Conrad Helm

Illuminating the base of the annelid tree using transcriptomics

Annelida is one of three animal groups possessing segmentation and is central in considerations about the evolution of different character traits. It has even been proposed that the bilaterian ancestor resembled an annelid. However, a robust phylogeny of Annelida, especially with respect to the basal relationships, has been lacking. Our study based on transcriptomic data comprising 68,750-170,497 amino acid sites from 305 to 622 proteins resolves annelid relationships, including Chaetopteridae, Amphinomidae, Sipuncula, Oweniidae, and Magelonidae in the basal part of the tree. Myzostomida, which have been indicated to belong to the basal radiation as well, are now found deeply nested within Annelida as sister group to Errantia in most analyses. On the basis of our reconstruction of a robust annelid phylogeny, we show that the basal branching taxa include a huge variety of life styles such as tube dwelling and deposit feeding, endobenthic and burrowing, tubicolous and filter feeding, and errant and carnivorous forms. Ancestral character state reconstruction suggests that the ancestral annelid possessed a pair of either sensory or grooved palps, bicellular eyes, biramous parapodia bearing simple chaeta, and lacked nuchal organs. Because the oldest fossil of Annelida is reported for Sipuncula (520 Ma), we infer that the early diversification of annelids took place at least in the Lower Cambrian.

Exploiting gene families for phylogenomic analysis of myzostomid transcriptome data

Background In trying to understand the evolutionary relationships of organisms, the current flood of sequence data offers great opportunities, but also reveals new challenges with regard to data quality, the selection of data for subsequent analysis, and the automation of steps that were once done manually for single-gene analyses. Even though genome or transcriptome data is available for representatives of most bilaterian phyla, some enigmatic taxa still have an uncertain position in the animal tree of life. This is especially true for myzostomids, a group of symbiotic (or parasitic) protostomes that are either placed with annelids or flatworms. Methodology Based on similarity criteria, Illumina-based transcriptome sequences of one myzostomid were compared to protein sequences of one additional myzostomid and 29 reference metazoa and clustered into gene families. These families were then used to investigate the phylogenetic position of Myzostomida using different approaches: Alignments of 989 sequence families were concatenated, and the resulting superalignment was analyzed under a Maximum Likelihood criterion. We also used all 1,878 gene trees with at least one myzostomid sequence for a supertree approach: the individual gene trees were computed and then reconciled into a species tree using gene tree parsimony. Conclusions Superalignments require strictly orthologous genes, and both the gene selection and the widely varying amount of data available for different taxa in our dataset may cause anomalous placements and low bootstrap support. In contrast, gene tree parsimony is designed to accommodate multilocus gene families and therefore allows a much more comprehensive data set to be analyzed. Results of this supertree approach showed a well-resolved phylogeny, in which myzostomids were part of the annelid radiation, and major bilaterian taxa were found to be monophyletic.

Early divergence, broad distribution and high diversity of animal chitin synthases

Even though chitin is one of the most abundant biopolymers in nature, current knowledge on chitin formation is largely based only on data from fungi and insects. This study reveals unanticipated broad taxonomic distribution and extensive diversification of chitin synthases (CSs) in Metazoa, shedding new light on the relevance of chitin in animals and suggesting unforeseen complexity of chitin synthesis in many groups. We uncovered robust orthologs to insect type CSs in several representatives of deuterostomes, which generally are not thought to possess chitin. This suggests a broader distribution and function of chitin in this branch of the animal kingdom. We characterize a new CS type present not only in basal metazoans such as sponges and cnidarians but also in several bilaterian representatives. The most extensive diversification of CSs took place during emergence of lophotrochozoans, the third large group of protostomes next to arthropods and nematodes, resulting in coexistence of up to ten CS paralogs in molluscs. Independent fusion to different kinds of myosin motor domains in fungi and lophotrochozoans points toward high relevance of CS interaction with the cytoskeleton for fine-tuned chitin secretion. Given the fundamental role that chitin plays in the morphology of many animals, the here presented CS diversification reveals many evolutionary complexities. Our findings strongly suggest a very broad and multifarious occurrence of chitin and question an ancestral role as cuticular component. The molecular mechanisms underlying regulation of animal chitin synthesis are most likely far more complex and diverse than existing data from insects suggest.

