Hidden aliens: Application of digital PCR to track an exotic foraminifer across the Skagerrak (North Sea) correlates well with traditional morphospecies analysis.

The problem of invasive species is a well-studied one, but knowledge of free-living unicellular eukaryotic invasive species is lacking. A potentially invasive foraminifer (Rhizaria), Nonionella sp. T1, was recently discovered in the Skagerrak and its fjords. Digital polymerase chain reaction (dPCR) was applied to track the spread of this non-indigenous species using a new dPCR assay (T1-1). The use of dPCR appears highly complementary to traditional hand picking of foraminiferal shells from the sediment, and is far less time-consuming. This study indicates that Nonionella sp. T1 has bypassed the outer Skagerrak strait, instead becoming established in Swedish west coast fjords, constituting up to half of the living foraminiferal community in fjord mouth areas. The ecology of Nonionella sp. T1 and its potential invasive impacts are still largely unknown, but it appears to be an opportunist using several energy sources such as nitrate respiration and kleptoplasty along with a possibly more efficient reproductive strategy to gain an advantage over the native foraminiferal species. Future ecological studies of Nonionella sp. T1 could be aided by dPCR and the novel Nonionella sp. T1-specific T1-1 assay.

Conservation and turnover of miRNAs and their highly complementary targets in early branching animals.

MicroRNAs (miRNAs) are crucial post-transcriptional regulators that have been extensively studied in Bilateria, a group comprising the majority of extant animals, where more than 30 conserved miRNA families have been identified. By contrast, bilaterian miRNA targets are largely not conserved. Cnidaria is the sister group to Bilateria and thus provides a unique opportunity for comparative studies. Strikingly, like their plant counterparts, cnidarian miRNAs have been shown to predominantly have highly complementary targets leading to transcript cleavage by Argonaute proteins. Here, we assess the conservation of miRNAs and their targets by small RNA sequencing followed by miRNA target prediction in eight species of Anthozoa (sea anemones and corals), the earliest-branching cnidarian class. We uncover dozens of novel miRNAs but only a few conserved ones. Further, given their high complementarity, we were able to computationally identify miRNA targets in each species. Besides evidence for conservation of specific miRNA target sites, which are maintained between sea anemones and stony corals across 500 Myr of evolution, we also find indications for convergent evolution of target regulation by different miRNAs. Our data indicate that cnidarians have only few conserved miRNAs and corresponding targets, despite their high complementarity, suggesting a high evolutionary turnover.

Evaluating the Utility of Single-Locus DNA Barcoding for the Identification of Ribbon Worms (Phylum Nemertea).

Whereas many nemerteans (ribbon worms; phylum Nemertea) can be identified from external characters if observed alive, many are still problematic. When it comes to preserved specimens (as in e.g. marine inventories), there is a particular need for specimen identifier alternatives. Here, we evaluate the utility of COI (cytochrome c oxidase subunit I) as a single-locus barcoding gene. We sequenced, data mined, and compared gene fragments of COI for 915 individuals representing 161 unique taxonomic labels for 71 genera, and subjected different constellations of these to both distance-based and character-based DNA barcoding approaches, as well as species delimitation analyses. We searched for the presence or absence of a barcoding gap at different taxonomic levels (phylum, subclass, family and genus) in an attempt to understand at what level a putative barcoding gap presents itself. This was performed both using the taxonomic labels as species predictors and using objectively inferred species boundaries recovered from our species delimitation analyses. Our data suggest that COI works as a species identifier for most groups within the phylum, but also that COI data are obscured by misidentifications in sequence databases. Further, our results suggest that the number of predicted species within the dataset is (in some cases substantially) higher than the number of unique taxonomic labels-this highlights the presence of several cryptic lineages within well-established taxa and underscores the urgency of an updated taxonomic backbone for the phylum.

The Bacterial (Vibrio alginolyticus) Production of Tetrodotoxin in the Ribbon Worm Lineus longissimus-Just a False Positive?

