Microbial associations of shallow-water Mediterranean marine cave Solenogastres (Mollusca).

The first cave-dwelling Solenogastres-marine shell-less worm-like mollusks-were sampled from Mediterranean marine caves floor silt in the Marseille area. The mollusks were 1.5 mm in length, had a transparent body with shiny spicules and appear to represent a new Tegulaherpia species. Electron microscopy revealed a high number of microbial cells, located on the surface of the spicules as well as in the cuticle of Tegulaherpia sp. The observed microbial cells varied in morphology and were unequally distributed through the cuticle, reaching a highest density on the dorsal and lateral sides and being practically absent on the ventral side. Next Generation Sequencing (NGS) of V4 region of 16S rRNA gene amplicons, obtained from the DNA samples of whole bodies of Tegulaherpia sp. revealed three dominating microorganisms, two of which were bacteria of Bacteroidetes and Nitrospirae phyla, while the third one represented archaea of Thaumarchaeota phylum. The Operational Taxonomic Unit (OTU), affiliated with Bacteroidetes was an uncultured bacteria of the family Saprospiraceae (93-95% of Bacteroidetes and 25-44% of the total community, depending on sample), OTU, affiliated with Nitrospirae belonged to the genus Nitrospira (8-30% of the community), while the thaumarchaeal OTU was classified as Candidatus Nitrosopumilus (11-15% of the community). Members of these three microbial taxa are known to form associations with various marine animals such as sponges or snails where they contribute to nitrogen metabolism or the decomposition of biopolymers. A similar role is assumed to be played by the microorganisms associated with Tegulaherpia sp.

Phylogenomics of Aplacophora (Mollusca, Aculifera) and a solenogaster without a foot.

Recent molecular phylogenetic investigations strongly supported the placement of the shell-less, worm-shaped aplacophoran molluscs (Solenogastres and Caudofoveata) and chitons (Polyplacophora) in a clade called Aculifera, which is the sister taxon of all other molluscs. Thus, understanding the evolutionary history of aculiferan molluscs is important for understanding early molluscan evolution. In particular, fundamental questions about evolutionary relationships within Aplacophora have long been unanswered. Here, we supplemented the paucity of available data with transcriptomes from 25 aculiferans and conducted phylogenomic analyses on datasets with up to 525 genes and 75 914 amino acid positions. Our results indicate that aplacophoran taxonomy requires revision as several traditionally recognized groups are non-monophyletic. Most notably, Cavibelonia, the solenogaster taxon defined by hollow sclerites, is polyphyletic, suggesting parallel evolution of hollow sclerites in multiple lineages. Moreover, we describe Apodomenia enigmatica sp. nov. , a bizarre new species that appears to be a morphological intermediate between Solenogastres and Caudofoveata. This animal is not a missing link, however; molecular and morphological studies show that it is a derived solenogaster that lacks a foot, mantle cavity and radula. Taken together, these results shed light on the evolutionary history of Aplacophora and reveal a surprising degree of morphological plasticity within the group.

Molecular phylogeny of Caudofoveata (Mollusca) challenges traditional views.

The shell-less, worm-shaped Caudofoveata (=Chaetodermomorpha) is one of the least known groups of molluscs. The taxon consists of 141 recognized species found from intertidal environments to the deep-sea where they live burrowing in sediment. Evolutionary relationships of the group have been debated, but few studies based on morphological or molecular data have investigated the phylogeny of the group. Here we use molecular phylogenetics to resolve relationships among and within families of Caudofoveata. Phylogenetic analyses were performed using selected mitochondrial and nuclear genes from species from all recognized families of Caudofoveata. In resulting trees and contrary to traditional views, Prochaetodermatidae forms the sister clade to a clade containing the other two currently recognized families, Chaetodermatidae and Limifossoridae. The monophyly of Prochaetodermatidae is highly supported, but Limifossoridae and Chaetodermatidae are not recovered as monophyletic. Most of the caudofoveate genera are also not recovered as monophyletic in our analyses. Thus results from our molecular data suggest that the current classification of Caudofoveata is in need of revision, and indicate evolutionary scenarios that differ from previously proposed hypotheses based on morphology.

Expression of six3 and otx in Solenogastres (Mollusca) supports an ancestral role in bilaterian anterior-posterior axis patterning.

