Transcriptomic landscape of posterior regeneration in the annelid Platynereis dumerilii.

BACKGROUND: Restorative regeneration, the capacity to reform a lost body part following amputation or injury, is an important and still poorly understood process in animals. Annelids, or segmented worms, show amazing regenerative capabilities, and as such are a crucial group to investigate. Elucidating the molecular mechanisms that underpin regeneration in this major group remains a key goal. Among annelids, the nereididae Platynereis dumerilii (re)emerged recently as a front-line regeneration model. Following amputation of its posterior part, Platynereis worms can regenerate both differentiated tissues of their terminal part as well as a growth zone that contains putative stem cells. While this regeneration process follows specific and reproducible stages that have been well characterized, the transcriptomic landscape of these stages remains to be uncovered. RESULTS: We generated a high-quality de novo Reference transcriptome for the annelid Platynereis dumerilii. We produced and analyzed three RNA-sequencing datasets, encompassing five stages of posterior regeneration, along with blastema stages and non-amputated tissues as controls. We included two of these regeneration RNA-seq datasets, as well as embryonic and tissue-specific datasets from the literature to produce a Reference transcriptome. We used this Reference transcriptome to perform in depth analyzes of RNA-seq data during the course of regeneration to reveal the important dynamics of the gene expression, process with thousands of genes differentially expressed between stages, as well as unique and specific gene expression at each regeneration stage. The study of these genes highlighted the importance of the nervous system at both early and late stages of regeneration, as well as the enrichment of RNA-binding proteins (RBPs) during almost the entire regeneration process. CONCLUSIONS: In this study, we provided a high-quality de novo Reference transcriptome for the annelid Platynereis that is useful for investigating various developmental processes, including regeneration. Our extensive stage-specific transcriptional analysis during the course of posterior regeneration sheds light upon major molecular mechanisms and pathways, and will foster many specific studies in the future.

On the hormonal control of posterior regeneration in the annelid Platynereis dumerilii.

Regeneration is the process by which many animals are able to restore lost or injured body parts. After amputation of the posterior part of its body, the annelid Platynereis dumerilii is able to regenerate the pygidium, the posteriormost part of its body that bears the anus, and a subterminal growth zone containing stem cells that allows the subsequent addition of new segments. The ability to regenerate their posterior part (posterior regeneration) is promoted, in juvenile worms, by a hormone produced by the brain and is lost when this hormonal activity becomes low at the time the worms undergo their sexual maturation. By characterizing posterior regeneration at the morphological and molecular levels in worms that have been decapitated, we show that the presence of the head is essential for multiple aspects of posterior regeneration, as well as for the subsequent production of new segments. We also show that methylfarnesoate, the molecule proposed to be the brain hormone, can partially rescue the posterior regeneration defects observed in decapitated worms. Our results are therefore consistent with a key role of brain hormonal activity in the control of regeneration and growth in P. dumerilii, and support the hypothesis of the involvement of methylfarnesoate in this control.

Studying Annelida Regeneration Using Platynereis dumerilii.

Regeneration, the ability to restore body parts after an injury or an amputation, is a widespread property in the animal kingdom. This chapter describes methods used to study this fascinating process in the annelid Platynereis dumerilii. During most of its life, this segmented worm is able to regenerate upon amputation the posterior part of its body, including its pygidium (terminal non-segmented body region bearing the anus) and a subterminal posterior growth zone which contains stem cells required for the formation of new segments. Detailed description of Platynereis worm culture and how to obtain large quantity of regenerating worms is provided. We also describe the staging system that we established and three important methods to study regeneration: whole mount in situ hybridization to study gene expression, 5-ethynyl-2'-deoxyuridine (EdU) labeling to characterize cell proliferation, and use of pharmacological treatments to establish putative roles of defined signaling pathways and processes.

Digital Marine: An online platform for blended learning in a marine experimental biology module, the Schmid Training Course.

