relationships deduced by Maximum Likelihood analysis, although not supported by bootstrap values, suggested that the SOX family started to diversify in the metazoan stem branch prior to the divergence of demosponges, and that further diversification occurred in the eumetazoan branch, as well as later in calcisponges, ctenophores, cnidarians and vertebrates. In contrast, gene loss appears to have occurred in the nematode and probably in other protostome lineages, explaining their lower number of SOX genes.     

Swimming movements of ctenophores,and the mechanics of propulsion by ctene rows

This study focuses on the mechanics of ciliary movement of ctenophores in relation to locomotion and feeding, with field and laboratory observations documented with 35 mm photographs and video sequences. Movement through the water is strongly modified by subtleties of body morphology. Whereas the entire ctenophore moves in a flow regime where the Reynolds numbers range from 100 to 6000, the cilia on the surface of the ctenophores move in a flow regime where the Reynolds numbers range only from 10 to 300. The water flow patterns seen by use of fluorescein dye do not match any current model of ciliary flow and assumptions for a new model are postulated. Ctenophores exhibit a wide variety of morphological adaptations that reduce drag, and a variety of behaviours that exploit fine-scale water movements for prey capture.     

Eoandromeda and the origin of Ctenophora

The Ediacaran fossil Eoandromeda octobrachiata had a high conical body with eight arms in helicospiral arrangement along the flanks. The arms carried transverse bands proposed to be homologous to ctenophore ctenes (comb plates). Eoandromeda is interpreted as an early stem‐group ctenophore, characterized by the synapomorphies ctenes, comb rows, and octoradial symmetry but lacking crown‐group synapomorphies such as tentacles, statoliths, polar fields, and biradial symmetry. It probably had a pelagic mode of life. The early appearance in the fossil record of octoradial ctenophores is most consistent with the Planulozoa hypothesis (Ctenophora is the sister group of Cnidaria + Bilateria) of metazoan phylogeny.     

A review of cnidarians and ctenophores feeding on competitors in the plankton

Predation among pelagic cnidarians and ctenophores is reviewed. The diets of semaeostome scyphomedusae and hydromedusae commonly include other gelatinous zooplanktivores. However, few species of siphonophores and ctenophores are known to consume other gelatinous species. Most of these species can be said to exhibit intraguild predation, since they consume species that potentially compete with them for food. In addition, some hydromedusan and ctenophore species may consume other gelatinous zooplanktivores exclusively. Characteristics of cnidarians and ctenophores as predators and as prey of other gelatinous species are discussed.     

Ctenophore kairomones and modified aminosugar disaccharides alter the shadow response in a larval crab

Zoea larvae of the estuarine crab Rhithropanopeus harrisii (Gould)descend in the water column upon sudden decreases in irradiance,which serves as a shadow response for the avoidance of zooplanktonpredators such as ctenophores. This study tested the hypothesesthat (i) kairomones from either the ctenophore Mnemiopsis leidyior the fish Fundulus heteroclitus increase the shadow-responsesensitivity by reducing the minimum decrease in irradiance necessaryto evoke the response (i.e. threshold); and (ii) a similar increasein shadow-response sensitivity as occurs with ctenophore kairomonesis also induced by the external mucus of ctenophores and modifiedaminosugar disaccharide hydrolysis products of acidic mucopolysaccharides.Light-adapted R. harrisii Stage I zoea were conditioned to chemicaltreatments for 3 h, and their photoresponses were tested inan apparatus that mimicked the underwater angular light distribution.In clean sea water, the shadow response was induced by a 50%reduction in irradiance (threshold). The threshold decreasedto 30% in sea water with fish kairomones and 9% in sea waterwith ctenophore kairomones. A minimum concentration of 1.0 gwet weight ctenophore mucus l^-1 was required to reduce the shadow-responsethreshold. Chondroitin sulfate A disaccharides had an effectsimilar to kairomones and mucus in reducing the threshold, withthe greatest effect at a concentration of 10^-6 M. Tests withother disaccharides indicated that only acetylamine-containingaminosugars were effective. The test hypotheses were supported;predator kairomones, ctenophore external mucus, and purifiedaminosugar disaccharides having acetylamine functionality reducedthe threshold decrease in irradiance needed to evoke shadowresponses in crab larvae.     

