Use of axonal projection patterns for the homologisation of cerebral nerves in Opisthobranchia, Mollusca and Gastropoda

Introduction: Gastropoda 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. Results: Nickel-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. Conclusions: As 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

Opisthobranchia (Mollusca, Gastropoda) – more than just slimy slugs. Shell reduction and its implications on defence and foraging

BACKGROUND: In general shell-less slugs are considered to be slimy animals with a rather dull appearance and a pest to garden plants. But marine slugs usually are beautifully coloured animals belonging to the less-known Opisthobranchia. They are characterized by a large array of interesting biological phenomena, usually related to foraging and/or defence. In this paper our knowledge of shell reduction, correlated with the evolution of different defensive and foraging strategies is reviewed, and new results on histology of different glandular systems are included. RESULTS: Based on a phylogeny obtained by morphological and histological data, the parallel reduction of the shell within the different groups is outlined. Major food sources are given and glandular structures are described as possible defensive structures in the external epithelia, and as internal glands. CONCLUSION: According to phylogenetic analyses, the reduction of the shell correlates with the evolution of defensive strategies. Many different kinds of defence structures, like cleptocnides, mantle dermal formations (MDFs), and acid glands, are only present in shell-less slugs. In several cases, it is not clear whether the defensive devices were a prerequisite for the reduction of the shell, or reduction occurred before. Reduction of the shell and acquisition of different defensive structures had an implication on exploration of new food sources and therefore likely enhanced adaptive radiation of several groups

Neuromuscular development of Aeolidiella stephanieae Valdéz, 2005 (Mollusca, Gastropoda, Nudibranchia)

Background: Studies on the development of the nervous system and the musculature of invertebrates have become more sophisticated and numerous within the last decade and have proven to provide new insights into the evolutionary history of organisms. In order to provide new morphogenetic data on opisthobranch gastropods we investigated the neuromuscular development in the nudibranch Aeolidiella stephanieae Valdez, 2005 using immunocytochemistry as well as F-actin labelling in conjunction with confocal laser scanning microscopy (cLSM). Results: The ontogenetic development of Aeolidiella stephanieae can be subdivided into 8 stages, each recognisable by characteristic morphological and behavioural features as well as specific characters of the nervous system and the muscular system, respectively. The larval nervous system of A. stephanieae includes an apical organ, developing central ganglia, and peripheral neurons associated with the velum, foot and posterior, visceral part of the larva. The first serotonergic and FMRFamidergic neural structures appear in the apical organ that exhibits an array of three sensory, flask-shaped and two non-sensory, round neurons, which altogether disappear prior to metamorphosis. The postmetamorphic central nervous system (CNS) becomes concentrated, and the rhinophoral ganglia develop together with the anlage of the future rhinophores whereas oral tentacle ganglia are not found. The myogenesis in A. stephanieae begins with the larval retractor muscle followed by the accessory larval retractor muscle, the velar or prototroch muscles and the pedal retractors that all together degenerate during metamorphosis, and the adult muscle complex forms de novo. Conclusions: Aeolidiella stephanieae comprises features of the larval and postmetamorphic nervous as well as muscular system that represent the ground plan of the Mollusca or even the Trochozoa (e. g. presence of the prototrochal or velar muscle ring). On the one hand, A. stephanieae shows some features shared by all nudibranchs like the postmetamorphic condensation of the CNS, the possession of rhinophoral ganglia and the lack of oral tentacle ganglia as well as the de novo formation of the adult muscle complex. On the other hand, the structure and arrangement of the serotonergic apical organ is similar to other caenogastropod and opisthobranch gastropods supporting their sister group relationship

From sea to land and beyond : new insights into the evolution of euthyneuran Gastropoda (Mollusca)

