Thomas Stach

Morphological Differences between Larvae of the Ciona intestinalis Species Complex: Hints for a Valid Taxonomic Definition of Distinct Species.

The cosmopolitan ascidian Ciona intestinalis is the most common model species of Tunicata, the sister-group of Vertebrata, and widely used in developmental biology, genomics and evolutionary studies. Recently, molecular studies suggested the presence of cryptic species hidden within the C. intestinalis species, namely C. intestinalis type A and type B. So far, no substantial morphological differences have been identified between individuals belonging to the two types. Here we present morphometric, immunohistochemical, and histological analyses, as well as 3-D reconstructions, of late larvae obtained by cross-fertilization experiments of molecularly determined type A and type B adults, sampled in different seasons and in four different localities. Our data point to quantitative and qualitative differences in the trunk shape of larvae belonging to the two types. In particular, type B larvae exhibit a longer pre-oral lobe, longer and relatively narrower total body length, and a shorter ocellus-tail distance than type A larvae. All these differences were found to be statistically significant in a Discriminant Analysis. Depending on the number of analyzed parameters, the obtained discriminant function was able to correctly classify > 93% of the larvae, with the remaining misclassified larvae attributable to the existence of intra-type seasonal variability. No larval differences were observed at the level of histology and immunohistochemical localization of peripheral sensory neurons. We conclude that type A and type B are two distinct species that can be distinguished on the basis of larval morphology and molecular data. Since the identified larval differences appear to be valid diagnostic characters, we suggest to raise both types to the rank of species and to assign them distinct names.

Neurogenesis in directly and indirectly developing enteropneusts: of nets and cords

Concerning the evolution of deuterostomes, enteropneusts (acorn worms) occupy a pivotal role as they share some characteristics with chordates (e.g., tunicates and vertebrates) but are also closely related to echinoderms (e.g., sea urchin). The nervous system in particular can be a highly informative organ system for evolutionary inferences, and advances in fluorescent microscopy have revealed overwhelming data sets on neurogenesis in various clades. However, immunocytochemical descriptions of neurogenesis of juvenile enteropneusts are particularly scarce, impeding the reconstruction of nervous system evolution in this group. We followed morphogenesis of the nervous system in two enteropneust species, one with direct (Saccoglossus kowalevskii) and the other with indirect development (Balanoglossus misakiensis), using an antibody against serotonin and electron microscopy. We found that all serotonin-like immunoreactive (LIR) neurons in both species are bipolar ciliary neurons that are intercalated between other epidermal cells. Unlike the tornaria larva of B. misakiensis, the embryonic nervous system of S. kowalevskii lacks serotonin-LIR neurons in the apical region as well as an opisthotroch neurite ring. Comparative analysis of both species shows that the projections of the serotonin-LIR somata initially form a basiepidermal plexus throughout the body that disappears within the trunk region soon after settlement before the concentrated dorsal and ventral neurite bundles emerge. Our data reveal a highly conserved mode of neurogenesis in enteropneusts that is independent of the developing mode and is inferred to be a common feature for Enteropneusta. Moreover, all detected serotonin-LIR neurons are presumably receptor cells, and the absence of serotonin-LIR interneurons from the enteropneust nervous system, which are otherwise common in various bilaterian central nervous systems, is interpreted as a loss that might have occurred already in the last common ancestor of Ambulacraria.