Haeckelia (= Euchlora) and Hydroctena and the phylogenetic interrelationships of Cnidaria and Ctenophora

A scrutiny of the literature shows that the ctenophore Haeckelia (= Euchlora) ruba has only kleptocnidae and that Hydroctena salenskii is a ctenophore without special cnidarian affinities. The “missing links” between cnidarians and ctenophores have thus turned out to be based on misinterpretations and must be excluded from future discussions on phylogeny.     

Regulation and regeneration in the ctenophore Mnemiopsis leidyi

Lobate ctenophores (tentaculates) generally exhibit a remarkable ability to regenerate missing structures as adults. On the other hand, their embryos exhibit a highly mosaic behavior when cut into halves or when specific cells are ablated. These deficient embryos do not exhibit embryonic regulation, and generate incomplete adult body plans. Under certain conditions, however, these deficient animals are subsequently able to replace the missing structures during the adult phase in a process referred to as "post-regeneration." We have determined that successful post-regeneration can be predicted on the basis of a modified polar coordinate model, and the rules of intercalary regeneration, as defined by French et al. (V. French, P. J. Bryant, and S. V. Bryant, 1976, Science 193, 969-981.) The model makes certain assumptions about the organization of the ctenophore body plan that fit well with what we have determined on the basis of cell lineage fates maps, and their twofold rotational ("biradial") symmetry. The results suggest that cells composing the ctenophore adult body plan possess positional information, which is utilized to reconstruct the adult body plan. More specifically, we have found that the progeny of three specific cell lineages are required to support post-regeneration of the comb rows (the e(1), e(2), and m(1) micromeres). Furthermore, post-regeneration of the comb rows involves a suite of cell-cell inductive interactions, which are similar to those that take place during their embryonic formation. The significance of these findings is discussed in terms of the organization of the ctenophore body plan, and the mechanisms involved in cell fate specification. This situation is also contrasted with that of the atentaculate ctenophores, which are unable to undergo post-regeneration.     

Ancient connection between NKL genes and the mesoderm? Insights from <Emphasis Type="Italic">Tlx</Emphasis> expression in a ctenophore

In recent years, evo-devo studies on non-bilaterian metazoans have improved our understanding of the early evolution of animal body plans. In particular, works on cnidarians suggested that contrary to classical views, the mesoderm originated far before the emergence of the Bilateria. In this context, a synthesis of genomic and functional data concerning the Antennapedia (Antp) superclass of homeobox genes suggested that early in animal evolution, each of the three germ layers was under the control of one cluster of Antp genes. In particular, the patterning and differentiation of the mesoderm was under the control of the NKL cluster. The ctenophores stand as a key taxon with respect to such issues because unlike other non-bilaterian phyla, their intermediate germ layer satisfies the strict embryological definition of a mesoderm. For that reason, we investigated the only known member of the NKL group in Ctenophora, a gene previously isolated from Pleurobrachia and attributed to the Tlx family. In our analysis of the NKL group, this ctenophore gene branches as the sister-group of bilaterian Tlx genes, but without statistical support. The expression pattern of this gene was revealed by in situ hybridisation in the adult ctenophore. The expression territories of PpiTlx are predominantly ectodermal, in two distinct types of ciliated epidermal cells and in one category of gland cells. We also identified a probable endodermal site of expression. Because we failed to detect any mesodermal expression, the results do not provide support to the hypothesis of an ancient functional association between the NKL group and the mesoderm.     