We test previous claims that the bacteria Vibrio alginolyticus produces tetrodotoxin (TTX) when living in symbiosis with the nemertean Lineus longissimus by a setup with bacteria cultivation for TTX production. Toxicity experiments on the shore crab, Carcinus maenas, demonstrated the presence of a paralytic toxin, but evidence from LC-MS and electrophysiological measurements of voltage-gated sodium channel-dependent nerve conductance in male Wistar rat tissue showed conclusively that this effect did not originate from TTX. However, a compound of similar molecular weight was found, albeit apparently non-toxic, and with different LC retention time and MS/MS fragmentation pattern than those of TTX. We conclude that C. maenas paralysis and death likely emanate from a compound <5 kDa, and via a different mechanism of action than that of TTX. The similarity in mass between TTX and the Vibrio-produced low-molecular-weight, non-toxic compound invokes that thorough analysis is required when assessing TTX production. Based on our findings, we suggest that re-examination of some published claims of TTX production may be warranted.

Thirty-Five Years of Nemertean (Nemertea) Research--Past, Present, and Future.

Developments in nemertean research over the last 35+ years are reviewed from a systematist's perspective. Nemertean systematics and classification, until fairly recently, was not based on explicit phylogenetic hypotheses, but rather on subjective assessment of "important characters". The first cladistic analyses appeared in the 1980s and were criticized at the time by leading researchers in nemertean systematics for not taking into account convergent evolution in ribbon worm morphology. The first molecular study involving the phylum Nemertea appeared in 1992, followed by reports later in the 1990s and early 2000s. Molecular information is now commonplace in nemertean research, and has changed our understanding of evolutionary relationships within the phylum, as well as our view on species and intraspecific variation. Challenges in nemertean systematics and taxonomy are discussed, with special emphasis on future species descriptions, and how to deal with a number of species names that in all likelihood never will be encountered again. Suggestions for how to deal with these challenges are discussed.

Species Diversity of Ramphogordius sanguineus/Lineus ruber-Like Nemerteans (Nemertea: Heteronemertea) and Geographic Distribution of R. sanguineus.

Heteronemerteans, such as Lineus ruber, L. viridis, Ramphogordius sanguineus, R. lacteus, Riseriellus occultus, and Micrura varicolor, share many similar external characters. Although several internal characters useful for distinguishing these nemertean species have been documented, their identification is based mostly on coloration, the shape of the head, and how they contract, which may not be always reliable. We sequenced the mitochondrial COI gene for 160 specimens recently collected from 27 locations around the world (provisionally identified as the above species, according to external characters and contraction patterns, with most of them as R. sanguineus). Based on these specimens, together with sequences of 16 specimens from GenBank, we conducted a DNA-based species delimitation/identification by means of statistical parsimony and phylogenetic analyses. Our results show that the analyzed specimens may contain nine species, which can be separated by large genetic gaps; heteronemerteans with an external appearance similar to R. sanguineus/Lineus ruber/L. viridis have high species diversity in European waters from where eight species can be discriminated. Our 42 individuals from Vancouver Island (Canada) are revealed to be R. sanguineus, which supports an earlier argument that nemerteans reported as L. ruber or L. viridis from the Pacific Northwest may refer to this species. We report R. sanguineus from Chile, southern China, and the species is also distributed on the Atlantic coast of South America (Argentina). In addition, present analyses reveal the occurrence of L. viridis in Qingdao, which is the first record of the species from Chinese waters.

DNA barcoding supports identification of Malacobdella species (Nemertea: Hoplonemertea).

BACKGROUND: Nemerteans of the genus Malacobdella live inside of the mantle cavity of marine bivalves. The genus currently contains only six species, five of which are host-specific and usually found in a single host species, while the sixth species, M. grossa, has a wide host range and has been found in 27 different bivalve species to date. The main challenge of Malacobdella species identification resides in the similarity of the external morphology between species (terminal sucker, gut undulations number, anus position and gonad colouration), and thus, the illustrations provided in the original descriptions do not allow reliable identification. In this article, we analyse the relationships amongthree species of Malacobdella:M.arrokeana,M.japonica andM.grossa,adding new data for the M.grossa and reporting the first for M. japonica, analysing 658 base pairs of the mitochondrial cytochrome c oxidase subunit I gene(COI).Based on these analyses, we present and discuss the potential of DNA barcoding for Malacobdellaspecies identification. RESULTS: Sixty-four DNA barcoding fragments of the mitochondrial COI gene from three different Malacobdella species (M. arrokeana, M. japonica and M. grossa) are analysed (24 of them newly sequenced for this study, along with four outgroup specimens) and used to delineate species. Divergences, measured as uncorrected differences, between the three species were M.arrokeana-M. grossa11.73%,M.arrokeana-M.japonica 10.62%and M.grossa-M. japonica 10.97%. The mean intraspecific divergence within the ingroup species showed a patent gap with respect to the interspecific ones: 0.18% for M.arrokeana,0.13% for M.grossa and0.02% for M.japonica (rangesfrom 0 to 0.91%). CONCLUSIONS: We conclude that there is a clear correspondence between the molecular data and distinguishing morphological characters. Our results thus indicate that some morphological characters are useful for species identification and support the potential of DNA barcoding for species identification in a taxonomic group with subtle morphological external differences.