For over 20 years, the Schmid Training Course (STC) has offered unique opportunities for marine biology students from European universities to learn about marine model organisms. While the topics of the course have continuously changed over the years with the advent of new research techniques and discoveries, the pedagogical approach has remained largely the same - a combination of lectures, lab practicals, and field excursions. Several life science researchers, who have taught in the STC for many years, sought to bring the course's pedagogical approach into the 21st century, and with the support of Erasmus+ Programme of the European Community funding, the Digital Marine project was developed. Digital Marine began in 2018 as an international partnership between the six research centers from which the STC instructors hail, and its main objective was to introduce a flipped, blended approach to learning and teaching with respect to established and emerging marine biological model systems. The Digital Marine platform, which covers 12 marine model organisms, is now publicly available.

A pan-metazoan concept for adult stem cells: the wobbling Penrose landscape.

Adult stem cells (ASCs) in vertebrates and model invertebrates (e.g. Drosophila melanogaster) are typically long-lived, lineage-restricted, clonogenic and quiescent cells with somatic descendants and tissue/organ-restricted activities. Such ASCs are mostly rare, morphologically undifferentiated, and undergo asymmetric cell division. Characterized by 'stemness' gene expression, they can regulate tissue/organ homeostasis, repair and regeneration. By contrast, analysis of other animal phyla shows that ASCs emerge at different life stages, present both differentiated and undifferentiated phenotypes, and may possess amoeboid movement. Usually pluri/totipotent, they may express germ-cell markers, but often lack germ-line sequestering, and typically do not reside in discrete niches. ASCs may constitute up to 40% of animal cells, and participate in a range of biological phenomena, from whole-body regeneration, dormancy, and agametic asexual reproduction, to indeterminate growth. They are considered legitimate units of selection. Conceptualizing this divergence, we present an alternative stemness metaphor to the Waddington landscape: the 'wobbling Penrose' landscape. Here, totipotent ASCs adopt ascending/descending courses of an 'Escherian stairwell', in a lifelong totipotency pathway. ASCs may also travel along lower stemness echelons to reach fully differentiated states. However, from any starting state, cells can change their stemness status, underscoring their dynamic cellular potencies. Thus, vertebrate ASCs may reflect just one metazoan ASC archetype.

The Nereid on the rise: Platynereis as a model system.

The Nereid Platynereis dumerilii (Audouin and Milne Edwards (Annales des Sciences Naturelles 1:195-269, 1833) is a marine annelid that belongs to the Nereididae, a family of errant polychaete worms. The Nereid shows a pelago-benthic life cycle: as a general characteristic for the superphylum of Lophotrochozoa/Spiralia, it has spirally cleaving embryos developing into swimming trochophore larvae. The larvae then metamorphose into benthic worms living in self-spun tubes on macroalgae. Platynereis is used as a model for genetics, regeneration, reproduction biology, development, evolution, chronobiology, neurobiology, ecology, ecotoxicology, and most recently also for connectomics and single-cell genomics. Research on the Nereid started with studies on eye development and spiralian embryogenesis in the nineteenth and early twentieth centuries. Transitioning into the molecular era, Platynereis research focused on posterior growth and regeneration, neuroendocrinology, circadian and lunar cycles, fertilization, and oocyte maturation. Other work covered segmentation, photoreceptors and other sensory cells, nephridia, and population dynamics. Most recently, the unique advantages of the Nereid young worm for whole-body volume electron microscopy and single-cell sequencing became apparent, enabling the tracing of all neurons in its rope-ladder-like central nervous system, and the construction of multimodal cellular atlases. Here, we provide an overview of current topics and methodologies for P. dumerilii, with the aim of stimulating further interest into our unique model and expanding the active and vibrant Platynereis community.

DNA methylation atlas and machinery in the developing and regenerating annelid Platynereis dumerilii.