The homeodomain transcription factors six3 and otx are involved in patterning the anterior body and parts of the central nervous system (CNS) in bilaterians. Their similar expression patterns have been used as an argument for homology of heads, brains, segmentation, and ciliated larvae. We investigated the developmental expression of six3 and otx in the aplacophoran mollusk Wirenia argentea. Six3 is expressed in subepithelial cells delimiting the apical organ of the solenogaster pericalymma larva. Otx is expressed in cells of the prototroch and adjacent regions as well as in posterior extensions of the prototrochal expression domain. Advanced larvae also show pretrochal otx expression in the developing CNS. Comparative analysis of six3 and otx expression in bilaterians argues for an ancestral function in anterior-posterior body axis patterning but, due to its presence in animals lacking a head and/or a brain, not necessarily for the presence of these morphological structures in the last common ancestor (LCA) of bilaterians. Likewise, the hypothesis that the posterior border of otx expression corresponds to the border between the unsegmented head and the segmented trunk of the LCA of protostomes is not supported, since otx is extensively expressed in the trunk in W. argentea and numerous other protostomes.

Abyssal fauna of the UK-1 polymetallic nodule exploration area, Clarion-Clipperton Zone, central Pacific Ocean: Mollusca.

We present the first DNA taxonomy publication on abyssal Mollusca from the Clarion-Clipperton Zone (CCZ), central Pacific ocean, using material collected as part of the Abyssal Baseline (ABYSSLINE) environmental survey cruise 'AB01' to the UK Seabed Resources Ltd (UKSRL) polymetallic-nodule exploration area 'UK-1' in the eastern CCZ. This is the third paper in a series to provide regional taxonomic data for a region that is undergoing intense deep-sea mineral exploration for high-grade polymetallic nodules. Taxonomic data are presented for 21 species from 42 records identified by a combination of morphological and genetic data, including molecular phylogenetic analyses. These included 3 heterodont bivalves, 5 protobranch bivalves, 4 pteriomorph bivalves, 1 caudofoveate, 1 monoplacophoran, 1 polyplacophoran, 4 scaphopods and 2 solenogastres. Gastropoda were recovered but will be the subject of a future study. Seven taxa matched published morphological descriptions for species with deep Pacific type localities, and our sequences provide the first genetic data for these taxa. One taxon morphologically matched a known cosmopolitan species but with a type locality in a different ocean basin and was assigned the open nomenclature 'cf' as a precautionary approach in taxon assignments to avoid over-estimating species ranges. One taxon is here described as a new species, Ledella knudseni sp. n. For the remaining 12 taxa, we have determined them to be potentially new species, for which we make the raw data, imagery and vouchers available for future taxonomic study. The Clarion-Clipperton Zone is a region undergoing intense exploration for potential deep-sea mineral extraction. We present these data to facilitate future taxonomic and environmental impact study by making both data and voucher materials available through curated and accessible biological collections.

Brain regionalization genes are co-opted into shell field patterning in Mollusca.

The 'brain regionalization genes' Six3/6, Otx, Pax2/5/8, Gbx, and Hox1 are expressed in a similar fashion in the deuterostome, ecdysozoan, and the cephalopod brain, questioning whether this holds also true for the remaining Mollusca. We investigated developmental Gbx-expression in representatives of both molluscan sister groups, the Aculifera and Conchifera. Gbx is expressed in the posterior central nervous system of an aculiferan polyplacophoran and solenogaster but not in a conchiferan bivalve suggesting that Gbx, together with Six3/6, Otx, Pax2/5/8, and Hox1, is involved in central nervous system regionalization as reported for other bilaterians. Gbx is, however, also expressed in the anterior central nervous system, i.e. the anlagen of the cerebral ganglia, in the solenogaster, a condition not reported for any other bilaterian so far. Strikingly, all Gbx-orthologs and the other 'posterior brain regionalization genes' such as Pax2/5/8 and Hox1 are expressed in the mantle that secretes shell(s) and spicules of mollusks (except cephalopods). In bivalves, the ancestral condition has even been lost, with Gbx and Pax2/5/8 not being expressed in the developing central nervous system anymore. This suggests an additional role in the formation of the molluscan shell field(s) and spicule-bearing cells, key features of mollusks.