Description of a new syllid species as a model for evolutionary research of reproduction and regeneration in annelids

Syllids are one of the most speciose annelid taxa and characterized by their variety of reproductive modes. We provide the description of a new species of Syllidae (Annelida, Phyllodocida), Typosyllis antoni n. sp., which is characterized by its distinct color pattern consisting of transversal red lines on the dorsum of anterior segments; long antennae and dorsal cirri with strong alternation in length; bidentate chaetae falciger like with long spinulation on edge, one tiny and thin acicula appearing in posterior segments in addition to thicker and pointed one, and a long proventricle. A phylogenetic analysis of Syllinae based on three genes supports that T. antoni n. sp. is sister species to Typosyllis heronislandensis. This sister group relationship may indicate a common ancestor from the Pacific. Moreover, we recommend several steps to unify the taxonomy with phylogenetic knowledge of this group. Using immunocytochemistry coupled with confocal laser scanning microscopy (cLSM), we describe the internal morphology of this species. The body wall is composed of two dorsal and two ventral longitudinal muscle bundles that form a distinct inner layer. The outer or “circular layer” of body wall musculature is represented by prominent transverse muscle fibers that exhibit a semicircular arrangement. The musculature of the uniramous parapodia is characterized by distinct parapodial retractor muscles, acicular protractor muscles, as well as prominent acicular and chaetal flexor muscle bundles. T. antoni n. sp. reproduces by schizogamic scissiparity producing dicerous stolons. This species is able to regenerate the anterior end, including the prostomium, the first chaetae-less segment with all appendages, and some additional chaetigers, depending on the dissection side. Regeneration of the proventricle, ventricle, caeca, or pharyngeal tooth is not detectable. In contrast, regeneration of the posterior end appears to be complete. The available data makes T. antoni n. sp. to be one of the best investigated syllids, emphasizing its potential as model for the whole group. Our analysis establishes a framework for future studies on the evolution of reproductive modes in Syllidae, and we outline research questions how they are related to regeneration and development.

Structure and anterior regeneration of musculature and nervous system in Cirratulus cf. cirratus (Cirratulidae, Annelida)

Annelids provide suitable models for studying regeneration. By now, comprehensive information is restricted to only a few taxa. For many other annelids, comparative data are scarce or even missing. Here, we describe the regeneration of a member of the Cirratulus cirratus species complex. Using phalloidin‐labeling and antibody‐stainings combined with subsequent confocal laser scanning microscopy, we provide data about the organization of body wall musculature and nervous system of intact specimens, as well as about anteriorly regenerating specimens. Our analyses show that C. cf. cirratus exhibits a prominent longitudinal muscle layer forming a dorsal muscle plate, two ventral muscle strands and a ventral‐median muscle fiber. The circular musculature forms closed rings which are interrupted in the area of parapodia. The nervous system of C. cf. cirratus shows a typical rope‐ladder like arrangement and the circumesophageal connectives exhibit two separate roots leading to the brain. During regeneration, the nervous system redevelops remarkably earlier than the musculature, first constituting a tripartite loop‐like structure which later become the circumesophageal connectives. Regeneration of longitudinal musculature starts with diffuse ingrowth and subsequent structuring into the blastema. In contrast, circular musculature develops independently inside the blastema. Our findings constitute the first analysis of regeneration for a member of the Cirratuliformia on a structural level. Summarizing the regeneration process in C. cf. cirratus, five main phases can be subdivided: 1) wound closure, 2) blastema formation, 3) blastema differentiation, 4) resegmentation, and 5) growth, respectively elongation. Additionally, the described tripartite loop‐like structure of the regenerating nervous system has not been reported for any other annelid taxon. In contrast, the regeneration of circular and longitudinal musculature originating from different groups of cells seems to be a general pattern in annelid regeneration. J. Morphol. 275:1418–1430, 2014.