A transcriptomic approach to ribbon worm systematics (nemertea): resolving the pilidiophora problem.

Resolving the deep relationships of ancient animal lineages has proven difficult using standard Sanger-sequencing approaches with a handful of markers. We thus reassess the relatively well-studied phylogeny of the phylum Nemertea (ribbon worms)-for which the targeted gene approaches had resolved many clades but had left key phylogenetic gaps-by using a phylogenomic approach using Illumina-based de novo assembled transcriptomes and automatic orthology prediction methods. The analysis of a concatenated data set of 2,779 genes (411,138 amino acids) with about 78% gene occupancy and a reduced version with 95% gene occupancy, under evolutionary models accounting or not for site-specific amino acid replacement patterns results in a well-supported phylogeny that recovers all major accepted nemertean clades with the monophyly of Heteronemertea, Hoplonemertea, Monostilifera, being well supported. Significantly, all the ambiguous patterns inferred from Sanger-based approaches were resolved, namely the monophyly of Palaeonemertea and Pilidiophora. By testing for possible conflict in the analyzed supermatrix, we observed that concatenation was the best solution, and the results of the analyses should settle prior debates on nemertean phylogeny. The study highlights the importance, feasibility, and completeness of Illumina-based phylogenomic data matrices.

Complete mitochondrial genome sequences of two parasitic/commensal nemerteans, Gononemertes parasita and Nemertopsis tetraclitophila (Nemertea: Hoplonemertea).

BACKGROUND: Most nemerteans (phylum Nemertea) are free-living, but about 50 species are known to be firmly associated with other marine invertebrates. For example, Gononemertes parasita is associated with ascidians, and Nemertopsis tetraclitophila with barnacles. There are 12 complete or near-complete mitochondrial genome (mitogenome) sequences of nemerteans available in GenBank, but no mitogenomes of none free-living nemerteans have been determined so far. In the present paper complete mitogenomes of the above two parasitic/commensal nemerteans are reported. METHODS: The complete mitochondrial genomes (mitogenome) of G. parasita and N. tetraclitophila were amplified by conventional and long PCR. Phylogenetic analyses of maximum likelihood (ML) and Bayesian inference (BI) were performed with both concatenated nucleotide and amino acid sequences. RESULTS: Complete mitogenomes of G. parasita and N. tetraclitophila are 14742 bp and 14597 bp in size, respectively, which are within the range of published Hoplonemertea mitogenomes. Their gene orders are identical to that of published Hoplonemertea mitogenomes, but different from those of Palaeo- and Heteronemertea species. All the coding genes, as well as major non-coding regions (mNCRs), are AT rich, which is especially pronounced at the third codon position. The AT/GC skew pattern of the coding strand is the same among nemertean mitogenomes, but is variable in the mNCRs. Some slight differences are found between mitogenomes of the present species and other hoplonemerteans: in G. parasita the mNCR is biased toward T and C (contrary to other hoplonemerteans) and the rrnS gene has a unique 58-bp insertion at the 5' end; in N. tetraclitophila the nad3 gene starts with the ATT codon (ATG in other hoplonemerteans). Phylogenetic analyses of the nucleotide and amino acid datasets show early divergent positions of G. parasita and N. tetraclitophila within the analyzed Distromatonemertea species, and provide strong support for the close relationship between Hoplonemertea and Heteronemertea. CONCLUSION: Gene order is highly conserved within the order Monostilifera, particularly within the Distromatonemertea, and the special lifestyle of G. parasita and N. tetraclitophila does not bring significant variations to the overall structures of their mitogenomes in comparison with free-living hoplonemerteans.

Mutation and selection cause codon usage and bias in mitochondrial genomes of ribbon worms (Nemertea).

