Comparative Metabolomics Approach Detects Stress-Specific Responses during Coral Bleaching in Soft Corals.

Chronic exposure to ocean acidification and elevated sea-surface temperatures pose significant stress to marine ecosystems. This in turn necessitates costly acclimation responses in corals in both the symbiont and host, with a reorganization of cell metabolism and structure. A large-scale untargeted metabolomics approach comprising gas chromatography mass spectrometry (GC-MS) and ultraperformance liquid chromatography coupled to high resolution mass spectrometry (UPLC-MS) was applied to profile the metabolite composition of the soft coral Sarcophyton ehrenbergi and its dinoflagellate symbiont. Metabolite profiling compared ambient conditions with response to simulated climate change stressors and with the sister species, S. glaucum. Among ∼300 monitored metabolites, 13 metabolites were modulated. Incubation experiments providing four selected upregulated metabolites (alanine, GABA, nicotinic acid, and proline) in the culturing water failed to subside the bleaching response at temperature-induced stress, despite their known ability to mitigate heat stress in plants or animals. Thus, the results hint to metabolite accumulation (marker) during heat stress. This study provides the first detailed map of metabolic pathways transition in corals in response to different environmental stresses, accounting for the superior thermal tolerance of S. ehrenbergi versus S. glaucum, which can ultimately help maintain a viable symbiosis and mitigate against coral bleaching.

Salicylic acid and its derivatives elicit the production of diterpenes and sterols in corals and their algal symbionts: a metabolomics approach to elicitor SAR.

INTRODUCTION: The production of marine drugs in its normal habitats is often low and depends greatly on ecological conditions. Chemical synthesis of marine drugs is not economically feasible owing to their complex structures. Biotechnology application via elicitation represents a promising tool to enhance metabolites yield that has yet to be explored in soft corals. OBJECTIVES: Study the elicitation impact of salicylic acid (SA) and six analogues in addition to a systemic acquired resistance inducer on secondary metabolites accumulation in the soft coral Sarcophyton ehrenbergi along with the symbiont zooxanthellae and if SA elicitation effect is extended to other coral species S. glaucum and Lobophyton pauciliforum. METHODS: Post elicitation in the three corals and zooxanthella, metabolites were extracted and analyzed via UHPLC-MS coupled with chemometric tools. RESULTS: Multivariate data analysis of the UHPLC-MS data set revealed clear segregation of SA, amino-SA, and acetyl-SA elicited samples. An increased level ca. 6- and 8-fold of the diterpenes viz., sarcophytonolide I, sarcophine and a C28-sterol, was observed in SA and amino-SA groups, respectively. Post elicitation, the level of diepoxy-cembratriene increased 1.5-fold and 2.4-fold in 1 mM SA, and acetyl-SA (aspirin) treatment groups, respectively. S. glaucum and Lobophyton pauciliforum showed a 2-fold increase of diepoxy-cembratriene levels. CONCLUSION: These results suggest that SA could function as a general and somewhat selective diterpene inducing signaling molecule in soft corals. Structural consideration reveals initial structure-activity relationship (SAR) in SA derivatives that seem important for efficient diterpene and sterol elicitation.

Genetic population structure of the convict surgeonfish Acanthurus triostegus: a phylogeographic reassessment across its range.

This study investigates the genetic population structure and connectivity of Acanthurus triostegus in five Indo-Pacific biogeographic regions (western and eastern Indian Ocean, western, central and eastern Pacific Ocean), using a mitochondrial DNA marker spanning the ATPase8 and ATPase6 gene regions. In order to assess the phylogeography and genetic population structure of A. triostegus across its range, 35 individuals were sampled from five localities in the western Indian Ocean and complemented with 227 sequences from two previous studies. Results from the overall analysis of molecular variance (AMOVA) without a priori grouping showed evidence of significant differentiation in the Indo-Pacific, with 25 (8.3%) out of 300 pairwise ΦST comparisons being significant. However, the hierarchical AMOVA grouping of Indian and Pacific Ocean populations failed to support the vicariance hypothesis, showing a lack of a genetic break between the two ocean basins. Instead, the correlation between pairwise ΦST values and geographic distance showed that dispersal of A. triostegus in the Indo-Pacific Ocean follows an isolation-by-distance model. Three haplogroups could be deduced from the haplotype network and phylogenetic tree, with haplogroup 1 and 2 dominating the Indian and the Pacific Ocean, respectively, while haplogroup 3 exclusively occurring in the Hawaiian Archipelago of the central Pacific Ocean.