Background The Euthyneura are considered to be the most successful and diverse group of Gastropoda. Phylogenetically, they are riven with controversy. Previous morphology-based phylogenetic studies have been greatly hampered by rampant parallelism in morphological characters or by incomplete taxon sampling. Based on sequences of nuclear 18S rRNA and 28S rRNA as well as mitochondrial 16S rRNA and COI DNA from 56 taxa, we reconstructed the phylogeny of Euthyneura utilising Maximum Likelihood and Bayesian inference methods. The evolution of colonization of freshwater and terrestrial habitats by pulmonate Euthyneura, considered crucial in the evolution of this group of Gastropoda, is reconstructed with Bayesian approaches. Results We found several well supported clades within Euthyneura, however, we could not confirm the traditional classification, since Pulmonata are paraphyletic and Opistobranchia are either polyphyletic or paraphyletic with several clades clearly distinguishable. Sacoglossa appear separately from the rest of the Opisthobranchia as sister taxon to basal Pulmonata. Within Pulmonata, Basommatophora are paraphyletic and Hygrophila and Eupulmonata form monophyletic clades. Pyramidelloidea are placed within Euthyneura rendering the Euthyneura paraphyletic. Conclusion Based on the current phylogeny, it can be proposed for the first time that invasion of freshwater by Pulmonata is a unique evolutionary event and has taken place directly from the marine environment via an aquatic pathway. The origin of colonisation of terrestrial habitats is seeded in marginal zones and has probably occurred via estuaries or semi-terrestrial habitats such as mangroves

The taxonomist - an endangered race : a practical proposal for its survival

Background: Taxonomy or biological systematics is the basic scientific discipline of biology, postulating hypotheses of identity and relationships, on which all other natural sciences dealing with organisms relies. However, the scientific contributions of taxonomists have been largely neglected when using species names in scientific publications by not citing the authority on which they are based. Discussion: Consequences of this neglect is reduced recognition of the importance of taxonomy, which in turn results in diminished funding, lower interest from journals in publishing taxonomic research, and a reduced number of young scientists entering the field. This has lead to the so-called taxonomic impediment at a time when biodiversity studies are of critical importance. Here we emphasize a practical and obvious solution to this dilemma. We propose that whenever a species name is used, the author(s) of the species hypothesis be included and the original literature source cited, including taxonomic revisions and identification literature - nothing more than what is done for every other hypothesis or assumption included in a scientific publication. In addition, we postulate that journals primarily publishing taxonomic studies should be indexed in ISISM. Summary: The proposal outlined above would make visible the true contribution of taxonomists within the scientific community, and would provide a more accurate assessment for funding agencies impact and importance of taxonomy, and help in the recruitment of young scientists into the field, thus helping to alleviate the taxonomic impediment. In addition, it would also make much of the biological literature more robust by reducing or alleviating taxonomic uncertainty. Keywords: Taxonomy crisis; taxonomic impediment; impact factor; original species description; citation index; systematic

Evolution of microgastropods (Ellobioidea, Carychiidae): integrating taxonomic, phylogenetic and evolutionary hypotheses

BACKGROUND: Current biodiversity patterns are considered largely the result of past climatic and tectonic changes. In an integrative approach, we combine taxonomic and phylogenetic hypotheses to analyze temporal and geographic diversification of epigean (Carychium) and subterranean (Zospeum) evolutionary lineages in Carychiidae (Eupulmonata, Ellobioidea). We explicitly test three hypotheses: 1) morphospecies encompass unrecognized evolutionary lineages, 2) limited dispersal results in a close genetic relationship of geographical proximally distributed taxa and 3) major climatic and tectonic events had an impact on lineage diversification within Carychiidae. RESULTS: Initial morphospecies assignments were investigated by different molecular delimitation approaches (threshold, ABGD, GMYC and SP). Despite a conservative delimitation strategy, carychiid morphospecies comprise a great number of unrecognized evolutionary lineages. We attribute this phenomenon to historic underestimation of morphological stasis and phenotypic variability amongst lineages. The first molecular phylogenetic hypothesis for the Carychiidae (based on COI, 16S and H3) reveals Carychium and Zospeum to be reciprocally monophyletic. Geographical proximally distributed lineages are often closely related. The temporal diversification of Carychiidae is best described by a constant rate model of diversification. The evolution of Carychiidae is characterized by relatively few (long distance) colonization events. We find support for an Asian origin of Carychium. Zospeum may have arrived in Europe before extant members of Carychium. Distantly related Carychium clades inhabit a wide spectrum of the available bioclimatic niche and demonstrate considerable niche overlap. CONCLUSIONS: Carychiid taxonomy is in dire need of revision. An inferred wide distribution and variable phenotype suggest underestimated diversity in Zospeum. Several Carychium morphospecies are results of past taxonomic lumping. By collecting populations at their type locality, molecular investigations are able to link historic morphospecies assignments to their respective evolutionary lineage. We propose that rare founder populations initially colonized a continent or cave system. Subsequent passive dispersal into adjacent areas led to in situ pan-continental or mountain range diversifications. Major environmental changes did not influence carychiid diversification. However, certain molecular delimitation methods indicated a recent decrease in diversification rate. We attribute this decrease to protracted speciation