Evolutionary diversification of secondary mechanoreceptor cells in tunicata

BackgroundHair cells are vertebrate secondary sensory cells located in the ear and in the lateral line organ. Until recently, these cells were considered to be mechanoreceptors exclusively found in vertebrates that evolved within this group. Evidence of secondary mechanoreceptors in some tunicates, the proposed sister group of vertebrates, has recently led to the hypothesis that vertebrate and tunicate secondary sensory cells share a common origin. Secondary sensory cells were described in detail in two tunicate groups, ascidians and thaliaceans, in which they constitute an oral sensory structure called the coronal organ. Among thaliaceans, the organ is absent in salps and it has been hypothesised that this condition is due to a different feeding system adopted by this group of animals. No information is available as to whether a comparable structure exists in the third group of tunicates, the appendicularians, although different sensory structures are known to be present in these animals.ResultsWe studied the detailed morphology of appendicularian oral mechanoreceptors. Using light and electron microscopy we could demonstrate that the mechanosensory organ called the circumoral ring is composed of secondary sensory cells. We described the ultrastructure of the circumoral organ in two appendicularian species, Oikopleura dioica and Oikopleura albicans, and thus taxonomically completed the data collection of tunicate secondary sensory cells. To understand the evolution of secondary sensory cells in tunicates, we performed a cladistic analysis using morphological data. We constructed a matrix consisting of 19 characters derived from detailed ultrastructural studies in 16 tunicate species and used a cephalochordate and three vertebrate species as outgroups.ConclusionsOur study clearly shows that the circumoral ring is the appendicularian homologue of the coronal organ of other tunicate taxa. The cladistic analysis enabled us to reconstruct the features of the putative ancestral hair cell in tunicates, represented by a simple monociliated cell. This cell successively differentiated into the current variety of oral mechanoreceptors in the various tunicate lineages. Finally, we demonstrated that the inferred evolutionary changes coincide with major transitions in the feeding strategies in each respective lineage.

Cell Lineages and Fate Maps in Tunicates: Conservation and Modification

Comparison of features of the cell lineages and fate maps of early embryos between related species is useful in inferring developmental mechanisms and amenable to evolutionary considerations. We present cleavage patterns, cell lineage trees, and fate maps of ascidian and appendicularian embryos side by side to facilitate comparison. This revealed a number of significant differences in cleavage patterns and cell lineage trees, whereas the fate maps were found to be conserved. This fate map similarity can be extended to vertebrates, thus representing the fate map characteristics of chordates. Cleavage patterns and cell lineages may have been modified during evolution without any drastic changes in fate maps. Selective pressures that constrain developmental mechanisms at early embryonic stages might not be so strong as long as embryos are still able to generate a chordate-type fate map. Aquatic chordates share similar fate maps and morphogenetic movements during gastrulation and neurulation, eventually developing into tadpole-shaped larvae. As swimming by tail beats, and not by cilia, is advantageous, selective pressure may maintain the basic elements of the tadpole shape. We also discuss the evolutionary origin of the vertebrate neural crest and the embryonic origin of the appendicularian heart to illustrate the usefulness of cell lineage data. From an evolutionary standpoint, cell lineages behave like other characteristics such as morphology or protein sequences. Both novel and primitive features are present in extant organisms, and it is of interest to identify the relative degree of evolutionary conservation as well as the level at which homology is inferred.

The central and peripheral nervous system of Cephalodiscus gracilis (Pterobranchia, Deuterostomia)

Nervous systems are important in assessing interphyletic phylogenies because they are conservative and complex. Regarding nervous system evolution within deuterostomes, two contrasting hypotheses are currently discussed. One that argues in favor of a concentrated, structured, central nervous system in the last common ancestor of deuterostomes (LCAD); the other reconstructing a decentralized nerve net as the nervous system of the LCAD. Here, we present a morphological analysis of the nervous system of the pterobranch deuterostome Cephalodiscus gracilis Harmer, 1905 based on transmission electron microscopy, confocal laser scanning microscopy, immunohistochemistry, and computer-assisted 3D reconstructions based on complete serial histological sections. The entire nervous system constitutes a basiepidermal plexus. The prominent dorsal brain at the base of the mesosomal tentacles contains an anterior concentration of serotonergic neurons and a posterior net of neurites. Predominant neurite directions differ between brain regions and synapses are present, indicating that the brain constitutes a centralized portion of the nervous system. Main structures of the peripheral nervous system are the paired branchial nerves, tentacle nerves, and the ventral stalk nerve. Serotonergic neurites are scattered throughout the epidermis and are present as concentrations along the anterior border of the branchial nerves. Serotonergic neurons line each tentacle and project into the brain. We argue that the presence of a centralized brain in C. gracilis supports the hypothesis that a nerve center was present in the LCAD. Moreover, based on positional and structural similarity, we suggest that the branchial nerves in C. gracilis could be homologous to branchial nerves in craniates, a hypothesis that should be further investigated.