DISMS2: A flexible algorithm for direct proteome- wide distance calculation of LC-MS/MS runs.

BACKGROUND: The classification of samples on a molecular level has manifold applications, from patient classification regarding cancer treatment to phylogenetics for identifying evolutionary relationships between species. Modern methods employ the alignment of DNA or amino acid sequences, mostly not genome-wide but only on selected parts of the genome. Recently proteomics-based approaches have become popular. An established method for the identification of peptides and proteins is liquid chromatography-tandem mass spectrometry (LC-MS/MS). First, protein sequences from MS/MS spectra are identified by means of database searches, given samples with known genome-wide sequence information, then sequence based methods are applied. Alternatively, de novo peptide sequencing algorithms annotate MS/MS spectra and deduce peptide/protein information without a database. A newer approach independent of additional information is to directly compare unidentified tandem mass spectra. The challenge then is to compute the distance between pairwise MS/MS runs consisting of thousands of spectra. METHODS: We present DISMS2, a new algorithm to calculate proteome-wide distances directly from MS/MS data, extending the algorithm compareMS2, an approach that also uses a spectral comparison pipeline. RESULTS: Our new more flexible algorithm, DISMS2, allows for the choice of the spectrum distance measure and includes different spectra preprocessing and filtering steps that can be tailored to specific situations by parameter optimization. CONCLUSIONS: DISMS2 performs well for samples from species with and without database annotation and thus has clear advantages over methods that are purely based on database search.

How to improve parameter estimates in GLM-based fMRI data analysis: cross-validated Bayesian model averaging.

In functional magnetic resonance imaging (fMRI), model quality of general linear models (GLMs) for first-level analysis is rarely assessed. In recent work (Soch et al., 2016: "How to avoid mismodelling in GLM-based fMRI data analysis: cross-validated Bayesian model selection", NeuroImage, vol. 141, pp. 469-489; http://dx.doi.org/10.1016/j.neuroimage.2016.07.047), we have introduced cross-validated Bayesian model selection (cvBMS) to infer the best model for a group of subjects and use it to guide second-level analysis. While this is the optimal approach given that the same GLM has to be used for all subjects, there is a much more efficient procedure when model selection only addresses nuisance variables and regressors of interest are included in all candidate models. In this work, we propose cross-validated Bayesian model averaging (cvBMA) to improve parameter estimates for these regressors of interest by combining information from all models using their posterior probabilities. This is particularly useful as different models can lead to different conclusions regarding experimental effects and the most complex model is not necessarily the best choice. We find that cvBMS can prevent not detecting established effects and that cvBMA can be more sensitive to experimental effects than just using even the best model in each subject or the model which is best in a group of subjects.

Phylogenomic analyses unravel annelid evolution.

Annelida, the ringed worms, is a highly diverse animal phylum that includes more than 15,000 described species and constitutes the dominant benthic macrofauna from the intertidal zone down to the deep sea. A robust annelid phylogeny would shape our understanding of animal body-plan evolution and shed light on the bilaterian ground pattern. Traditionally, Annelida has been split into two major groups: Clitellata (earthworms and leeches) and polychaetes (bristle worms), but recent evidence suggests that other taxa that were once considered to be separate phyla (Sipuncula, Echiura and Siboglinidae (also known as Pogonophora)) should be included in Annelida. However, the deep-level evolutionary relationships of Annelida are still poorly understood, and a robust reconstruction of annelid evolutionary history is needed. Here we show that phylogenomic analyses of 34 annelid taxa, using 47,953 amino acid positions, recovered a well-supported phylogeny with strong support for major splits. Our results recover chaetopterids, myzostomids and sipunculids in the basal part of the tree, although the position of Myzostomida remains uncertain owing to its long branch. The remaining taxa are split into two clades: Errantia (which includes the model annelid Platynereis), and Sedentaria (which includes Clitellata). Ancestral character trait reconstructions indicate that these clades show adaptation to either an errant or a sedentary lifestyle, with alteration of accompanying morphological traits such as peristaltic movement, parapodia and sensory perception. Finally, life history characters in Annelida seem to be phylogenetically informative.