Phylogenetic support values are not necessarily informative: the case of the Serialia hypothesis (a mollusk phylogeny)

Background: Molecular phylogenies are being published increasingly and many biologists rely on the most recent topologies. However, different phylogenetic trees often contain conflicting results and contradict significant background data. Not knowing how reliable traditional knowledge is, a crucial question concerns the quality of newly produced molecular data. The information content of DNA alignments is rarely discussed, as quality statements are mostly restricted to the statistical support of clades. Here we present a case study of a recently published mollusk phylogeny that contains surprising groupings, based on five genes and 108 species, and we apply new or rarely used tools for the analysis of the information content of alignments and for the filtering of noise (masking of random-like alignment regions, split decomposition, phylogenetic networks, quartet mapping). Results: The data are very fragmentary and contain contaminations. We show that that signal-like patterns in the data set are conflicting and partly not distinct and that the reported strong support for a "rather surprising result" (monoplacophorans and chitons form a monophylum Serialia) does not exist at the level of primary homologies. Split-decomposition, quartet mapping and neighbornet analyses reveal conflicting nucleotide patterns and lack of distinct phylogenetic signal for the deeper phylogeny of mollusks. Conclusion: Even though currently a majority of molecular phylogenies are being justified with reference to the 'statistical' support of clades in tree topologies, this confidence seems to be unfounded. Contradictions between phylogenies based on different analyses are already a strong indication of unnoticed pitfalls. The use of tree-independent tools for exploratory analyses of data quality are highly recommended. Concerning the new mollusk phylogeny more convincing evidence is needed

Groping through the black box of variability: An integrative taxonomic and nomenclatural re-evaluation of Zospeum isselianum Pollonera, 1887 and allied species using new imaging technology (Nano-CT, SEM), conchological, histological and molecular data (Ellobioidea, Carychiidae).

The minute troglobitic species, Zospeum isselianum Pollonera, 1887 (Eupulmonata: Ellobioidea, Carychiidae) is widely distributed within its Southern Alpine-Dinaric range. Its broad distribution and highly variable shell has caused this species to be historically lumped into its current taxonomic state of ambiguity. In an integrative taxonomic approach, phenotypic and genotypic data are synthesized to assess the intraspecific variability recently inferred for this taxon. We collected 16 Zospeum specimens in the Slovenian Alpine Arc encompassing the type locality for Z. isselianum. The material comprises five morphologically recognized (sub)species. The species are re-evaluated using SEM, X-ray nanotomography (nano-CT), conchological, histological and molecular data. Four well-defined lineages are present, which can be attributed to i) Z. isselianum s.str. from its new type locality (Turjeva jama), ii) a highly morphologically variable lineage that so far cannot be attributed to a single morphospecies, iii) Z. kupitzense A. Stummer, 1984 (raised to species rank) and iv) a lineage comprising the two subspecies Z. alpestre alpestre (Freyer, 1855) and Z. alpestre bolei Slapnik, 1991 plus Z. isselianum individuals. The latter is treated as a single taxon Z. alpestre. After considering the severely degraded syntype material of Zospeum isselianum, we provide a taxonomic re-description and propose aneotype for this species. Furthermore, new diagnostic information is revealed regarding the columella of Zospeum isselianum and allied species. Detailed anatomical study reveals new structural aspects of Zospeum morphology and provides groundwork for future investigations

Alpine Crossroads or Origin of Genetic Diversity? Comparative Phylogeography of Two Sympatric Microgastropod Species