The phenomenon of codon usage bias is known to exist in many genomes and it is mainly determined by mutation and selection. To understand the patterns of codon usage in nemertean mitochondrial genomes, we use bioinformatic approaches to analyze the protein-coding sequences of eight nemertean species. Neutrality analysis did not find a significant correlation between GC12 and GC3. ENc-plot showed a few genes on or close to the expected curve, but the majority of points with low-ENc values are below it. ENc-plot suggested that mutational bias plays a major role in shaping codon usage. The Parity Rule 2 plot (PR2) analysis showed that GC and AT were not used proportionally and we propose that codons containing A or U at third position are used preferentially in nemertean species, regardless of whether corresponding tRNAs are encoded in the mitochondrial DNA. Context-dependent analysis indicated that the nucleotide at the second codon position slightly affects synonymous codon choices. These results suggested that mutational and selection forces are probably acting to codon usage bias in nemertean mitochondrial genomes.

Taxonomic identity of a tetrodotoxin-accumulating ribbon-worm Cephalothrix simula (Nemertea: Palaeonemertea): a species artificially introduced from the Pacific to Europe.

We compared the anatomy of the holotype of the palaeonemertean Cephalothrix simula ( Iwata, 1952 ) with that of the holotypes of Cephalothrix hongkongiensis Sundberg, Gibson and Olsson, 2003 and Cephalothrix fasciculus ( Iwata, 1952 ), as well as additional specimens from Fukue (type locality of C. simula) and Hiroshima, Japan. While there was no major morphological discordance between these specimens, we found discrepancies between the actual morphology and some statements in the original description of C. simula with respect to supposedly species-specific characters. Our observation indicates that these three species cannot be discriminated by the anatomical characters so far used to distinguish congeners. For objectivity of scientific names, topogenetypes of the mitochondrial cytochrome c oxidase subunit I (COI) sequences are designated for C. simula, C. hongkongiensis, and C. fasciculus. Analysis of COI sequence showed that the Hiroshima population can be identified as C. simula, which has been found in previous studies from Trieste, Italy, and also from both the Mediterranean and Atlantic coasts of the Iberian Peninsula, indicating an artificial introduction via (1) ballast water, (2) ship-fouling communities, or (3) the commercially cultured oyster Crassostrea gigas ( Thunberg, 1793 ) brought from Japan to France in 1970s. Cephalothrix simula is known to be toxic, as it contains large amounts of tetrodotoxin (TTX). We report here that the grass puffer Takifugu niphobles ( Jordan and Snyder, 1901 )-also known to contain TTX- consumes C. simula. We suggest that the puffer may be able to accumulate TTX by eating C. simula.

A comparative study of nemertean complete mitochondrial genomes, including two new ones for Nectonemertes cf. mirabilis and Zygeupolia rubens, may elucidate the fundamental pattern for the phylum Nemertea.

BACKGROUND: The mitochondrial genome is important for studying genome evolution as well as reconstructing the phylogeny of organisms. Complete mitochondrial genome sequences have been reported for more than 2200 metazoans, mainly vertebrates and arthropods. To date, from a total of about 1275 described nemertean species, only three complete and two partial mitochondrial DNA sequences from nemerteans have been published. Here, we report the entire mitochondrial genomes for two more nemertean species: Nectonemertes cf. mirabilis and Zygeupolia rubens. RESULTS: The sizes of the entire mitochondrial genomes are 15365 bp for N. cf. mirabilis and 15513 bp for Z. rubens. Each circular genome contains 37 genes and an AT-rich non-coding region, and overall nucleotide composition is AT-rich. In both species, there is significant strand asymmetry in the distribution of nucleotides, with the coding strand being richer in T than A and in G than C. The AT-rich non-coding regions of the two genomes have some repeat sequences and stem-loop structures, both of which may be associated with the initiation of replication or transcription. The 22 tRNAs show variable substitution patterns in nemerteans, with higher sequence conservation in genes located on the H strand. Gene arrangement of N. cf. mirabilis is identical to that of Paranemertes cf. peregrina, both of which are Hoplonemertea, while that of Z. rubens is the same as in Lineus viridis, both of which are Heteronemertea. Comparison of the gene arrangements and phylogenomic analysis based on concatenated nucleotide sequences of the 12 mitochondrial protein-coding genes revealed that species with closer relationships share more identical gene blocks. CONCLUSION: The two new mitochondrial genomes share many features, including gene contents, with other known nemertean mitochondrial genomes. The tRNA families display a composite substitution pathway. Gene order comparison to the proposed ground pattern of Bilateria and some lophotrochozoans suggests that the nemertean ancestral mitochondrial gene order most closely resembles the heteronemertean type. Phylogenetic analysis proposes a sister-group relationship between Hetero- and Hoplonemertea, which supports one of two recent alternative hypotheses of nemertean phylogeny.