Phylogenomics reveals deep molluscan relationships.

Evolutionary relationships among the eight major lineages of Mollusca have remained unresolved despite their diversity and importance. Previous investigations of molluscan phylogeny, based primarily on nuclear ribosomal gene sequences or morphological data, have been unsuccessful at elucidating these relationships. Recently, phylogenomic studies using dozens to hundreds of genes have greatly improved our understanding of deep animal relationships. However, limited genomic resources spanning molluscan diversity has prevented use of a phylogenomic approach. Here we use transcriptome and genome data from all major lineages (except Monoplacophora) and recover a well-supported topology for Mollusca. Our results strongly support the Aculifera hypothesis placing Polyplacophora (chitons) in a clade with a monophyletic Aplacophora (worm-like molluscs). Additionally, within Conchifera, a sister-taxon relationship between Gastropoda and Bivalvia is supported. This grouping has received little consideration and contains most (>95%) molluscan species. Thus we propose the node-based name Pleistomollusca. In light of these results, we examined the evolution of morphological characters and found support for advanced cephalization and shells as possibly having multiple origins within Mollusca.

Cytotoxic Effects of Sarcophyton sp. Soft Corals-Is There a Correlation to Their NMR Fingerprints?

Sarcophyton sp. soft corals are rich in cembranoid diterpenes, which represent the main chemical defense of corals against their natural predators in addition to their myriad biological effects in humans. Quantitative NMR (qNMR) was applied for assessing the diterpene variation in 16 soft coral specimens in the context of their genotype, origin, and growing habitat. qNMR revealed high diterpene levels in Sarcophyton sp. compared to Sinularia and Lobophyton, with (ent)sarcophines as major components (17-100 µg/mg) of the coral tissues. Multivariate data analysis was employed to classify samples based on the quantified level of diterpenes, and compared to the untargeted NMR approach. Results revealed that qNMR provided a stronger classification model of Sarcophyton sp. than untargeted NMR fingerprinting. Additionally, cytotoxicity of soft coral crude extracts was assessed against androgen-dependent prostate cancer cell lines (PC3) and androgen-independent colon cancer cell lines (HT-29), with IC50 values ranging from 10-60 µg/mL. No obvious correlation between the extracts' IC50 values and their diterpene levels was found using either Spearman or Pearson correlations. This suggests that this type of bioactivity may not be easily predicted by NMR metabolomics in soft corals, or is not strongly correlated to measured diterpene levels.

Effect of Oxylipins, Terpenoid Precursors and Wounding on Soft Corals' Secondary Metabolism as Analyzed via UPLC/MS and Chemometrics.

The effect of three oxylipin analogues, a terpenoid intermediate and wounding on the secondary metabolism of the soft corals Sarcophyton glaucum and Lobophyton pauciflorum was assessed. Examined oxylipins included prostaglandin (PG-E1), methyl jasmonate (MeJA), and arachidonic acid (AA) in addition to the diterpene precursor geranylgeranylpyrophosphate (GGP). Post-elicitation, metabolites were extracted from coral heads and analyzed via UPLC-MS followed by multivariate data analyses. Both supervised and unsupervised data analyses were used for sample classification. Multivariate data analysis revealed clear segregation of PG-E1 and MeJA elicited S. glaucum at 24 and 48 h post elicitation from other elicitor samples and unelicited control group. PG-E1 was found more effective in upregulating S. glaucum terpene/sterol levels compared to MeJA. Metabolites showing upregulation in S. glaucum include campestene-triol and a cembranoid, detected at ca. 30- and 2-fold higher levels compared to unelicited corals. Such an elicitation effect was less notable in the other coral species L. pauciflorum, suggesting a differential oxylipin response in soft corals. Compared to MeJA and PG, no elicitation effect was observed for GGP, AA or wounding on the metabolism of either coral species.