An immunocytochemical window into the development of Platynereis massiliensis (Annelida, Nereididae).

The nereidid annelid Platynereis dumerilii emerged as a well-understood model organism. P. dumerilii and P. massiliensis are sister taxa, which are morphologically indistinguishable as adults. Interestingly, they exhibit highly contrasting life-histories: while P. dumerilii is a gonochorostic species with planktonic feeding larvae, P. massiliensis is a protandric hermaphrodite with lecitotrophic semi-direct -development in brood tubes. Using light microscopy and immunohistochemical methods coupled with confocal laser scanning microscopy, we describe the development of P. massiliensis. Musculature was stained with phalloidin-rhodamine. FMRFamide, acetylated α-tubulin, and serotonin were targeted by antibodies for the staining of neuronal structures. Additionally, eye development was investigated with the specific 22C10-antibody. The development of P. massiliensis is characterized by the absence of a free-swimming stage, a late development of food uptake, and the presence of a large amount of yolk even in late juvenile stages. Most notably, early juvenile stages already exhibit an organization of several organ systems that resembles those of adults. Larval characters present in the free-swimming feeding larvae of P. dumerilii, as e.g. the apical organ and larval eyes, are absent and regarded to be lost in developing stages of P. massiliensis. Many of the differences found in the development of these two species can be described in the context of heterochronic changes. We strongly advocate expanding evolutionary developmental studies from the well-established model annelid P. dumerilii to the closely related P. massiliensis to study the evolutionary conservation and divergence of genetic pathways involved in developmental processes.

Comparative analyses of morphological characters in Sphaerodoridae and allies (Annelida) revealed by an integrative microscopical approach

Sphaerodoridae is a group of benthic marine worms (Annelida) characterized by the presence of spherical tubercles covering their whole surface. They are commonly considered as belonging to Phyllodocida although sistergroup relationships are still far from being understood. Primary homology assessment of their morphological features are lacking, hindering the appraisal of evolutionary relationships between taxa. Therefore, our detailed morphological investigation focuses on different Sphaerodoridae as well as on other members of Phyllodocida using an integrative approach combining scanning electron microscopy (SEM) as well as immunohistochemistry with standard neuronal (anti-5-HT) and muscular (phalloidin-rhodamine) markers and subsequent CLSM analysis of whole mounts and sections. Furthermore, we provide histological (HES) and light microscopical data to shed light on the structures and hypothetical function of sphaerodorid key morphological features. We provide fundamental details into the sphaerodorid morphology supporting a Phyllodocida ancestry of these enigmatic worms. However, the muscular arrangement and the presence of an axial muscular pharynx is similar to conditions observed in other members of the Errantia too. Furthermore, nervous system and muscle staining as well as SEM and histological observations of different types of tubercles indicate a homology of the so called microtubercles, present in the long-bodied sphaerodorids, to the dorsal cirri of other Errantia. The macrotubercles seem to represent a sphaerodorid autapomorphy based on our investigations. Therefore, our results allow comparisons concerning morphological patterns between Sphaerodoridae and other Phyllodocida and constitute a starting point for further comparative investigations to reveal the evolution of the remarkable Sphaerodoridae.

Myoanatomy of Myzostoma cirriferum (Annelida, Myzostomida): implications for the evolution of the myzostomid body plan.

Studies of rare genomic marker systems suggest that Myzostomida are a subgroup of Annelida and phylogenomic analyses indicate an early divergence of this taxon within annelids. However, adult myzostomids show a highly specialized body plan, which lacks typical annelid features, such as external body annulation, coelomic cavities with metanephridia, and segmental ganglia of the nervous system. The putative loss of these features might be due to the parasitic/symbiotic lifestyle of myzostomids associated with echinoderms. In contrast, the larval anatomy and adult locomotory system resemble those of annelids. To clarify whether the myoanatomy of myzostomids reflects their relationship to annelids, we analyzed the distribution of f‐actin, a common component of muscle fibers, in specimens of Myzostoma cirriferum using phalloidin‐rhodamine labeling in conjunction with confocal laser‐scanning microscopy. Our data reveal that the musculature of the myzostomid body comprises an outer circular layer, an inner longitudinal layer, numerous dorsoventral muscles, and prominent muscles of the parapodial complex. These features correspond well with the common organization of the muscular system in Annelida. In contrast to other annelids, however, several elements of the muscular system in M. cirriferum, including the musculature of the body wall, and the parapodial flexor muscles, exhibit radial symmetry overlaying a bilateral body plan. These findings are in line with the annelid affinity of myzostomids and suggest that the apparent partial radial symmetry of M. cirriferum arose secondarily in this species. Based on our data, we provide a scenario on the rearrangements of muscle fibers that might have taken place in the lineage leading to this species. J. Morphol., 2013.