Disentangling ribbon worm relationships: multi-locus analysis supports traditional classification of the phylum Nemertea.

The phylogenetic relationships of selected members of the phylum Nemertea are explored by means of six markers amplified from the genomic DNA of freshly collected specimens (the nuclear 18S rRNA and 28S rRNA genes, histones H3 and H4, and the mitochondrial genes 16S rRNA and cytochrome c oxidase subunit I). These include all previous markers and regions used in earlier phylogenetic analyses of nemerteans, therefore acting as a scaffold to which one could pinpoint any previously published study. Our results, based on analyses of static and dynamic homology concepts under probabilistic and parsimony frameworks, agree in the non-monophyly of Palaeonemertea and in the monophyly of Heteronemerta and Hoplonemertea. The position of Hubrechtella and the Pilidiophora hypothesis are, however, sensitive to analytical method, as is the monophyly of the non-hubrechtiid palaeonemerteans. Our results are, however, consistent with the main division of Hoplonemertea into Polystilifera and Monostilifera, the last named being divided into Cratenemertea and Distromatonemertea, as well as into the main division of Heteronemertea into Baseodiscus and the remaining species. The study also continues to highlight the deficient taxonomy at the family and generic level within Nemertea and sheds light on the areas of the tree that require further refinement. © The Willi Hennig Society 2011.

The mitochondrial genomes of two nemerteans, Cephalothrix sp. (Nemertea: Palaeonemertea) and Paranemertes cf. peregrina (Nemertea: Hoplonemertea).

The mitochondrial genome sequences were determined for two species of nemerteans, Cephalothrix sp. (15,800 bp sequenced, near-complete) and Paranemertes cf. peregrina (14,558 bp, complete). As seen in most metazoans, the genomes encode 13 protein, 2 ribosomal RNA and 22 transfer RNA genes. The nucleotide composition is strongly biased toward A and T, as is typical for metazoan mtDNAs. There is also a significant strand skew in the distribution of these nucleotides, with the coding strand being richer in T than A and in G than C. All genes are transcribed in the same direction except for trnP and trnT, which is consistent with that reported for Cephalothrix hongkongiensis and Lineus viridis. Gene arrangement of Cephalothrix sp. is identical to that of C. hongkongiensis, while in P. cf. peregrina it is similar to L. viridis, but differs significantly from the three Cephalothrix species in the position of four protein-coding genes and seven tRNAs. Some protein-coding genes have 3' end stem-loop structures, which may allow mRNA processing without flanking tRNAs. The major non-coding regions observed in the two genomes with potential to form stem-loop structures may be involved in the initiation of replication or transcription. The average Ka/Ks values, varying from 0.12 to 0.89, are markedly different among the 13 mitochondrial protein-coding genes, suggesting that there may exist different selective pressure among mitochondrial genes of nemerteans.

Statistical parsimony networks and species assemblages in Cephalotrichid nemerteans (nemertea).

BACKGROUND: It has been suggested that statistical parsimony network analysis could be used to get an indication of species represented in a set of nucleotide data, and the approach has been used to discuss species boundaries in some taxa. METHODOLOGY/PRINCIPAL FINDINGS: Based on 635 base pairs of the mitochondrial protein-coding gene cytochrome c oxidase I (COI), we analyzed 152 nemertean specimens using statistical parsimony network analysis with the connection probability set to 95%. The analysis revealed 15 distinct networks together with seven singletons. Statistical parsimony yielded three networks supporting the species status of Cephalothrix rufifrons, C. major and C. spiralis as they currently have been delineated by morphological characters and geographical location. Many other networks contained haplotypes from nearby geographical locations. Cladistic structure by maximum likelihood analysis overall supported the network analysis, but indicated a false positive result where subnetworks should have been connected into one network/species. This probably is caused by undersampling of the intraspecific haplotype diversity. CONCLUSIONS/SIGNIFICANCE: Statistical parsimony network analysis provides a rapid and useful tool for detecting possible undescribed/cryptic species among cephalotrichid nemerteans based on COI gene. It should be combined with phylogenetic analysis to get indications of false positive results, i.e., subnetworks that would have been connected with more extensive haplotype sampling.