Growth dynamics of a ctenophore (Mnemiopsis) in relation to variable food supply. II. Carbon budgets and growth model

Our goal was to test our understanding of ingestion, assimilationefficiency and metabolism for Mnemiopsis mccradyi by formulatingand validating a simulation model of growth under differentconditions of food availability. The model was based on a carbonbudget approach using formulations derived from empirical results,including how each process was affected by food availabilityand ctenophore size. An experimentally measured carbon budgetfor pulsed food availability indicated that, relative to totalingestion, growth was high (17–48%), respiration plusorganic release was relatively low (24–48%) and little(<10%) of the ingested carbon was unaccounted for. New laboratoryinvestigations of feeding and assimilation efficiency were necessaryto refine the formulations so that model predictions comparedfavorably with a variety of laboratory measurements of growth,and growth efficiency, as well as the complete experimentallymeasured carbon budget. The refined model predicted a high ratioof growth to metabolism (>2) and a high gross growth efficiency(>30%) for smaller ctenophores at high food concentrations(>20 prey l^–1). Both growth rates and growth efficiencieswere predicted to decrease for larger ctenophores. Model predictionswere generally consistent with experimental results, includinginvestigations using pulsed food availability to simulate environmentalpatchiness. Although the model underpredicted ctenophore growthin some experiments at low food densities, the model predictionof a minimum prey concentration of about 8 l^–1 (24 µgC l^–1) for sustaining a ctenophore population of reproductivesize agreed with field observations.     

大黄鱼16SrRNA和18SrRNA基因的测定与序列分析

利用多对引物,扩增并测定出大黄鱼16SrRNA基因和18SrRNA基因的部分序列,其长度分别为1202bp和1275bp,16SrRNA基因序列的GC含量为46．12％,18SrRNA基因的Gc含量为53．oo％。将大黄鱼16SrRNA基因序列与GenBank中15种硬骨鱼类的同源序列结合,同时将其18SrRNA基因序列与GenBank中9种脊索动物的同源序列相结合,运用软件获得各自序列间差异百分比,转换和颠换数值等信息。基于这两种基因序列,利用NJ法和BI法,分别构建16种硬骨鱼类和10种脊索动物的分子系统树。18SrRNA构建的系统树包括三大支,一支为哺乳类、鸟类和爬行类共6个物种,一支为两栖类的1个物种,另一支为2种硬骨鱼类。16SrRNA构建的系统树显示大黄鱼所在的石首鱼科与鲈科和盖刺鱼科亲缘关系较近。此外还讨论了这两个基因的序列特征。     

Understanding the trophic role of the Antarctic ctenophore,Callianira antarctica,using lipid biomarkers

To better understand the trophic role of ctenophores in Antarctica during austral fall and winter, a major species of cydippid ctenophore, Callianira antarctica, was collected during April/May (fall) and August/September (winter) 2002 in the vicinity of Marguerite Bay. Lipid content, lipid classes, fatty acids, fatty alcohols and sterols were analyzed in animals, together with lipid biomarkers in krill and copepod species representing potential ctenophore prey. Lipid content in ctenophores collected in winter was slightly higher than from animals in fall (4.8 and 3.5% of dry weight, respectively). Polar lipids were the dominant lipid class in ctenophores, accounting for over half of the lipid content, with significant amounts of free fatty alcohols (more than 10% of total lipid content) detected. Lipid-class composition, however, differed significantly between seasons, with significant amounts of neutral lipid (wax esters and triacylglycerols) only detected in animals from fall. Although the dominant lipid classes in ctenophores varied between fall and winter, individual lipids (i.e., fatty acids, alcohols and sterols) showed only minor changes between seasons. Specifically, long-chain polyunsaturated fatty acids [20:5(n-3) and 22:6(n-3)] found in high abundance in larval krill were also elevated in ctenophores collected in winter. Very high amounts of monounsaturated fatty alcohols, particularly 20:1(n-9) and 22:1(n-11), known to be important components of wax esters in calanoid copepods, were also observed. Multivariate analysis using the suite of lipids found indicated that copepods are an important diet item for ctenophores in the study area. Results further suggest that C. antarctica feed actively year-round, with larval krill providing a food resource during austral winter.     