BACKGROUND: Methylation of cytosines in DNA (5mC methylation) is a major epigenetic modification that modulates gene expression and constitutes the basis for mechanisms regulating multiple aspects of embryonic development and cell reprogramming in vertebrates. In mammals, 5mC methylation of promoter regions is linked to transcriptional repression. Transcription regulation by 5mC methylation notably involves the nucleosome remodeling and deacetylase complex (NuRD complex) which bridges DNA methylation and histone modifications. However, less is known about regulatory mechanisms involving 5mC methylation and their function in non-vertebrate animals. In this paper, we study 5mC methylation in the marine annelid worm Platynereis dumerilii, an emerging evolutionary and developmental biology model capable of regenerating the posterior part of its body post-amputation. RESULTS: Using in silico and experimental approaches, we show that P. dumerilii displays a high level of DNA methylation comparable to that of mammalian somatic cells. 5mC methylation in P. dumerilii is dynamic along the life cycle of the animal and markedly decreases at the transition between larval to post-larval stages. We identify a full repertoire of mainly single-copy genes encoding the machinery associated with 5mC methylation or members of the NuRD complex in P. dumerilii and show that this repertoire is close to the one inferred for the last common ancestor of bilaterians. These genes are dynamically expressed during P. dumerilii development and regeneration. Treatment with the DNA hypomethylating agent Decitabine impairs P. dumerilii larval development and regeneration and has long-term effects on post-regenerative growth. CONCLUSIONS: Our data reveal high levels of 5mC methylation in the annelid P. dumerilii, highlighting that this feature is not specific to vertebrates in the bilaterian clade. Analysis of DNA methylation levels and machinery gene expression during development and regeneration, as well as the use of a chemical inhibitor of DNA methylation, suggest an involvement of 5mC methylation in P. dumerilii development and regeneration. We also present data indicating that P. dumerilii constitutes a promising model to study biological roles and mechanisms of DNA methylation in non-vertebrate bilaterians and to provide new knowledge about evolution of the functions of this key epigenetic modification in bilaterian animals.

[A trio of mechanisms involved in regeneration initiation in animals]. / Un trio de mécanismes au cœur de l'initiationde la régénération chez les animaux.

Whole-body and complex structure regeneration is a widespread phenomenon in animals. While regenerative abilities vary greatly from one species to another, a number of mechanisms appear essential for regeneration in distantly related phylogenetic groups. In this review, we synthetize the knowledge gathered on the implication of three mechanisms that appear to be important for the initiation of regeneration in animals. Reactive Oxygen Species (ROS) are metabolic by-products involved in cell signalling, which are produced shortly after amputation in several species. ROS production may be responsible for triggering apoptosis, another recurring mechanism involved in regeneration initiation. In turn, apoptosis causes compensatory proliferation by setting off cellular division, thus contributing to the reconstitution of tissues. Inhibiting either ROS production, apoptosis or cellular proliferation impairs regeneration in a variety of model species.

TITLE: Un trio de mécanismes au cœur de l'initiationde la régénération chez les animaux. ABSTRACT: La régénération d'un membre ou du corps entier est un processus largement répandu chez les animaux. Même si les capacités régénératives varient d'une espèce à l'autre, trois mécanismes sont observés de façon récurrente lors de l'initiation de la régénération : la production de dérivés réactifs de l'oxygène (DRO), l'apoptose, et la prolifération cellulaire. De nombreux travaux ont mis en évidence le fait que ces trois mécanismes étaient nécessaires au bon déroulement de la régénération chez des espèces pourtant phylogénétiquement éloignées. Comprendre en détail les mécanismes moléculaires et cellulaires qui sous-tendent la coordination de ces trois processus dans l'initiation de la régénération pourrait aider à développer des thérapies pro-régénératives.

Animal regeneration in the era of transcriptomics.