The first internal molecular phylogeny of the animal phylum Entoprocta (Kamptozoa).

This article provides the first molecular phylogenetic study of the enigmatic invertebrate phylum Entoprocta and was designed to resolve the internal phylogenetic relationships of the taxon. The study is based on partial and combined analyses of the mitochondrial gene cytochrome c oxidase subunit I (COI), as well as the nuclear ribosomal genes 28S rDNA and 18S rDNA. A short morphological character matrix was constructed to trace character evolution along the combined molecular phylogenetic tree. The combined analyses of all three genes strongly support the monophyly of the phylum Entoprocta and a sister group relationship of Entoprocta and Cycliophora, a result which is consistent with a number of previous morphological and molecular assessments. We find evidence for two separate lineages within the Entoprocta, one lineage leading to all recent colonial taxa, Coloniales, another representing the clade of solitary entoprocts, Solitaria. Our study suggests that Loxosomella is a paraphyletic assembly with regard to the genera Loxomitra, Loxosoma, and Loxocorone. The results imply that the ancestral entoproct was a solitary, marine organism with an epizoic life style. The groundplan of the entoproct adult stage probably included a bilobed centralized nervous system, and the larva was assumedly planktonic, with a gut and a ciliated creeping sole.

The Lanius excubitor (Aves, Passeriformes) conundrum--Taxonomic dilemma when molecular and non-molecular data tell different stories.

The phylogeny of 18 taxa in the Lanius excubitor complex, and the related species L. sphenocercus, L. ludovicianus and L. somalicus, was estimated based on the mitochondrial cytochrome b gene and the non-coding D-loop (in total approximately 1.3 kb). According to the mitochondrial gene tree, Lanius excubitor s.l. is non-monophyletic, with some of its subspecies being more closely related to L. sphenocercus, L. ludovicianus, and L. somalicus. Also the division of the L. excubitor complex into a northern (L. excubitor) and a southern (L. meridionalis) species, as has been proposed based on morphological and ecological similarity and geographical distributions, is not compatible with the mitochondrial tree. Overall, genetic divergences among the ingroup taxa are small, indicating a recent radiation. A tree based on the nuclear ornithine decarboxylase (ODC) introns 6-7 is unresolved with respect to the ingroup, but provides strong support for a clade containing the Lanius excubitor complex, L. sphenocercus, L. ludovicianus and L. somalicus. We discuss the incongruence between the current taxonomy and the mitochondrial gene tree, and conclude that based on the latter the Lanius excubitor complex may be treated as at least six species, L. borealis, L. elegans, L. excubitor, L. lahtora, L. meridionalis, and L. uncinatus, but that other taxonomic treatments are also possible. However, uncertainty regarding to which extent the mitochondrial gene tree reflects the species phylogeny prevents us from recommending taxonomic change without further investigation. This study highlights the possible danger of relying on a single molecular marker, such as mitochondrial DNA, in taxonomic revisions and phylogenetic inference.

The first comprehensive molecular phylogeny of Bryozoa (Ectoprocta) based on combined analyses of nuclear and mitochondrial genes.

Bryozoa is one of the most puzzling phyla in the animal kingdom and little is known about their evolutionary history. Its phylogenetic position among the Metazoa remains unsettled, as well as its intra-phylum relationships. Here, we present the first comprehensive molecular phylogeny of Bryozoa based on the mitochondrial gene COI and two nuclear genes 18S rDNA and 28S rDNA including 32 species from 23 families. We show that the monophyletic status is supported for the phylum as well as for previously defined bryozoan classes. The 28S rDNA supports a close relationship of Phylactolaemata and Stenolaemata, while partial COI and 18S rDNA show the freshwater Phylactolaemata as basal bryozoans. The Gymnolaemata have generally been divided into soft-bodied forms (Ctenostomata) and hard-bodied species (Cheilostomata). In our analyses all three genes conflict with this assumption and show hard body forms having evolved within Gymnolaemata several times.