Soft Corals Biodiversity in the Egyptian Red Sea: A Comparative MS and NMR Metabolomics Approach of Wild and Aquarium Grown Species.

Marine life has developed unique metabolic and physiologic capabilities and advanced symbiotic relationships to survive in the varied and complex marine ecosystems. Herein, metabolite composition of the soft coral genus Sarcophyton was profiled with respect to its species and different habitats along the coastal Egyptian Red Sea via (1)H NMR and ultra performance liquid chromatography-mass spectrometry (UPLC-MS) large-scale metabolomics analyses. The current study extends the application of comparative secondary metabolite profiling from plants to corals revealing for metabolite compositional differences among its species via a comparative MS and NMR approach. This was applied for the first time to investigate the metabolism of 16 Sarcophyton species in the context of their genetic diversity or growth habitat. Under optimized conditions, we were able to simultaneously identify 120 metabolites including 65 diterpenes, 8 sesquiterpenes, 18 sterols, and 15 oxylipids. Principal component analysis (PCA) and orthogonal projection to latent structures-discriminant analysis (OPLS) were used to define both similarities and differences among samples. For a compound based classification of coral species, UPLC-MS was found to be more effective than NMR. The main differentiations emanate from cembranoids and oxylipids. The specific metabolites that contribute to discrimination between soft corals of S. ehrenbergi from the three different growing habitats also belonged to cembrane type diterpenes, with aquarium S. ehrenbergi corals being less enriched in cembranoids compared to sea corals. PCA using either NMR or UPLC-MS data sets was found equally effective in predicting the species origin of unknown Sarcophyton. Cyclopropane containing sterols observed in abundance in corals may act as cellular membrane protectant against the action of coral toxins, that is, cembranoids.

Time-Dependent Inhibition of CYP2C19 by Isoquinoline Alkaloids: In Vitro and In Silico Analysis.

The cytochrome P450 2C19 (CYP2C19) enzyme plays an important role in the metabolism of many commonly used drugs. Relatively little is known about CYP2C19 inhibitors, including compounds of natural origin, which could inhibit CYP2C19, potentially causing clinically relevant metabolism-based drug interactions. We evaluated a series (N = 49) of structurally related plant isoquinoline alkaloids for their abilities to interact with CYP2C19 enzyme using in vitro and in silico methods. We examined several common active alkaloids found in herbal products such as apomorphine, berberine, noscapine, and papaverine, as well as the previously identified mechanism-based inactivators bulbocapnine, canadine, and protopine. The IC50 values of the alkaloids ranged from 0.11 to 210 µM, and 42 of the alkaloids were confirmed to be time-dependent inhibitors of CYP2C19. Molecular docking and three-dimensional quantitative structure-activity relationship analysis revealed key interactions of the potent inhibitors with the enzyme active site. We constructed a comparative molecular field analysis model that was able to predict the inhibitory potency of a series of independent test molecules. This study revealed that many of these isoquinoline alkaloids do have the potential to cause clinically relevant drug interactions. These results highlight the need for studying more profoundly the potential interactions between drugs and herbal products. When further refined, in silico methods can be useful in the high-throughput prediction of P450 inhibitory potential of pharmaceutical compounds.

New phylogenomic data support the monophyly of Lophophorata and an Ectoproct-Phoronid clade and indicate that Polyzoa and Kryptrochozoa are caused by systematic bias.