Spatial structure of the novel light-sensitive photoprotein berovin from the ctenophore Beroe abyssicola in the Ca-loaded apoprotein conformation state

The bright bioluminescence of ctenophores, found in oceans worldwide, is determined by Ca^2 +-regulated photoproteins, functionally identical to and sharing many properties of hydromedusan photoproteins. In contrast, however, the ctenophore photoproteins are extremely sensitive to UV and visible light over the range of their absorption spectrum. The spatial structure of a novel light-sensitive photoprotein from the ctenophore Beroe abyssicola in its apoform bound with three calcium ions is determined at 2.0 Å. We demonstrate that the apoberovin is a slightly asymmetrical compact globular protein formed by two domains with a cavity in the center, which exactly retains the fold architecture characteristic of hydromedusan photoproteins despite their low amino acid sequence identity. However, the structural alignment of these two photoprotein classes clearly shows that despite the high similarity of shape and geometry of their coelenterazine-binding cavities, their interiors differ drastically. The key residues appearing to be crucial for stabilizing the 2-hydroperoxycoelenterazine and for formation of the emitter in hydromedusan photoproteins, are replaced in berovin by amino acid residues having completely different side chain properties. Evidently, these replacements must be responsible for the distinct properties of ctenophore photoproteins such as sensitivity to light or the fact that the formation of active photoprotein from apophotoprotein, coelenterazine, and oxygen is more effective at alkaline pH.     

Should we be worried about long-branch attraction in real data sets? Investigations using metazoan 18S rDNA

Although long-branch attraction (LBA) is frequently cited as the cause of anomalous phylogenetic groupings, few examples of LBA involving real sequence data are known. We have found several cases of probable LBA by analyzing subsamples from an alignment of 18S rDNA sequences for 133 metazoans. In one example, maximum parsimony analysis of sequences from two rotifers, a ctenophore, and a polychaete annelid resulted in strong support for a tree grouping two "long-branch taxa" (a rotifer and the ctenophore). Maximum-likelihood analysis of the same sequences yielded strong support for a more biologically reasonable "rotifer monophyly" tree. Attempts to break up long branches for problematic subsamples through increased taxon sampling reduced, but did not eliminate, LBA problems. Exhaustive analyses of all quartets for a subset of 50 sequences were performed in order to compare the performance of maximum likelihood, equal-weights parsimony, and two additional variants of parsimony; these methods do differ substantially in their rates of failure to recover trees consistent with well established, but highly unresolved phylogenies. Power analyses using simulations suggest that some incorrect inferences by maximum parsimony are due to statistical inconsistency and that when estimates of central branch lengths for certain quartets are very low, maximum-likelihood analyses have difficulty recovering accepted phylogenies even with large amounts of data. These examples demonstrate that LBA problems can occur in real data sets, and they provide an opportunity to investigate causes of incorrect inferences.     

Cloning,Sequencing, Expression and Structural Investigation of Mnemiopsin from <Emphasis Type="Italic">Mnemiopsis leidyi</Emphasis>: An Attempt Toward Understanding Ca<Superscript>2+</Superscript>-Regulated Photoproteins

A comparison of the two most famous groups of calcium-regulated photoproteins, cnidarians and ctenophores, showed unexpectedly high degree of structural similarity regardless of their low sequence identity. It was suggested these photoproteins can play an important role in understanding the structural basis of bioluminescence activity. Based on this postulate, in this study the cDNA of mnemiopsin from luminous ctenophore Mnemiopsis leidyi was cloned, expressed, purified and sequenced. The purified cDNA, with 621 base pairs, coded a 206 residues protein. Sequence of mnemiopsin showed 93.5 and 51% similarity to other ctenophore proteins and cnidarians, respectively. The cDNA encoding apo-mnemiopsin of M. leidyi was expressed in Escherichia coli. The purified apo-protein showed a single band on SDS-PAGE (molecular weight ~27 kDa). A semi-synthetic mnemiopsin was prepared using coelenterazine and EDTA and its luminescence activity was measured in the presence of CaCl₂. The results showed an optimum pH of 9.0 and lower calcium sensitivity compared to aequorin. Comparison of amino acid residues in substrate binding site indicated that binding pocket of ctenophores contains less aromatic residues than cnidarians. This can lead to a decline in the number of stacking interactions between substrate and protein which can affect the stability of coelenterazine in binding cavity. Structural comparison of photoproteins with low sequence identity and high 3D structural similarity, can present a new insight into the mechanism of light emission in photoproteins.