The Alpine Region, constituting the Alps and the Dinaric Alps, has played a major role in the formation of current patterns of biodiversity either as a contact zone of postglacial expanding lineages or as the origin of genetic diversity. In our study, we tested these hypotheses for two widespread, sympatric microgastropod taxa – Carychium minimum O.F. Müller, 1774 and Carychium tridentatum (Risso, 1826) (Gastropoda, Eupulmonata, Carychiidae) – by using COI sequence data and species potential distribution models analyzed in a statistical phylogeographical framework. Additionally, we examined disjunct transatlantic populations of those taxa from the Azores and North America. In general, both Carychium taxa demonstrate a genetic structure composed of several differentiated haplotype lineages most likely resulting from allopatric diversification in isolated refugial areas during the Pleistocene glacial periods. However, the genetic structure of Carychium minimum is more pronounced, which can be attributed to ecological constraints relating to habitat proximity to permanent bodies of water. For most of the Carychium lineages, the broader Alpine Region was identified as the likely origin of genetic diversity. Several lineages are endemic to the broader Alpine Region whereas a single lineage per species underwent a postglacial expansion to (re)colonize previously unsuitable habitats, e.g. in Northern Europe. The source populations of those expanding lineages can be traced back to the Eastern and Western Alps. Consequently, we identify the Alpine Region as a significant ‘hot-spot’ for the formation of genetic diversity within European Carychium lineages. Passive dispersal via anthropogenic means best explains the presence of transatlantic European Carychium populations on the Azores and in North America. We conclude that passive (anthropogenic) transport could mislead the interpretation of observed phylogeographical patterns in general

Comparative histology and ultrastructure of the nidamental glands and egg masses of the Opisthobranchia (Mollusca, Gastropoda) : a functional and evolutionary approach

Klussmann-Kolb A. Comparative histology and ultrastructure of the nidamental glands and egg masses of the Opisthobranchia (Mollusca, Gastropoda) : a functional and evolutionary approach. Bielefeld (Germany): Bielefeld University; 1999.In the present study the nidamental glands of 32 species of opisthobranchs, belonging to the "Cephalaspidea s. l.", Anaspidea, Sacoglossa, Tylodinoidea, Pleurobranchoidea and Nudibranchia and of five species from outgroup taxa (Pyramidelloidea, Pulmonata and Gymnomorpha) have been investigated histologically, histochemically and ultrastructurally. The egg masses of nineteen opisthobranch taxa and one pulmonate species have also been studied with the same techniques. Generally speaking, the structure of the nidamental glands is rather uniform in the investigated species: all species possess three distinct glandular parts, namely an albumen or a capsule gland, a membrane gland and a mucous gland. An albumen gland is present in some "Cephalaspidea s. l.", all Anaspidea and all Sacoglossa, as well as in the outgroup taxa whereas some "Cephalaspidea s. l.", all Tylodinoidea, all Pleurobranchoidea and all Nudibranchia possess a capsule gland instead. A membrane and a mucous gland are found in all investigated species. The epithelia of all glandular parts have the typical arrangement of alternating glandular cells and supporting cells. The glandular cells contain the secretory products whereas the supporting cells bear cilia. The secretory products can have the form of distinct granules (albumen and capsule glands) or be of heterogeneous, filamentous shape (membrane and mucous glands). The contents of the secretory products are neutral mucopolysaccharides and proteins in the albumen und capsule glands and acidic, sulphated as well as neutral mucopolysaccharides in the membrane and mucous glands. These substances can also be found in according layers in the egg masses. The egg masses also have a uniform structure throughout the taxa. Generally the embryos are encapsulated in a distinct capsule, which is composed of one membrane layer in those species which possess an albumen gland in their nidamental system. In these species a viscous albumen fluid is also present in the egg capsules. Those species which possess a capsule gland have two layered egg capsules, one outer membrane layer and one inner albumnineous layer. Albumen fluid is missing in the egg capsule of these species. In all investigated taxa the egg capsules themselves are surrounded by an inner mucous layer, are embedded in a mucous matrix and the whole egg mass is surrounded by a multi-layered outer mucous cover. Differences in this fine structure are found in the number of embryos per capsule, the number of capsules per egg mass and the fine structure of the various mucous layers. In this study the possible homologies of the different glandular parts of the nidamental glandular system as well as the homologies of the different layers in the egg masses throughout the Opisthobranchia are discussed. Aspects of functional morphology are presented. Moreover a hypothetical scenario of the evolution of the nidamental glandular system is proposed and the phylogenetic implications are discussed