Ancestral and novel roles of Pax family genes in mollusks.

BACKGROUND: Pax genes are transcription factors with significant roles in cell fate specification and tissue differentiation during animal ontogeny. Most information on their tempo-spatial mode of expression is available from well-studied model organisms where the Pax-subfamilies Pax2/5/8, Pax6, and Paxα/ß are mainly involved in the development of the central nervous system (CNS), the eyes, and other sensory organs. In certain taxa, Pax2/5/8 seems to be additionally involved in the development of excretion organs. Data on expression patterns in lophotrochozoans, and in particular in mollusks, are very scarce for all the above-mentioned Pax-subfamilies, which hampers reconstruction of their putative ancestral roles in bilaterian animals. Thus, we studied the developmental expression of Pax2/5/8, Pax6, and the lophotrochozoan-specific Paxß in the worm-shaped mollusk Wirenia argentea, a member of Aplacophora that together with Polyplacophora forms the Aculifera, the proposed sister taxon to all primarily single-shelled mollusks (Conchifera). RESULTS: All investigated Pax genes are expressed in the developing cerebral ganglia and in the ventral nerve cords, but not in the lateral nerve cords of the tetraneural nervous system. Additionally, Pax2/5/8 is expressed in epidermal spicule-secreting or associated cells of the larval trunk and in the region of the developing protonephridia. We found no indication for an involvement of the investigated Pax genes in the development of larval or adult sensory organs of Wirenia argentea. CONCLUSIONS: Pax2/5/8 seems to have a conserved role in the development of the CNS, whereas expression in the spicule-secreting tissues of aplacophorans and polyplacophorans suggests co-option in aculiferan skeletogenesis. The Pax6 expression pattern in Aculifera largely resembles the common bilaterian expression during CNS development. All data available on Paxß expression argue for a common role in lophotrochozoan neurogenesis.

Phylogenomics of Lophotrochozoa with Consideration of Systematic Error.

Phylogenomic studies have improved understanding of deep metazoan phylogeny and show promise for resolving incongruences among analyses based on limited numbers of loci. One region of the animal tree that has been especially difficult to resolve, even with phylogenomic approaches, is relationships within Lophotrochozoa (the animal clade that includes molluscs, annelids, and flatworms among others). Lack of resolution in phylogenomic analyses could be due to insufficient phylogenetic signal, limitations in taxon and/or gene sampling, or systematic error. Here, we investigated why lophotrochozoan phylogeny has been such a difficult question to answer by identifying and reducing sources of systematic error. We supplemented existing data with 32 new transcriptomes spanning the diversity of Lophotrochozoa and constructed a new set of Lophotrochozoa-specific core orthologs. Of these, 638 orthologous groups (OGs) passed strict screening for paralogy using a tree-based approach. In order to reduce possible sources of systematic error, we calculated branch-length heterogeneity, evolutionary rate, percent missing data, compositional bias, and saturation for each OG and analyzed increasingly stricter subsets of only the most stringent (best) OGs for these five variables. Principal component analysis of the values for each factor examined for each OG revealed that compositional heterogeneity and average patristic distance contributed most to the variance observed along the first principal component while branch-length heterogeneity and, to a lesser extent, saturation contributed most to the variance observed along the second. Missing data did not strongly contribute to either. Additional sensitivity analyses examined effects of removing taxa with heterogeneous branch lengths, large amounts of missing data, and compositional heterogeneity. Although our analyses do not unambiguously resolve lophotrochozoan phylogeny, we advance the field by reducing the list of viable hypotheses. Moreover, our systematic approach for dissection of phylogenomic data can be applied to explore sources of incongruence and poor support in any phylogenomic data set. [Annelida; Brachiopoda; Bryozoa; Entoprocta; Mollusca; Nemertea; Phoronida; Platyzoa; Polyzoa; Spiralia; Trochozoa.].

Cell Proliferation Pattern and Twist Expression in an Aplacophoran Mollusk Argue Against Segmented Ancestry of Mollusca.