Comparative study of serotonin-like immunoreactivity in the branchial basket, digestive tract, and nervous system in tunicates

Abstract As one of the three major chordate taxa, the highly diverse taxon, Tunicata has always played a key role in considerations of evolutionary origins of vertebrates, especially since several larger-scaled molecular phylogenetic analyses support the hypothesis that tunicates are the sister group to vertebrates. Molecular phylogenetic studies of the relationships within Tunicata are highly contradictory, and cladistic analyses of morphological characters for Tunicata remained limited. In order to come to a better understanding of chordate evolution, we comparatively investigated nine different tunicate species belonging to five different higher tunicate taxa: Phlebobranchiata, Aplousobranchiata, Stolidobranchiata, Thaliacea, and Appendicularia using immunofluorescence labeling with antibodies against serotonin in conjunction with confocal laser scanning microscopy. We found that adult ascidians are comparable in regard to their respective patterns of serotonin-like immunoreactive (serotonin-lir) cells, whereas the planktonic tunicates differ in several aspects. The distribution patterns of serotonin-lir cells in tunicates suggest that serotonin-like immunoreactivity can behave as an independent character during evolution. While the distribution pattern of serotonin-lir cells agrees well with classical taxonomic groupings, phylogenetic interpretation remains inconclusive, as ad hoc hypotheses are always necessary to explain contradictory character distribution. Based on light-microscopically observed morphology, we could distinguish three different types of serotonin-lir cells, most probably functionally distinctive. These were approximately spherical serotonin-lir cells, possibly involved in the control of ciliary beating and mucus secretion, elongated serotonin-lir cells potentially involved in hormonal regulation of feeding, and serotonin-lir neurons that might be implicated in the initiation of locomotory behavior.

Structure and ultrastructure of eyes of tornaria larvae of Glossobalanus marginatus

Enteropneusts or acorn worms are marine deuterostomes that have retained many plesiomorphic characters. Thus, enteropneusts are of prime interest in evolutionary comparisons between deuterostomes and protostomes. In the present study, the larval eyes of Glossobalanus marginatus were reconstructed and described based on serial sectioning for transmission electron microscopy. The everse eyes of the late Metschnikoff/early Krohn-stage tornaria larvae of G. marginatus are epidermal structures consisting of two rows of in total 13 shading pigment cells and another two rows of 13 photoreceptor cells. The pigment cells form a shallow cup with a relatively wide opening, making the cup-shaped eye optically unsuitable for picture generation. We demonstrate that the photosensitive cells possess numerous enlarged microvilli and an unmodified apical cilium. Our ultrastructural studies thus corroborate the photoreceptor cells in the eye of G. marginatus to be of a clearly rhabdomeric type. Preliminary immunohistochemical experiments support those findings by demonstrating immunopositive reaction of the tornarian eye photoreceptors with an antibody designed against rhabdomeric sea urchin photopigment (Sp-Opsin4). Observations of living animals indicate that Late Metschnikoff/early Krohn-stage tornaria larvae are negatively phototactic, probably concordant with imminent metamorphosis.

Larval anatomy of the pterobranch Cephalodiscus gracilis supports secondarily derived sessility concordant with molecular phylogenies

Pterobranchs have been interpreted as “missing links” combining primitive invertebrate features with advanced vertebrate-like characteristics. The first detailed morphological description of an ontogenetic stage of a pterobranch, based on digital 3D-reconstruction at electron microscopic resolution, reveals a triploblastic animal with monociliated epithelia, an extensive coelomic cavity, a through gut with an asymmetrically developed gill slit but no signs of planktonic specializations, such as ciliated bands. Therefore, this crawling larva supports the hypothesis proposed in previous molecular phylogenetic studies that pterobranchs could be derived within enteropneusts rather than being “missing links”.

