BackgroundInvertebrate nervous systems are highly disparate between different taxa. This is reflected in the terminology used to describe them, which is very rich and often confusing. Even very general terms such as 'brain', 'nerve', and 'eye' have been used in various ways in the different animal groups, but no consensus on the exact meaning exists. This impedes our understanding of the architecture of the invertebrate nervous system in general and of evolutionary transformations of nervous system characters between different taxa.ResultsWe provide a glossary of invertebrate neuroanatomical terms with a precise and consistent terminology, taxon-independent and free of homology assumptions. This terminology is intended to form a basis for new morphological descriptions. A total of 47 terms are defined. Each entry consists of a definition, discouraged terms, and a background/comment section.ConclusionsThe use of our revised neuroanatomical terminology in any new descriptions of the anatomy of invertebrate nervous systems will improve the comparability of this organ system and its substructures between the various taxa, and finally even lead to better and more robust homology hypotheses.

BackgroundArthropod diversity is unparalleled in the animal kingdom. The study of ontogeny is pivotal to understand which developmental processes underlie the incredible morphological disparity of arthropods and thus to eventually unravel evolutionary transformations leading to their success. Work on laboratory model organisms has yielded in-depth data on numerous developmental mechanisms in arthropods. Yet, although the range of studied taxa has increased noticeably since the advent of comparative evolutionary developmental biology (evo-devo), several smaller groups remain understudied. This includes the bizarre Pycnogonida (sea spiders) or “no-bodies”, a taxon occupying a crucial phylogenetic position for the interpretation of arthropod development and evolution.ResultsPycnogonid development is variable at familial and generic levels and sometimes even congeneric species exhibit different developmental modes. Here, we summarize the available data since the late 19th century. We clarify and resolve terminological issues persisting in the pycnogonid literature and distinguish five developmental pathways, based on (1) type of the hatching stage, (2) developmental-morphological features during postembryonic development and (3) selected life history characteristics. Based on phylogenetic analyses and the fossil record, we discuss plausible plesiomorphic features of pycnogonid development that allow comparison to other arthropods. These features include (1) a holoblastic, irregular cleavage with equal-sized blastomeres, (2) initiation of gastrulation by a single bottle-shaped cell, (3) the lack of a morphologically distinct germ band during embryogenesis, (4) a parasitic free-living protonymphon larva as hatching stage and (5) a hemianamorphic development during the postlarval and juvenile phases. Further, we propose evolutionary developmental trajectories within crown-group Pycnogonida.ConclusionsA resurgence of studies on pycnogonid postembryonic development has provided various new insights in the last decades. However, the scarcity of modern-day embryonic data – including the virtual lack of gene expression and functional studies – needs to be addressed in future investigations to strengthen comparisons to other arthropods and arthropod outgroups in the framework of evo-devo. Our review may serve as a basis for an informed choice of target species for such studies, which will not only shed light on chelicerate development and evolution but furthermore hold the potential to contribute important insights into the anamorphic development of the arthropod ancestor.

Within the last decade, gene expression patterns and neuro-anatomical data have led to a new consensus concerning the long-debated association between anterior limbs and neuromeres in the arthropod head. According to this new view, the first appendage in all extant euarthropods is innervated by the second neuromere, the deutocerebrum, whereas the anterior-most head region bearing the protocerebrum lacks an appendage. This stands in contrast to the clearly protocerebrally targeted "antennae" of Onychophora and to some evidence for protocerebral limbs in fossil euarthropod representatives. Yet, the latter "frontal appendages" or "primary antennae" have most likely been reduced or lost in the lineage, leading to extant taxa. Surprisingly, a recent neuro-anatomical study on a pycnogonid challenged this evolutionary scenario, reporting a protocerebral innervation of the first appendages, the chelifores. However, this interpretation was soon after questioned by Hox gene expression data. To re-evaluate the unresolved controversy, we analyzed neuro-anatomy and neurogenesis in four pycnogonid species using immunohistochemical techniques. We clearly show the postprotocerebral innervation of the chelifores, which is resolved as the plesiomorphic condition in pycnogonids when evaluated against a recently published comprehensive phylogeny. By providing direct morphological support for the deutocerebral status of the cheliforal ganglia, we reconcile morphological and gene expression data and argue for a corresponding position between the anterior-most appendages in all extant euarthropods. Consequently, other structures have to be scrutinized to illuminate the fate of a presumptive protocerebral appendage in recent euarthropods. The labrum and the "frontal filaments" of some crustaceans are possible candidates for this approach.