The study of aplacophoran mollusks (i.e., Solenogastres or Neomeniomorpha and Caudofoveata or Chaetodermomorpha) has traditionally been regarded as crucial for reconstructing the morphology of the last common ancestor of the Mollusca. Since their proposed close relatives, the Polyplacophora, show a distinct seriality in certain organ systems, the aplacophorans are also in the focus of attention with regard to the question of a potential segmented ancestry of mollusks. To contribute to this question, we investigated cell proliferation patterns and the expression of the twist ortholog during larval development in solenogasters. In advanced to late larvae, during the outgrowth of the trunk, a pair of longitudinal bands of proliferating cells is found subepithelially in a lateral to ventrolateral position. These bands elongate during subsequent development as the trunk grows longer. Likewise, expression of twist occurs in two laterally positioned, subepithelial longitudinal stripes in advanced larvae. Both, the pattern of proliferating cells and the expression domain of twist demonstrate the existence of extensive and long-lived mesodermal bands in a worm-shaped aculiferan, a situation which is similar to annelids but in stark contrast to conchiferans, where the mesodermal bands are usually rudimentary and ephemeral. Yet, in contrast to annelids, neither the bands of proliferating cells nor the twist expression domain show a separation into distinct serial subunits, which clearly argues against a segmented ancestry of mollusks. Furthermore, the lack of twist expression during the development of the ventromedian muscle argues against homology of a ventromedian longitudinal muscle in protostomes with the notochord of chordates.

Ancestral role of Pax2/5/8 in molluscan brain and multimodal sensory system development.

BACKGROUND: Mollusks represent the largest lophotrochozoan phylum and exhibit highly diverse body plans. Previous studies have demonstrated that transcription factors such as Pax genes play important roles during their development. Accordingly, in ecdysozoan and vertebrate model organisms, orthologs of Pax2/5/8 are among others involved in the formation of the midbrain/hindbrain boundary, the auditory/geosensory organ systems, and the excretory system. METHODS: Pax2/5/8 expression was investigated by in situ hybridization during the development of representatives of the two major molluscan subclades, Aculifera and Conchifera. RESULTS: Compared to the investigated polyplacophoran and bivalve species that lack larval statocysts as geosensory organs and elaborate central nervous systems (CNS), cephalopods possess highly centralized brains and statocysts. Pax2/5/8 is expressed in regions where sensory cells develop subsequently during ontogenesis. Expression domains include esthetes and the ampullary system in polyplacophorans as well as the eyes of cephalopods. No Pax2/5/8 expression was observed in the less centralized CNS of bivalve, polyplacophoran, and gastropod embryos, thus arguing for a loss of Pax2/5/8 involvement in CNS development in these lineages. In contrast, Pax2/5/8 is expressed among others in brain lobes along the trajectory of the esophagus that divides the cephalopod brain. CONCLUSIONS: Our results, along with those on Otx- and Hox-gene expression, demonstrate that the cephalopod condition is similar to that in mouse and fruit fly, with Otx being expressed in the anterior-most brain region (except for the vertical lobe) and a Pax2/5/8 expression domain separating the Otx-domain from a Hox-gene expressing posterior brain region. Thus, Pax2/5/8 appears to have been recruited independently into regionalization of non-homologous complex brains of organisms as different as squid, fruit fly, and mouse. In addition, Pax2/5/8 is expressed in multimodal sensory systems in mollusks such as the esthetes and the ampullary system of polyplacophorans as well as the eyes of cephalopods. Pax2/5/8-expressing cells are present in regions where the future sensory cells such as the polyplacophoran esthetes are situated and hence Pax2/5/8 expression probably predates sensory cell development during ontogeny. In mollusks, Pax2/5/8 is only expressed in derivatives of the ectoderm and hence an ancestral role in molluscan ectoderm differentiation is inferred.

From complex to simple: myogenesis in an aplacophoran mollusk reveals key traits in aculiferan evolution.