BACKGROUND: Within the complex metazoan phylogeny, the relationships of the three lophophorate lineages, ectoprocts, brachiopods and phoronids, are particularly elusive. To shed further light on this issue, we present phylogenomic analyses of 196 genes from 58 bilaterian taxa, paying particular attention to the influence of compositional heterogeneity. RESULTS: The phylogenetic analyses strongly support the monophyly of Lophophorata and a sister-group relationship between Ectoprocta and Phoronida. Our results contrast previous findings based on rDNA sequences and phylogenomic datasets which supported monophyletic Polyzoa (= Bryozoa sensu lato) including Ectoprocta, Entoprocta and Cycliophora, Brachiozoa including Brachiopoda and Phoronida as well as Kryptrochozoa including Brachiopoda, Phoronida and Nemertea, thus rendering Lophophorata polyphyletic. Our attempts to identify the causes for the conflicting results revealed that Polyzoa, Brachiozoa and Kryptrochozoa are supported by character subsets with deviating amino acid compositions, whereas there is no indication for compositional heterogeneity in the character subsets supporting the monophyly of Lophophorata. CONCLUSION: Our results indicate that the support for Polyzoa, Brachiozoa and Kryptrochozoa gathered so far is likely an artifact caused by compositional bias. The monophyly of Lophophorata implies that the horseshoe-shaped mesosomal lophophore, the tentacular feeding apparatus of ectoprocts, phoronids and brachiopods is, indeed, a synapomorphy of the lophophorate lineages. The same may apply to radial cleavage. However, among phoronids also spiral cleavage is known. This suggests that the cleavage pattern is highly plastic and has changed several times within lophophorates. The sister group relationship of ectoprocts and phoronids is in accordance with the interpretation of the eversion of a ventral invagination at the beginning of metamorphosis as a common derived feature of these taxa.

Excess labile carbon promotes diazotroph abundance in heat-stressed octocorals.

Nitrogen limitation is the foundation of stable coral-algal symbioses. Diazotrophs, prokaryotes capable of fixing N2 into ammonia, support the productivity of corals in oligotrophic waters, but could contribute to the destabilization of holobiont functioning when overstimulated. Recent studies on reef-building corals have shown that labile dissolved organic carbon (DOC) enrichment or heat stress increases diazotroph abundance and activity, thereby increasing nitrogen availability and destabilizing the coral-algal symbiosis. However, the (a)biotic drivers of diazotrophs in octocorals are still poorly understood. We investigated diazotroph abundance (via relative quantification of nifH gene copy numbers) in two symbiotic octocorals, the more mixotrophic soft coral Xenia umbellata and the more autotrophic gorgonian Pinnigorgia flava, under (i) labile DOC enrichment for 21 days, followed by (ii) combined labile DOC enrichment and heat stress for 24 days. Without heat stress, relative diazotroph abundances in X. umbellata and P. flava were unaffected by DOC enrichment. During heat stress, DOC enrichment (20 and 40 mg glucose l-1) increased the relative abundances of diazotrophs by sixfold in X. umbellata and fourfold in P. flava, compared with their counterparts without excess DOC. Our data suggest that labile DOC enrichment and concomitant heat stress could disrupt the nitrogen limitation in octocorals by stimulating diazotroph proliferation. Ultimately, the disruption of nitrogen cycling may further compromise octocoral fitness by destabilizing symbiotic nutrient cycling. Therefore, improving local wastewater facilities to reduce labile DOC input into vulnerable coastal ecosystems may help octocorals cope with ocean warming.

Collecting and Culturing Kamptozoans for Regenerative Studies.

Kamptozoa, also known as Entoprocta, are small aquatic filter-feeders that belong to the Lophotrochozoan superphylum, which also contains other acoelomate phyla including Annelida, Nemertea, and Mollusca. The study of Kamptozoa is thus of great interest to understand the early Lophotrochozoan evolution. Moreover, many kamptozoans have been shown to possess great regeneration capacities, including whole-body regeneration. In addition, and in particular for colonial cosmopolitan species such as Barentsia benedeni, kamptozoans are highly suitable as laboratory model organisms because of their simple culture, low space requirements, transparency and rapid life cycle. This chapter provides a brief introduction into field collection, culturing techniques for both the animals as well as the algae required for their feeding, fixation, staining, and sequencing.

