Serotonin-immunoreactive neurons in the ventral nerve cord of Remipedia (Crustacea): support for a sister group relationship of Remipedia and Hexapoda?

BACKGROUND: Remipedia were initially seen as a primitive taxon within Pancrustacea based on characters considered ancestral, such as the homonomously segmented trunk. Meanwhile, several morphological and molecular studies proposed a more derived position of Remipedia within Pancrustacea, including a sister group relationship to Hexapoda. Because of these conflicting hypotheses, fresh data are crucial to contribute new insights into euarthropod phylogeny. The architecture of individually identifiable serotonin-immunoreactive neurons has successfully been used for phylogenetic considerations in Euarthropoda. Here, we identified neurons in three species of Remipedia with an antiserum against serotonin and compared our findings to reconstructed ground patterns in other euarthropod taxa. Additionally, we traced neurite connectivity and neuropil outlines using antisera against acetylated α-tubulin and synapsin. RESULTS: The ventral nerve cord of Remipedia displays a typical rope-ladder-like arrangement of separate metameric ganglia linked by paired longitudinally projecting connectives. The peripheral projections comprise an intersegmental nerve, consisting of two branches that fuse shortly after exiting the connectives, and the segmental anterior and posterior nerve. The distribution and morphology of serotonin-immunoreactive interneurons in the trunk segments is highly conserved within the remipede species we analyzed, which allows for the reconstruction of a ground pattern: two posterior and one anterior pair of serotonin-immunoreactive neurons that possess a single contralateral projection. Additionally, three pairs of immunoreactive neurons are found in the medial part of each hemiganglion. In one species (Cryptocorynetes haptodiscus), the anterior pair of immunoreactive neurons is missing. CONCLUSIONS: The anatomy of the remipede ventral nerve cord with its separate metameric ganglia mirrors the external morphology of the animal's trunk. The rope-ladder-like structure and principal architecture of the segmental ganglia in Remipedia corresponds closely to that of other Euarthropoda. A comparison of the serotonin-immunoreactive cell arrangement of Remipedia to reconstructed ground patterns of major euarthropod taxa supports a homology of the anterior and posterior neurons in Pancrustacea. These neurons in Remipedia possess unbranched projections across the midline, pointing towards similarities to the hexapod pattern. Our findings are in line with a growing number of phylogenetic investigations proposing Remipedia to be a rather derived crustacean lineage that perhaps has close affinities to Hexapoda.

Serotonin immunoreactive interneurons in the brain of the Remipedia: new insights into the phylogenetic affinities of an enigmatic crustacean taxon.

BACKGROUND: Remipedia, a group of homonomously segmented, cave-dwelling, eyeless arthropods have been regarded as basal crustaceans in most early morphological and taxonomic studies. However, molecular sequence information together with the discovery of a highly differentiated brain led to a reconsideration of their phylogenetic position. Various conflicting hypotheses have been proposed including the claim for a basal position of Remipedia up to a close relationship with Malacostraca or Hexapoda. To provide new morphological characters that may allow phylogenetic insights, we have analyzed the architecture of the remipede brain in more detail using immunocytochemistry (serotonin, acetylated α-tubulin, synapsin) combined with confocal laser-scanning microscopy and image reconstruction techniques. This approach allows for a comprehensive neuroanatomical comparison with other crustacean and hexapod taxa. RESULTS: The dominant structures of the brain are the deutocerebral olfactory neuropils, which are linked by the olfactory globular tracts to the protocerebral hemiellipsoid bodies. The olfactory globular tracts form a characteristic chiasm in the center of the brain. In Speleonectes tulumensis, each brain hemisphere contains about 120 serotonin immunoreactive neurons, which are distributed in distinct cell groups supplying fine, profusely branching neurites to 16 neuropilar domains. The olfactory neuropil comprises more than 300 spherical olfactory glomeruli arranged in sublobes. Eight serotonin immunoreactive neurons homogeneously innervate the olfactory glomeruli. In the protocerebrum, serotonin immunoreactivity revealed several structures, which, based on their position and connectivity resemble a central complex comprising a central body, a protocerebral bridge, W-, X-, Y-, Z-tracts, and lateral accessory lobes. CONCLUSIONS: The brain of Remipedia shows several plesiomorphic features shared with other Mandibulata, such as deutocerebral olfactory neuropils with a glomerular organization, innervations by serotonin immunoreactive interneurons, and connections to protocerebral neuropils. Also, we provided tentative evidence for W-, X-, Y-, Z-tracts in the remipedian central complex like in the brain of Malacostraca, and Hexapoda. Furthermore, Remipedia display several synapomorphies with Malacostraca supporting a sister group relationship between both taxa. These homologies include a chiasm of the olfactory globular tract, which connects the olfactory neuropils with the lateral protocerebrum and the presence of hemiellipsoid bodies. Even though a growing number of molecular investigations unites Remipedia and Cephalocarida, our neuroanatomical comparison does not provide support for such a sister group relationship.

