Abstract. To re-evaluate the relationships of the major bivalve lineages, we amassed detailed morpho-anatomical, ultrastructural and molecular sequence data for a targeted selection of exemplar bivalves spanning the phylogenetic diversity of the class. We included molecular data for 103 bivalve species (up to five markers) and also analysed a subset of taxa with four additional nuclear protein-encoding genes. Novel as well as historically employed morphological characters were explored, and we systematically disassembled widely used descriptors such as gill and stomach 'types'. Phylogenetic analyses, conducted using parsimony direct optimisation and probabilistic methods on static alignments (maximum likelihood and Bayesian inference) of the molecular data, both alone and in combination with morphological characters, offer a robust test of bivalve relationships. A calibrated phylogeny also provided insights into the tempo of bivalve evolution. Finally, an analysis of the informativeness of morphological characters showed that sperm ultrastructure characters are among the best morphological features to diagnose bivalve clades, followed by characters of the shell, including its microstructure. Our study found support for monophyly of most broadly recognised higher bivalve taxa, although support was not uniform for Protobranchia. However, monophyly of the bivalves with protobranchiate gills was the best-supported hypothesis with incremental morphological and/or molecular sequence data. Autobranchia,

The phylogeny and potential mode of speciation of the river limpet Ancylus fluviatilis (Basommatophora) was examined using mitochondrial DNA sequences from 16S ribosomal RNA, cytochrome c oxidase subunit I (COI) and nuclear DNA from internal transcribed spacer (ITS-1) regions from 103 populations across Europe. Four highly divergent lineages were observed within Ancylus. Clade 1, representing the nominal taxon Ancylus fluviatilis (O.F. Müller, 1774), is mainly found in central and northern Europe, Clade 2 is present in a single Portuguese population, Clade 3 is distributed on the Canary islands, North Africa and the eastern Mediterranean region, whereas Clade 4 inhabits the Northern Mediterranean coasts. Phylogenetic analyses revealed an overall consistent topology of nuclear and mitochondrial gene trees. Based on a molecular clock, we estimated that the basic radiation occurred in the late Pliocene. Although clades differ significantly in size independent shell shape, morphological differentiation of lineages is not feasible without genetic data. Environmental data related to climate (precipitation, temperature, etc.) showed a significant differentiation of clades. Clade 1 dwells in relatively colder and more stable habitats than Clades 3 and 4, whose habitats in turn differ in a low or high amount of precipitation during spring and autumn, respectively. Based on the combined data sets on mitochondrial DNA, nuclear DNA, morphological and ecological differentiation, we conclude that Ancylus represents a cryptic species complex of reproductively and genetically isolated lineages. In addition, the joint analysis suggests that ecological speciation is probable to explain current patterns.

The structure and function of the central nervous systems of opisthobranch gastropods have been studied extensively. However, the organisation and function of the peripheral nervous system are poorly understood. The cephalic sensory organs (CSOs) are known to be chemosensory structures in the head region of opisthobranchs. In the present study, we used immunohistochemical methods and confocal laserscanning microscopy to comparatively examine the CSOs of diVerent opisthobranchs, namely Acteon tornatilis, Aplysia punctata, Archidoris pseudoargus and Haminoea hydatis. We wanted to characterise sensory epithelia in order to infer the function of sensory structures and the organs they constitute. Immunoreactivity against the three antigens tyrosine hydroxylase, FMRFamide and serotonin was very similar in the CSOs of all investigated species. Tyrosine hydroxylase-like immunoreactivity was detected primarily in subepidermal sensory cell bodies, which were much more abundant in the anteriorly situated CSOs. This observation indicates that these cells and the respective organs may be involved in contact chemoreception and mechanoreception. The dominant features of FMRFamide-like immunoreactivity, especially in the posterior CSOs, were tightly knotted Wbres which reveal glomerulus-like structures. This suggests an olfactory role for these organs. Serotonin-like immunoreactivity was detected in an extensive network of eVerent Wbres, but was not found within any peripheral cell bodies. Serotonin-like immunoreactivity was found in the same glomerulus-like structures as FMRFamide-like immunoreactivity, indicating a function of serotonin in the eVerent control of olfactory inputs. Besides this functional implication, this study could also add some knowledge on the doubtful homology of the CSOs in opisthobranch gastropods.

IntroductionGastropoda are guided by several sensory organs in the head region, referred to as cephalic sensory organs (CSOs). These CSOs are innervated by distinct nerves. This study proposes a unified terminology for the cerebral nerves and the categories of CSOs and then investigates the neuroanatomy and cellular innervation patterns of these cerebral nerves, in order to homologise them. The homologisation of the cerebral nerves in conjunction with other data, e.g. ontogenetic development or functional morphology, may then provide insights into the homology of the CSOs themselves.ResultsNickel-lysine axonal tracing (“backfilling”) was used to stain the somata projecting into specific nerves in representatives of opisthobranch Gastropoda. Tracing patterns revealed the occurrence, size and relative position of somata and their axons and enabled these somata to be mapped to specific cell clusters. Assignment of cells to clusters followed a conservative approach based primarily on relative location of the cells. Each of the four investigated cerebral nerves could be uniquely identified due to a characteristic set of soma clusters projecting into the respective nerves via their axonal pathways.ConclusionsAs the described tracing patterns are highly conserved morphological characters, they can be used to homologise nerves within the investigated group of gastropods. The combination of adequate number of replicates and a comparative approach allows us to provide preliminary hypotheses on homologies for the cerebral nerves. Based on the hypotheses regarding cerebral nerve homology together with further data on ultrastructure and immunohistochemistry of CSOs published elsewhere, we can propose preliminary hypotheses regarding homology for the CSOs of the Opisthobranchia themselves.

This study describes the innervation patterns for the cerebral nerves which project to the cephalic sensory organs (CSOs) in the opisthobranch Haminoea hydatis (Linnaeus 1758) and uses axonal tracing techniques (backWlling) to reveal the central cellular origins for these cerebral nerves. Cell clusters projecting into the cerebral nerves can be deWned by their positions in the ganglion relative to other clusters, nerve roots and lobes. The number of cell clusters and the relative sizes of somata are constant in a given cluster, whereas the absolute number of somata and absolute sizes of single somata in a given cluster increase with the size of the animal. Additionally, the invariable morphological characteristics of the cell clusters are used to deWne criteria for the assessment of possible homology for the clusters innervating the CSOs in Opisthobranchia. The data suggest two diVerent strategies to accommodate the increasing body size; Wrst, the additions of nerve cells and second, the growth of nerve cells.