Animal regeneration, the ability to restore a lost body part, is a process that has fascinated scientists for centuries. In this review, we first present what regeneration is and how it relates to development, as well as the widespread and diverse nature of regeneration in animals. Despite this diversity, animal regeneration includes three common mechanistic steps: initiation, induction and activation of progenitors, and morphogenesis. In this review article, we summarize and discuss, from an evolutionary perspective, the recent data obtained for a variety of regeneration models which have allowed to identify key shared mechanisms that control these main steps of animal regeneration. This review also synthesizes the wealth of high-throughput mRNA sequencing data (bulk mRNA-seq) concerning regeneration which have been obtained in recent years, highlighting the major advances in the regeneration field that these studies have revealed. We stress out that, through a comparative approach, these data provide opportunities to further shed light on the evolution of regeneration in animals. Finally, we point out how the use of single-cell mRNA-seq technology and integration with epigenomic approaches may further help researchers to decipher mechanisms controlling regeneration and their evolution in animals.

7th Euro Evo Devo meeting: Report on the "Evolution of regeneration in Metazoa" symposium.

Regeneration, the ability to restore lost parts of the body, is a widespread phenomenon in animals. While this ability is somehow limited in classical developmental model organisms, a variety of animals are able to regenerate complex structures such as limbs or important parts of their body, upon injury. Despite the recent emergence of regenerative studies using a large variety of metazoans, we still lack a general view of the evolution of animal regeneration. In the context of the 7th EvoDevo meeting that took place in June 2018 in Galway, Ireland, the "Evolution of regeneration in Metazoa" symposium gathered scientists studying the regenerative potential of evolutionarily distant animal species.

Morphological, cellular and molecular characterization of posterior regeneration in the marine annelid Platynereis dumerilii.

Regeneration, the ability to restore body parts after an injury or an amputation, is a widespread but highly variable and complex phenomenon in animals. While having fascinated scientists for centuries, fundamental questions about the cellular basis of animal regeneration as well as its evolutionary history remain largely unanswered. Here, we present a study of regeneration of the marine annelid Platynereis dumerilii, an emerging comparative developmental biology model, which, like many other annelids, displays important regenerative abilities. When P. dumerilii worms are amputated, they are able to regenerate the posteriormost differentiated part of their body and a stem cell-rich growth zone that allows the production of new segments replacing the amputated ones. We show that posterior regeneration is a rapid process that follows a well reproducible path and timeline, going through specific stages that we thoroughly defined. Wound healing is achieved one day after amputation and a regeneration blastema forms one day later. At this time point, some tissue specification already occurs, and a functional posterior growth zone is re-established as early as three days after amputation. Regeneration timing is only influenced, in a minor manner, by worm size. Comparable regenerative abilities are found for amputations performed at different positions along the antero-posterior axis of the worm, except when amputation planes are very close to the pharynx. Regenerative abilities persist upon repeated amputations without important alterations of the process. We also show that intense cell proliferation occurs during regeneration and that cell divisions are required for regeneration to proceed normally. Finally, 5-ethynyl-2'-deoxyuridine (EdU) pulse and chase experiments suggest that blastemal cells mostly derive from the segment immediately abutting the amputation plane. The detailed characterization of P. dumerilii posterior body regeneration presented in this article provides the foundation for future mechanistic and comparative studies of regeneration in this species.

The Notch pathway in the annelid Platynereis: insights into chaetogenesis and neurogenesis processes.