Ovary structure and early oogenesis in the remipede, Godzilliognomus frondosus (Crustacea, Remipedia): phylogenetic implications.

Remipedia are enigmatic crustaceans of uncertain phylogenetic position with the general consensus that they are crucial for understanding the crustacean/arthropod evolution. It has been demonstrated previously that the features of the ovary organization and subcellular aspects of oogenesis are useful in resolving phylogenetic relationships in arthropods such as hexapods and onychophorans. The structure of the female gonads in Remipedia remains largely unknown; therefore, we examined the gross morphology and ultrastructural details of the ovary in a remipede, Godzilliognomus frondosus, with special emphasis on characters relevant to phylogenetic reconstructions. The ovaries of G. frondosus are located in the anterior part of the body and are composed of a single anterior proliferative zone (the germarium) and paired ovarian tubes (the vitellarium). The oocytes undergo subsequent stages of development within the lumen of the ovarian tubes, hence the remipede ovaries can be classified as endogenous. During oogenesis, each oocyte is enveloped by a set of characteristic somatic follicular cells, which results in the formation of distinct ovarian follicles. Here, we demonstrate that Remipedia share significant similarities in the ovary organization with Cephalocarida, including the anterior location of the ovary, the anterior-most position of the germarium and the endogenous type of oocyte development. Phylogenetic implications of our findings are discussed.

Pancrustacean phylogeny in the light of new phylogenomic data: support for Remipedia as the possible sister group of Hexapoda.

Remipedes are a small and enigmatic group of crustaceans, first described only 30 years ago. Analyses of both morphological and molecular data have recently suggested a close relationship between Remipedia and Hexapoda. If true, the remipedes occupy an important position in pancrustacean evolution and may be pivotal for understanding the evolutionary history of crustaceans and hexapods. However, it is important to test this hypothesis using new data and new types of analytical approaches. Here, we assembled a phylogenomic data set of 131 taxa, incorporating newly generated 454 expressed sequence tag (EST) data from six species of crustaceans, representing five lineages (Remipedia, Laevicaudata, Spinicaudata, Ostracoda, and Malacostraca). This data set includes all crustacean species for which EST data are available (46 species), and our largest alignment encompasses 866,479 amino acid positions and 1,886 genes. A series of phylogenomic analyses was performed to evaluate pancrustacean relationships. We significantly improved the quality of our data for predicting putative orthologous genes and for generating data subsets by matrix reduction procedures, thereby improving the signal to noise ratio in the data. Eight different data sets were constructed, representing various combinations of orthologous genes, data subsets, and taxa. Our results demonstrate that the different ways to compile an initial data set of core orthologs and the selection of data subsets by matrix reduction can have marked effects on the reconstructed phylogenetic trees. Nonetheless, all eight data sets strongly support Pancrustacea with Remipedia as the sister group to Hexapoda. This is the first time that a sister group relationship of Remipedia and Hexapoda has been inferred using a comprehensive phylogenomic data set that is based on EST data. We also show that selecting data subsets with increased overall signal can help to identify and prevent artifacts in phylogenetic analyses.