Temporal Plasticity in Annelid Development—Ontogeny of Phyllodoce groenlandica (Phyllodocidae, Annelida) Reveals Heterochronous Patterns

The variety of annelid larval types and developmental modes reflects the high diversity and variability within these lophotrochozoans. However, our knowledge of pattern formation and tissue development in annelids and allies is scarce. In order to gain more data concerning neurogenesis and myogenesis in annelid trochophores, we analyzed different larval stages of Phyllodoce groenlandica using immunohistochemical staining techniques and subsequent confocal laser scanning microscopy (clsm). Focusing on pre-metamorphic stages, we reconstruct the process of tissue and body formation within planktonic polychaetous trochophore larvae. Our investigations revealed detailed knowledge of general developmental modes in Annelida and exhibit ontogenetic heterochrony of tissue development between two closely related annelid species. As such, P. groenlandica shows a delayed onset of nervous system development when compared with P. maculata. In contrast to the latter species, we were not able to detect the posterior sensory organ in larval P. groenlandica. We draw conclusions concerning general development of annelid trochophores and provide data showing new insights into the plasticity of body plans in Annelida.

Owenia fusiformis – a basally branching annelid suitable for studying ancestral features of annelid neural development

BackgroundComparative investigations on bilaterian neurogenesis shed light on conserved developmental mechanisms across taxa. With respect to annelids, most studies focus on taxa deeply nested within the annelid tree, while investigations on early branching groups are almost lacking. According to recent phylogenomic data on annelid evolution Oweniidae represent one of the basally branching annelid clades. Oweniids are thought to exhibit several plesiomorphic characters, but are scarcely studied - a fact that might be caused by the unique morphology and unusual metamorphosis of the mitraria larva, which seems to be hardly comparable to other annelid larva. In our study, we compare the development of oweniid neuroarchitecture with that of other annelids aimed to figure out whether oweniids may represent suitable study subjects to unravel ancestral patterns of annelid neural development. Our study provides the first data on nervous system development in basally branching annelids.ResultsBased on histology, electron microscopy and immunohistochemical investigations we show that development and metamorphosis of the mitraria larva has many parallels to other annelids irrespective of the drastic changes in body shape during metamorphosis. Such significant changes ensuing metamorphosis are mainly from diminution of a huge larval blastocoel and not from major restructuring of body organization. The larval nervous system features a prominent apical organ formed by flask-shaped perikarya and circumesophageal connectives that interconnect the apical and trunk nervous systems, in addition to serially arranged clusters of perikarya showing 5-HT-LIR in the ventral nerve cord, and lateral nerves. Both 5-HT-LIR and FMRFamide-LIR are present in a distinct nerve ring underlying the equatorial ciliary band. The connections arising from these cells innervate the circumesophageal connectives as well as the larval brain via dorsal and ventral neurites. Notably, no distinct somata with 5-HT -LIR in the apical organ are detectable in the larval stages of Owenia.Most of the larval neural elements including parts of the apical organ are preserved during metamorphosis and contribute to the juvenile nervous system.ConclusionsOur studies in Owenia fusiformis strongly support that early branching annelids are comparable to other annelids with regard to larval neuroanatomy and formation of the juvenile nervous system. Therefore, Owenia fusiformis turns out to be a valuable study subject for comparative investigations and unravelling ancestral processes in neural development in Annelida and Bilateria in general.