Assessing divergence time of Spirulida and Sepiida (Cephalopoda) based on hemocyanin sequences.

The phylogenetic position of the mesopelagic decabrachian cephalopod Spirula is still a matter of debate. Since hemocyanin has successfully been used to calibrate a molecular clock for many molluscan species, a molecular clock was calculated based on this gene with special attention to the cephalopod genera Spirula and Sepia. The obtained partial sequence comprising ca., one third (3567 bp) of the complete gene is similar to that of Sepia officinalis. The molecular clock was calibrated using the splits of Gastropoda-Cephalopoda (ca. 550 ± 50 mya) and Heterobranchia-Vetigastropoda (ca. 380 ± 10 mya). The resulting hemocyanin-based molecular clock is stable, and the estimated divergence time of Spirulida and Sepiida, some 150 ± 30 million years ago, can be deemed reliable.

CYP2C19 progress curve analysis and mechanism-based inactivation by three methylenedioxyphenyl compounds.

Several in vitro criteria were used to assess whether three methylenedioxyphenyl (MDP) compounds, the isoquinoline alkaloids bulbocapnine, canadine, and protopine, are mechanism-based inactivators of CYP2C19. The recently reported fluorometric CYP2C19 progress curve analysis approach was applied first to determine whether these alkaloids demonstrate time-dependent inhibition. In this experiment, bulbocapnine, canadine, and protopine displayed time dependence and saturation in their inactivation kinetics with K(I) and k(inact) values of 72.4 ± 14.7 µM and 0.38 ± 0.036 min(-1), 2.1 ± 0.63 µM and 0.18 ± 0.015 min(-1), and 7.1 ± 2.3 µM and 0.24 ± 0.021 min(-1), respectively. Additional studies were performed to determine whether other specific criteria for mechanism-based inactivation were fulfilled: NADPH dependence, irreversibility, and involvement of a catalytic step in the enzyme inactivation. CYP2C19 activity was not significantly restored by dialysis when it had been inactivated by the alkaloids in the presence of a NADPH-regenerating system, and a metabolic-intermediate complex-associated increase in absorbance at approximately 455 nm was observed. In conclusion, the CYP2C19 progress curve analysis method revealed time-dependent inhibition by these alkaloids, and additional experiments confirmed its quasi-irreversible nature. This study revealed that the CYP2C19 progress curve analysis method is useful for identifying novel mechanism-based inactivators and yields a wealth of information in one run. The alkaloids bulbocapnine, canadine, and protopine, present in herbal medicines, are new mechanism-based inactivators and the first MDP compounds exhibiting quasi-irreversible inactivation of CYP2C19.

Benzylisoquinoline alkaloids from the papaveraceae: the heritage of Johannes Gadamer (1867-1928).

The substance archive of the laboratory of Johannes Gadamer (1867-1928), Marburg, Germany, was analyzed thoroughly with modern instrumental methods, with the samples purified when necessary, and the authenticity of the samples confirmed by historical and analytical evidence. Eight formerly unknown alkaloids of the benzylisoquinoline type were identified in the archive originally isolated from Corydalis cava or Fumaria vaillantii. This finding underscores the importance of the work of Johannes Gadamer and his group in stimulating overall progress in natural product chemistry. Several alkaloids were isolated by the group long before they were officially reported.

Bleaching effect in Sarcophyton spp. soft corals-is there a correlation to their diterpene content?