Despite significant advances in invertebrate phylogenomics over the past decade, the higher-level phylogeny of Pycnogonida (sea spiders) remains elusive. Due to the inaccessibility of some small-bodied lineages, few phylogenetic studies have sampled all sea spider families. Previous efforts based on a handful of genes have yielded unstable tree topologies. Here, we inferred the relationships of 89 sea spider species using targeted capture of the mitochondrial genome, 56 conserved exons, 101 ultraconserved elements, and three nuclear ribosomal genes. We inferred molecular divergence times by integrating morphological data for fossil species to calibrate 15 nodes in the arthropod tree of life. This integration of data classes resolved the basal topology of sea spiders with high support. The enigmatic family Austrodecidae was resolved as the sister group to the remaining Pycnogonida and the small-bodied family Rhynchothoracidae as the sister group of the robust-bodied family Pycnogonidae. Molecular divergence time estimation recovered a basal divergence of crown group sea spiders in the Ordovician. Comparison of diversification dynamics with other marine invertebrate taxa that originated in the Paleozoic suggests that sea spiders and some crustacean groups exhibit resilience to mass extinction episodes, relative to mollusk and echinoderm lineages.

BackgroundStudies on early neurogenesis have had considerable impact on the discussion of the phylogenetic relationships of arthropods, having revealed striking similarities and differences between the major lineages. In Hexapoda and crustaceans, neurogenesis involves the neuroblast, a type of neural stem cell. In each hemi-segment, a set of neuroblasts produces neural cells by repeated asymmetrical and interiorly directed divisions. In Euchelicerata and Myriapoda, neurogenesis lacks neural stem cells, featuring instead direct immigration of neural cell groups from fixed sites in the neuroectoderm. Accordingly, neural stem cells were hitherto assumed to be an evolutionary novelty of the Tetraconata (Hexapoda + crustaceans). To further test this hypothesis, we investigated neurogenesis in Pycnogonida, or sea spiders, a group of marine arthropods with close affinities to euchelicerates.ResultsWe studied neurogenesis during embryonic development of Pseudopallene sp. (Callipallenidae), using fluorescent histochemical staining and immunolabelling. Embryonic neurogenesis has two phases. The first phase shows notable similarities to euchelicerates and myriapods. These include i) the lack of morphologically different cell types in the neuroectoderm; ii) the formation of transiently identifiable, stereotypically arranged cell internalization sites; iii) immigration of predominantly post-mitotic ganglion cells; and iv) restriction of tangentially oriented cell proliferation to the apical cell layer. However, in the second phase, the formation of a central invagination in each hemi-neuromere is accompanied by the differentiation of apical neural stem cells. The latter grow in size, show high mitotic activity and an asymmetrical division mode. A marked increase of ganglion cell numbers follows their differentiation. Directly basal to the neural stem cells, an additional type of intermediate neural precursor is found.ConclusionsEmbryonic neurogenesis of Pseudopallene sp. combines features of central nervous system development that have been hitherto described separately in different arthropod taxa. The two-phase character of pycnogonid neurogenesis calls for a thorough reinvestigation of other non-model arthropods over the entire course of neurogenesis. With the currently available data, a common origin of pycnogonid neural stem cells and tetraconate neuroblasts remains unresolved. To acknowledge this, we present two possible scenarios on the evolution of arthropod neurogenesis, whereby Myriapoda play a key role in the resolution of this issue.