Global biodiversity and phylogenetic evaluation of remipedia (crustacea).

Remipedia is one of the most recently discovered classes of crustaceans, first described in 1981 from anchialine caves in the Bahamas Archipelago. The class is divided into the order Enantiopoda, represented by two fossil species, and Nectiopoda, which contains all known extant remipedes. Since their discovery, the number of nectiopodan species has increased to 24, half of which were described during the last decade. Nectiopoda exhibit a disjunct global distribution pattern, with the highest abundance and diversity in the Caribbean region, and isolated species in the Canary Islands and in Western Australia. Our review of Remipedia provides an overview of their ecological characteristics, including a detailed list of all anchialine marine caves, from which species have been recorded. We discuss alternative hypotheses of the phylogenetic position of Remipedia within Arthropoda, and present first results of an ongoing molecular-phylogenetic analysis that do not support the monophyly of several nectiopodan taxa. We believe that a taxonomic revision of Remipedia is absolutely essential, and that a comprehensive revision should include a reappraisal of the fossil record.

Arthropod phylogeny revisited, with a focus on crustacean relationships.

Higher-level arthropod phylogenetics is an intensely active field of research, not least as a result of the hegemony of molecular data. However, not all areas of arthropod phylogenetics have so far received equal attention. The application of molecular data to infer a comprehensive phylogeny of Crustacea is still in its infancy, and several emerging results are conspicuously at odds with morphology-based studies. In this study, we present a series of molecular phylogenetic analyses of 88 arthropods, including 57 crustaceans, representing all the major lineages, with Onychophora and Tardigrada as outgroups. Our analyses are based on published and new sequences for two mitochondrial markers, 16S rDNA and cytochrome c oxidase subunit I (COI), and the nuclear ribosomal gene 18S rDNA. We designed our phylogenetic analyses to assess the effects of different strategies of sequence alignment, alignment masking, nucleotide coding, and model settings. Our comparisons show that alignment optimization of ribosomal markers based on secondary structure information can have a radical impact on phylogenetic reconstruction. Trees based on optimized alignments recover monophyletic Arthropoda (excluding Onychophora), Pancrustacea, Malacostraca, Insecta, Myriapoda and Chelicerata, while Maxillopoda and Hexapoda emerge as paraphyletic groups. Our results are unable to resolve the highest-level relationships within Arthropoda, and none of our trees supports the monophyly of Myriochelata or Mandibulata. We discuss our results in the context of both the methodological variations between different analyses, and of recently proposed phylogenetic hypotheses. This article offers a preliminary attempt to incorporate the large diversity of crustaceans into a single molecular phylogenetic analysis, assessing the robustness of phylogenetic relationships under varying analysis parameters. It throws into sharp relief the relative strengths and shortcomings of the combined molecular data for assessing this challenging phylogenetic problem, and thereby provides useful pointers for future studies.

The post-embryonic development of Remipedia (Crustacea)--additional results and new insights.

The post-embryonic development of a species of the enigmatic crustacean group Remipedia is described in detail for the first time under various aspects. Applying a molecular approach, we can clearly prove the species identity of the larvae as belonging to Pleomothra apletocheles. We document the cellular level of several larval stages and the differentiation of segments, limbs, and the general body morphology applying the techniques of confocal laser scanning microscopy and scanning electron microscopy. In addition, we document the swimming behavior and the peculiar movements of the naupliar appendages. A comparison of our results with published data on other Crustacea and their larval development tentatively supports ideas about phylogenetic affinities of the Remipedia to the Malacostraca.

Post-embryonic development of remipede crustaceans.

During diving explorations of anchialine cave systems on Abaco Island, Bahamas, we collected five larvae that represent different developmental stages of remipede crustaceans. Based on four early naupliar stages and a post-naupliar larva, it is possible for the first time to reconstruct the postembryonic development of Remipedia some 25 years after their discovery. These specimens begin to fill in some critical gaps in our knowledge of this important group of crustaceans.