BACKGROUND: Recent studies suggest a bifurcation at the base of Mollusca, resulting in the primarily single-shelled Conchifera (Bivalvia, Gastropoda, Scaphopoda, Monoplacophora, Cephalopoda) and the spicule-bearing Aculifera (Polyplacophora, Neomeniomorpha, Chaetodermomorpha). A recent study revealed a complex larval musculature exclusively shared by Neomeniomorpha and Polyplacophora, supporting a close relationship of both taxa. However, the ontogenetic transition from the complex larval to the simple adult neomeniomorph musculature, which mainly consists of a three-layered body-wall musculature and serially iterated dorsoventral muscles, remains unknown. To close this gap in knowledge, we studied remodeling of the larval musculature during metamorphosis in the neomeniomorph Wirenia argentea. A comparative analysis with a novel data set of a polyplacophoran, Leptochiton asellus, allows us to infer the morphology of the last common ancestor of Aculifera and the evolution of its subclades therefrom. RESULTS: The complex larval musculature of Wirenia argentea persists through metamorphosis and becomes modified to form two of the three muscle layers of the adult body wall. The innermost longitudinal layer of the three-layered body wall musculature is generated by transformation and expansion of distinct larval longitudinal muscle bundles. The larval ventrolateral muscle strands are remodeled and eventually become the most ventral part of the adult longitudinal layer of the body wall musculature. The paired larval enrolling muscle forms the lateral parts and the former rectus muscle is destined to become the most dorsal part of the longitudinal layer of the body wall musculature. The transient ventromedian muscle is lost during postmetamorphic development. CONCLUSIONS: Postmetamorphic remodeling in W. argentea supports the hypothesis of a complex myoanatomy rather than a three-layered body wall musculature at the base of Aculifera, and thus argues against homology of the body wall musculature of adult Neomeniomorpha and other potential molluscan sister groups. Our data show that the neomeniomorph body wall musculature is a derived condition and not an aculiferan or molluscan plesiomorphy.

The complete mitochondrial genome of Scutopus ventrolineatus (Mollusca: Chaetodermomorpha) supports the Aculifera hypothesis.

BACKGROUND: With more than 100000 living species, mollusks are the second most diverse metazoan phylum. The current taxonomic classification of mollusks recognizes eight classes (Neomeniomorpha, Chaetodermomorpha, Polyplacophora, Monoplacophora, Cephalopoda, Gastropoda, Bivalvia, and Scaphopoda) that exhibit very distinct body plans. In the past, phylogenetic relationships among mollusk classes have been contentious due to the lack of indisputable morphological synapomorphies. Fortunately, recent phylogenetic analyses based on multi-gene data sets are rendering promising results. In this regard, mitochondrial genomes have been widely used to reconstruct deep phylogenies. For mollusks, complete mitochondrial genomes are mostly available for gastropods, bivalves, and cephalopods, whereas other less-diverse lineages have few or none reported. RESULTS: The complete DNA sequence (14662 bp) of the mitochondrial genome of the chaetodermomorph Scutopus ventrolineatus Salvini-Plawen, 1968 was determined. Compared with other mollusks, the relative position of protein-coding genes in the mitochondrial genome of S. ventrolineatus is very similar to those reported for Polyplacophora, Cephalopoda and early-diverging lineages of Bivalvia and Gastropoda (Vetigastropoda and Neritimorpha; but not Patellogastropoda). The reconstructed phylogenetic tree based on combined mitochondrial and nuclear sequence data recovered monophyletic Aplacophora, Aculifera, and Conchifera. Within the latter, Cephalopoda was the sister group of Gastropoda and Bivalvia + Scaphopoda. CONCLUSIONS: Phylogenetic analyses of mitochondrial sequences showed strong among-lineage rate heterogeneity that produced long-branch attraction biases. Removal of long branches (namely those of bivalves and patellogastropods) ameliorated but not fully resolved the problem. Best results in terms of statistical support were achieved when mitochondrial and nuclear sequence data were concatenated.

Development of the nervous system in Solenogastres (Mollusca) reveals putative ancestral spiralian features.