Structure and ultrastructure of eyes and brains of Thalia democratica (Thaliacea, Tunicata, Chordata)

Salps are marine planktonic chordates that possess an obligatory alternation of reproductive modes in subsequent generations. Within tunicates, salps represent a derived life cycle and are of interest in considerations of the evolutionary origin of complex anatomical structures and life history strategies. In the present study, the eyes and brains of both the sexual, aggregate blastozooid and the asexual, solitary oozooid stage of Thalia democratica (Forskål, ) were digitally reconstructed in detail based on serial sectioning for light and transmission electron microscopy. The blastozooid stage of T. democratica possesses three pigment cup eyes, situated in the anterior ventral part of the brain. The eyes are arranged in a way that the optical axes of each eye point toward different directions. Each eye is an inverse eye that consists of two different cell types: pigment cells (pigc) and rhabdomeric photoreceptor cells (prcs). The oozooid stage of T. democratica is equipped with a single horseshoe‐shaped eye, positioned in the anterior dorsal part of the brain. The opening of the horseshoe‐shaped eye points anteriorly. Similar to the eyes of the blastozooid, the eye of the oozooid consists of pigment cells and rhabdomeric photoreceptor cells. The rhabdomeric photoreceptor cells possess apical microvilli that form a densely packed presumably photosensitive receptor part adjacent to the concave side of the pigc. We suggest correspondences of the individual eyes in the blastozooid stage to respective parts of the single horseshoe‐shaped eye in the oozooid stage and hypothesize that the differences in visual structures and brain anatomies evolved as a result of the aggregate life style of the blastozooid as opposed to the solitary life style of the oozooid.

High-precision morphology: bifocal 4D-microscopy enables the comparison of detailed cell lineages of two chordate species separated for more than 525 million years

BackgroundUnderstanding the evolution of divergent developmental trajectories requires detailed comparisons of embryologies at appropriate levels. Cell lineages, the accurate visualization of cleavage patterns, tissue fate restrictions, and morphogenetic movements that occur during the development of individual embryos are currently available for few disparate animal taxa, encumbering evolutionarily meaningful comparisons. Tunicates, considered to be close relatives of vertebrates, are marine invertebrates whose fossil record dates back to 525 million years ago. Life-history strategies across this subphylum are radically different, and include biphasic ascidians with free swimming larvae and a sessile adult stage, and the holoplanktonic larvaceans. Despite considerable progress, notably on the molecular level, the exact extent of evolutionary conservation and innovation during embryology remain obscure.ResultsHere, using the innovative technique of bifocal 4D-microscopy, we demonstrate exactly which characteristics in the cell lineages of the ascidian Phallusia mammillata and the larvacean Oikopleura dioica were conserved and which were altered during evolution. Our accurate cell lineage trees in combination with detailed three-dimensional representations clearly identify conserved correspondence in relative cell position, cell identity, and fate restriction in several lines from all prospective larval tissues. At the same time, we precisely pinpoint differences observable at all levels of development. These differences comprise fate restrictions, tissue types, complex morphogenetic movement patterns, numerous cases of heterochronous acceleration in the larvacean embryo, and differences in bilateral symmetry.ConclusionsOur results demonstrate in extraordinary detail the multitude of developmental levels amenable to evolutionary innovation, including subtle changes in the timing of fate restrictions as well as dramatic alterations in complex morphogenetic movements. We anticipate that the precise spatial and temporal cell lineage data will moreover serve as a high-precision guide to devise experimental investigations of other levels, such as molecular interactions between cells or changes in gene expression underlying the documented structural evolutionary changes. Finally, the quantitative amount of digital high-precision morphological data will enable and necessitate software-based similarity assessments as the basis of homology hypotheses.