Mystacocarida is a species-poor group of minute crustaceans with unclear phylogenetic affinities. Previous studies have highlighted the putative "primitiveness" of several mystacocarid features, including the architecture of the nervous system. Recent studies on arthropod neuroarchitecture have provided a wealth of characters valuable for phylogenetic reconstructions. To permit and facilitate comparison with these data, we used immunohistochemical labeling (against acetylated alpha-tubulin, serotonin and FMRFamide) on the mystacocarid Derocheilocaris remanei, analyzing it with confocal laser-scanning microscopy and 3D reconstruction. The mystacocarid brain is fairly elongated, exhibiting a complicated stereotypic arrangement of neurite bundles. However, none of the applied markers provided evidence of structured neuropils such as a central body or olfactory glomeruli. A completely fused subesophageal ganglion is not present, all segmental soma clusters of the respective neuromeres still being delimitable. The distinct mandibular commissure comprises neurite bundles from more anterior regions, leading us to propose that it may have fused with an ancestral posterior tritocerebral commissure. The postcephalic ventral nervous system displays a typical ladder-like structure with separated ganglia which bears some resemblance to larval stages in other crustaceans. Ganglia and commissures are also present in the first three limbless "abdominal" segments, which casts doubt on the notion of a clear-cut distinction between thorax and abdomen. An unpaired longitudinal median neurite bundle is present and discussed as a potential tetraconate autapomorphy. Additionally, a paired latero-longitudinal neurite bundle extends along the trunk. It is connected to the intersegmental nerves and most likely fulfils neurohemal functions. We report the complete absence of serotonin-ir neurons in the ventral nervous system, which is a unique condition in arthropods and herein interpreted as a derived character.

Pycnogonida (sea spiders) are bizarre marine arthropods that are nowadays most frequently considered as being the sister group to all other chelicerates. The majority of pycnogonid species develops via a protonymphon larva with only three pairs of limbs affiliated with the future head region. Deviating from this, the hatching stage of some representatives shows already an advanced degree of trunk differentiation. Using scanning electron microscopy, fluorescent nucleic staining, and bright-field stereomicroscopy, postembryonic development of Pseudopallene sp. (Callipallenidae), a pycnogonid with an advanced hatching stage, is described. Based on external morphology, six postembryonic stages plus a sub-adult stage are distinguished. The hatching larva is lecithotrophic and bears the chelifores as only functional appendage pair and unarticulated limb buds of walking leg pairs 1 and 2. Palpal and ovigeral larval limbs are absent. Differentiation of walking leg pairs 3 and 4 is sequential. Apart from the first pair, each walking leg goes through a characteristic sequence of three externally distinct stages with two intermittent molts (limb bud-seven podomeres-nine podomeres). First external signs of oviger development are detectable in postembryonic stage 3 bearing three articulated walking leg pairs. Following three more molts, the oviger has attained adult podomere composition. The advanced hatching stages of different callipallenids are compared and the inclusive term "walking leg-bearing larva" is suggested, as opposed to the behavior-based name "attaching larva". Data on temporal and structural patterns of walking leg differentiation in other pycnogonids are reviewed and discussed. To facilitate comparisons of walking leg differentiation patterns across many species, we propose a concise notation in matrix fashion. Due to deviating structural patterns of oviger differentiation in another callipallenid species as well as within other pycnogonid taxa, evolutionary conservation of characteristic stages of oviger development is not apparent even in closely related species.

Embryonic development of Pycnogonida (sea spiders) is poorly understood in comparison to other euarthropod lineages with well-established model organisms. However, given that pycnogonids potentially represent the sister group to chelicerates or even to all other euarthropods, their development might yield important data for the reconstruction of arthropod evolution. Using scanning electron microscopy, fluorescent nucleic staining and immunohistochemistry, the general course of embryonic morphogenesis in Pseudopallene sp. (Callipallenidae), a pycnogonid with prolonged embryonic development, is described. A staging system comprising ten stages is presented, which can be used in future studies addressing specific developmental processes. The initially slit-like stomodeum anlage forms at the anterior end of an eight-shaped germ band and predates proboscis outgrowth. The latter process is characterized by the protrusion of three cell populations that are subsequently involved in pharynx formation. In later stages, the proboscis assumes distally a horseshoe-like shape. At no time, a structure corresponding to the euarthropod labrum is detectable. Based on the complete lack of palpal and ovigeral embryonic limbs and the early differentiation of walking leg segments 1 and 2, the existence of an embryonized protonymphon stage during callipallenid development is rejected. The evolution of pycnogonid hatching stages, especially within Callipallenidae and Nymphonidae, is re-evaluated in the light of recent phylogenetic analyses. Specifically, the re-emergence of the ancestral protonymphon larva (including re-development of palpal and ovigeral larval limbs) and a possible re-appearance of adult palps in the nymphonid lineage are discussed. This challenges the perception of pycnogonid head appendage evolution as being driven by reduction events alone.