BACKGROUND: The Solenogastres (or Neomeniomorpha) are a taxon of aplacophoran molluscs with contentious phylogenetic placement. Since available developmental data on non-conchiferan (that is, aculiferan) molluscs mainly stem from polyplacophorans, data on aplacophorans are needed to clarify evolutionary questions concerning the morphological features of the last common ancestor (LCA) of the Aculifera and the entire Mollusca. We therefore investigated the development of the nervous system in two solenogasters, Wirenia argentea and Gymnomenia pellucida, using immunocytochemistry and electron microscopy. RESULTS: Nervous system formation starts simultaneously from the apical and abapical pole of the larva with the development of a few cells of the apical organ and a posterior neurogenic domain. A pair of neurite bundles grows out from both the neuropil of the apical organ and the posterior neurogenic domain. After their fusion in the region of the prototroch, which is innervated by an underlying serotonin-like immunoreactive (-LIR) plexus, the larva exhibits two longitudinal neurite bundles - the future lateral nerve cords. The apical organ in its fully developed state exhibits approximately 8 to 10 flask-shaped cells but no peripheral cells. The entire ventral nervous system, which includes a pair of longitudinal neurite bundles (the future ventral nerve cords) and a serotonin-LIR ventromedian nerve plexus, appears simultaneously and is established after the lateral nervous system. During metamorphosis the apical organ and the prototrochal nerve plexus are lost. CONCLUSIONS: The development of the nervous system in early solenogaster larvae shows striking similarities to other spiralians, especially polychaetes, in exhibiting an apical organ with flask-shaped cells, a single pair of longitudinal neurite bundles, a serotonin-LIR innervation of the prototroch, and formation of these structures from an anterior and a posterior neurogenic domain. This provides evidence for an ancestral spiralian pattern of early nervous system development and a LCA of the Spiralia with a single pair of nerve cords. In later nervous system development, however, the annelids deviate from all other spiralians including solenogasters in forming a posterior growth zone, which initiates teloblastic growth. Since this mode of organogenesis is confined to annelids, we conclude that the LCA of both molluscs and spiralians was unsegmented.

Aplacophoran mollusks evolved from ancestors with polyplacophoran-like features.

Mollusca is an animal phylum with vast morphological diversity and includes worm-shaped aplacophorans, snails, bivalves, and the complex cephalopods. The interrelationships of these class-level taxa are still contentious, but recent phylogenomic analyses suggest a dichotomy at the base of Mollusca, resulting in a monophyletic Aculifera (comprising the shell-less, sclerite-bearing aplacophorans and the eight-shelled polyplacophorans) and Conchifera (all other, primarily univalved groups). The Aculifera concept has recently gained support via description of the fossil Kulindroplax, which shows both aplacophoran- and polyplacophoran-like features and suggests that the aplacophorans originated from a shelled ancestor, but the overall morphology of the last common aculiferan ancestor remains obscure. Here we show that larvae of the aplacophoran Wirenia argentea have several sets of muscles previously known only from polyplacophoran mollusks. Most of these are lost during metamorphosis, and we interpret them as ontogenetic remnants of an ancestor with a complex, polyplacophoran-like musculature. Moreover, we find that the first seven pairs of dorsoventral muscles develop synchronously in Wirenia, similar to juvenile polyplacophorans, which supports the conclusions based on the seven-shelled Kulindroplax. Accordingly, we argue that the simple body plan of recent aplacophorans is the result of simplification and does not represent a basal molluscan condition.

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.

Comparative neuroanatomy suggests repeated reduction of neuroarchitectural complexity in Annelida.

BACKGROUND: Paired mushroom bodies, an unpaired central complex, and bilaterally arranged clusters of olfactory glomeruli are among the most distinctive components of arthropod neuroarchitecture. Mushroom body neuropils, unpaired midline neuropils, and olfactory glomeruli also occur in the brains of some polychaete annelids, showing varying degrees of morphological similarity to their arthropod counterparts. Attempts to elucidate the evolutionary origin of these neuropils and to deduce an ancestral ground pattern of annelid cerebral complexity are impeded by the incomplete knowledge of annelid phylogeny and by a lack of comparative neuroanatomical data for this group. The present account aims to provide new morphological data for a broad range of annelid taxa in order to trace the occurrence and variability of higher brain centers in segmented worms. RESULTS: Immunohistochemically stained preparations provide comparative neuroanatomical data for representatives from 22 annelid species. The most prominent neuropil structures to be encountered in the annelid brain are the paired mushroom bodies that occur in a number of polychaete taxa. Mushroom bodies can in some cases be demonstrated to be closely associated with clusters of spheroid neuropils reminiscent of arthropod olfactory glomeruli. Less distinctive subcompartments of the annelid brain are unpaired midline neuropils that bear a remote resemblance to similar components in the arthropod brain. The occurrence of higher brain centers such as mushroom bodies, olfactory glomeruli, and unpaired midline neuropils seems to be restricted to errant polychaetes. CONCLUSIONS: The implications of an assumed homology between annelid and arthropod mushroom bodies are discussed in light of the 'new animal phylogeny'. It is concluded that the apparent homology of mushroom bodies in distantly related groups has to be interpreted as a plesiomorphy, pointing towards a considerably complex neuroarchitecture inherited from the last common ancestor, Urbilateria. Within the annelid radiation, the lack of mushroom bodies in certain groups is explained by widespread secondary reductions owing to selective pressures unfavorable for the differentiation of elaborate brains. Evolutionary pathways of mushroom body neuropils in errant polychaetes remain enigmatic.