Rising seawater temperature is one of the greatest threats to the persistence of coral reefs. While great efforts have been made to understand the metabolic costs of thermal acclimation, the exact roles of many secondary metabolites involved in the immediate response exhibited by soft corals remain largely unknown. Herein, an untargeted metabolomics approach using ultra-performance liquid chromatography coupled to high-resolution mass spectrometry (UPLC-MS) was employed to investigate thermal stress-induced modifications to the de novo synthesis of secondary metabolites in two soft coral species, Sarcophyton ehrenbergi and S. glaucum. Exposure to elevated temperature resulted in symbiont photoinhibition primarily via either damage to photosystem II (PSII) or the loss of algal symbionts during coral bleaching. This was suggested by a decrease in pulse amplitude modulated (PAM) measurements of corals incubated at different temperatures. Thermal stress was also found to impair the production of diterpenoid secondary metabolites in soft corals. Principally, reduction in the levels of a number of diterpenes, viz. sarcophytoxide and deoxysarcophytoxide, in heat stressed S. ehrenbergi and S. glaucum was observed indicative that thermal acclimation is energetically costly and will necessitate downstream changes in secondary metabolic pathways. Our data suggest that, while the host controls the production of ecologically important terpenes, when energetic contribution from the algal symbiont is reduced or absent as a result of a bleaching event, energy reserves may be insufficient to maintain the production of such energetically cost chemicals. This study provides for the first time a holistic assessment of secondary metabolite changes imposed in soft corals during exposure and acclimation to elevated temperatures.

Fast evolving 18S rRNA sequences from Solenogastres (Mollusca) resist standard PCR amplification and give new insights into mollusk substitution rate heterogeneity.

BACKGROUND: The 18S rRNA gene is one of the most important molecular markers, used in diverse applications such as molecular phylogenetic analyses and biodiversity screening. The Mollusca is the second largest phylum within the animal kingdom and mollusks show an outstanding high diversity in body plans and ecological adaptations. Although an enormous amount of 18S data is available for higher mollusks, data on some early branching lineages are still limited. Despite of some partial success in obtaining these data from Solenogastres, by some regarded to be the most "basal" mollusks, this taxon still remained problematic due to contamination with food organisms and general amplification difficulties. RESULTS: We report here the first authentic 18S genes of three Solenogastres species (Mollusca), each possessing a unique sequence composition with regions conspicuously rich in guanine and cytosine. For these GC-rich regions we calculated strong secondary structures. The observed high intra-molecular forces hamper standard amplification and appear to increase formation of chimerical sequences caused by contaminating foreign DNAs from potential prey organisms. In our analyses, contamination was avoided by using RNA as a template. Indication for contamination of previously published Solenogastres sequences is presented. Detailed phylogenetic analyses were conducted using RNA specific models that account for compensatory substitutions in stem regions. CONCLUSIONS: The extreme morphological diversity of mollusks is mirrored in the molecular 18S data and shows elevated substitution rates mainly in three higher taxa: true limpets (Patellogastropoda), Cephalopoda and Solenogastres. Our phylogenetic tree based on 123 species, including representatives of all mollusk classes, shows limited resolution at the class level but illustrates the pitfalls of artificial groupings formed due to shared biased sequence composition.

Mitochondrial genome sequence and gene order of Sipunculus nudus give additional support for an inclusion of Sipuncula into Annelida.

BACKGROUND: Mitochondrial genomes are a valuable source of data for analysing phylogenetic relationships. Besides sequence information, mitochondrial gene order may add phylogenetically useful information, too. Sipuncula are unsegmented marine worms, traditionally placed in their own phylum. Recent molecular and morphological findings suggest a close affinity to the segmented Annelida. RESULTS: The first complete mitochondrial genome of a member of Sipuncula, Sipunculus nudus, is presented. All 37 genes characteristic for metazoan mtDNA were detected and are encoded on the same strand. The mitochondrial gene order (protein-coding and ribosomal RNA genes) resembles that of annelids, but shows several derivations so far found only in Sipuncula. Sequence based phylogenetic analysis of mitochondrial protein-coding genes results in significant bootstrap support for Annelida sensu lato, combining Annelida together with Sipuncula, Echiura, Pogonophora and Myzostomida. CONCLUSION: The mitochondrial sequence data support a close relationship of Annelida and Sipuncula. Also the most parsimonious explanation of changes in gene order favours a derivation from the annelid gene order. These results complement findings from recent phylogenetic analyses of nuclear encoded genes as well as a report of a segmental neural patterning in Sipuncula.