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.

Of tests, trochs, shells, and spicules: Development of the basal mollusk Wirenia argentea (Solenogastres) and its bearing on the evolution of trochozoan larval key features.

BACKGROUND: The phylogenetic status of the aplacophoran mollusk taxon Solenogastres (Neomeniomorpha) is controversially discussed. Some authors propose the clade to represent the most basal branch within Mollusca, while others claim aplacophoran mollusks (Solenogastres and Caudofoveata) to be derived. Larval characters are central in these discussions, specifically the larval test (calymma, apical cap), the ontogeny of the epidermal scleritome, and the proposed absence of larval protonephridia. To date, developmental data are available for five solenogaster species, but most reports are incomplete and need confirmation. RESULTS: Wirenia argentea deposit small batches of uncleaved embryos that are tightly enclosed by a smooth and transparent egg hull. Cleavage is spiral and unequal. The ciliated larvae hatch about 45 hours after deposition and swim actively in the water column. Within 48-60 hours after hatching they become mushroom-shaped with a pronounced apical cap partly enclosing a posterior trunk. The cells covering the apical cap are large and cleavage arrested. On the apical cap there is a prominent prototrochal band of compound cilia and an apical ciliary tuft and the trunk bears a terminal ciliary band (telotroch). Obscured by the apical cap, a ciliary band originates in the stomodaeal pore and surrounds the trunk. As development is proceeding, the trunk elongates and becomes covered by cuticle with the exception of a ventral ciliary band, the future foot. The larvae have a pair of protonephridia. At 5 days after hatching they begin to settle and within the following 7-9 days the apical cap is gradually reduced. Scattered epidermal sclerites form under the cuticle. Wirenia argentea lack iterated groups of sclerites at any developmental stage. At 40 days after hatching, the postlarvae have a fully developed foregut, but the midgut and hindgut are not yet interconnected. CONCLUSIONS: Solenogastres develop via a trochophore-like lecitotrophic larva with a preoral apical cap that at least partly represents an enlarged prototrochal area. Homology of this larval type (pericalymma larva) to test cell larvae of other spiralian clades is doubtful. The ontogeny of W. argentea does not provide any evidence for a derived status of Solenogastres within Mollusca.

Structure and evolution of the pharynx simplex in acoel flatworms (Acoela).

The homology of pharynges within the mostly pharynx-less Acoela has been a matter of discussion for decades and even the basic question of whether a pharynx is a primitive trait within the Acoela and homologous to the pharynx of platyhelminth turbellarians is open. By using fluorescence staining of musculature, as well as conventional histological techniques and transmission electron microscopy, the present study sets focus on the mouth and pharynx (where present) of seven species of Acoela within Paratomellidae, Solenofilomorphidae, Hofsteniidae, Proporidae, and Convolutidae, as well as one species of Nemertodermatida and Catenulida, respectively. It is shown that among the investigated families of acoels there is a great variability in muscle systems associated with the mouth and pharynx and that pharynx histology and ultrastructural characters are widely diverse. There are no close similarities between the acoel pharynges and the catenulid pharynx but there is a general resemblance of the musculature associated with the mouth in the representatives of Paratomellidae and Nemertodermatida. On the basis of the profound differences in pharynx morphology, three major conclusions are drawn: 1) the pharynges as present in Recent acoels are not homologous to the pharynx simplex characteristic for Catenulida and Macrostomida within the Platyhelminthes; 2) the different muscular pharynx types of acoels are not homologous between higher taxa and thus a single acoel-type pharynx simplex cannot be defined; 3) the presence of a muscular pharynx most likely does not represent the ancestral state.