Notch is a key signalling pathway playing multiple and varied functions during development. Notch regulates the selection of cells with a neurogenic fate and maintains a pool of yet uncommitted precursors through lateral inhibition, both in insects and in vertebrates. Here, we explore the functions of Notch in the annelid Platynereis dumerilii (Lophotrochozoa). Conserved components of the pathway are identified and a scenario for their evolution in metazoans is proposed. Unexpectedly, neither Notch nor its ligands are expressed in the neurogenic epithelia of the larva at the time when massive neurogenesis begins. Using chemical inhibitors and neural markers, we demonstrate that Notch plays no major role in the general neurogenesis of larvae. Instead, we find Notch components expressed in nascent chaetal sacs, the organs that produce the annelid bristles. Impairing Notch signalling induces defects in chaetal sac formation, abnormalities in chaetae producing cells and a change of identity of chaeta growth accessory cells. This is the first bilaterian species in which the early neurogenesis processes appear to occur without a major involvement of the Notch pathway. Instead, Notch is co-opted to pattern annelid-specific organs, likely through a lateral inhibition process. These features reinforce the view that Notch signalling has been recruited multiple times in evolution due to its remarkable 'toolkit' nature.

The Conservation of the Germline Multipotency Program, from Sponges to Vertebrates: A Stepping Stone to Understanding the Somatic and Germline Origins.

The germline definition in metazoans was first based on few bilaterian models. As a result, gene function interpretations were often based on phenotypes observed in those models and led to the definition of a set of genes, considered as specific of the germline, named the "germline core". However, some of these genes were shown to also be involved in somatic stem cells, thus leading to the notion of germline multipotency program (GMP). Because Porifera and Ctenophora are currently the best candidates to be the sister-group to all other animals, the comparative analysis of gene contents and functions between these phyla, Cnidaria and Bilateria is expected to provide clues on early animal evolution and on the links between somatic and germ lineages. Our present bioinformatic analyses at the metazoan scale show that a set of 18 GMP genes was already present in the last common ancestor of metazoans and indicate more precisely the evolution of some of them in the animal lineage. The expression patterns and levels of 11 of these genes in the homoscleromorph sponge Oscarella lobularis show that they are expressed throughout their life cycle, in pluri/multipotent progenitors, during gametogenesis, embryogenesis and during wound healing. This new study in a nonbilaterian species reinforces the hypothesis of an ancestral multipotency program.

Oscarella lobularis (Homoscleromorpha, Porifera) Regeneration: Epithelial Morphogenesis and Metaplasia.

Sponges are known to possess remarkable reconstitutive and regenerative abilities ranging from common wounding or body part regeneration to more impressive re-building of a functional body from dissociated cells. Among the four sponge classes, Homoscleromorpha is notably the only sponge group to possess morphologically distinct basement membrane and specialized cell-junctions, and is therefore considered to possess true epithelia. The consequence of this peculiar organization is the predominance of epithelial morphogenesis during ontogenesis of these sponges. In this work we reveal the underlying cellular mechanisms used during morphogenesis accompanying ectosome regeneration in the homoscleromorph sponge model: Oscarella lobularis. We identified three main sources of novel exopinacoderm during the processes of its regeneration and the restoration of functional peripheral parts of the aquiferous system in O. lobularis: (1) intact exopinacoderm surrounding the wound surface, (2) the endopinacoderm from peripheral exhalant and inhalant canals, and (3) the intact choanoderm found on the wound surface. The basic morphogenetic processes during regeneration are the spreading and fusion of epithelial sheets that merge into one continuous epithelium. Transdifferentiation of choanocytes into exopinacocytes is also present. Epithelial-mesenchymal transition is absent during regeneration. Moreover, we cannot reveal any other morphologically distinct pluripotent cells. In Oscarella, neither blastema formation nor local dedifferentiation and proliferation have been detected, which is probably due to the high morphogenetic plasticity of the tissue. Regeneration in O. lobularis goes through cell transdifferentiation and through the processes, when lost body parts are replaced by the remodeling of the remaining tissue. Morphogenesis during ectosome regeneration in O. lobularis is correlated with its true epithelial organization. Knowledge of the morphological basis of morphogenesis during Oscarella regeneration could have important implications for our understanding of the diversity and evolution of regeneration mechanisms in metazoans, and is a strong basis for future investigations with molecular-biological approaches.

History of a prolific family: the Hes/Hey-related genes of the annelid Platynereis.

BACKGROUND: The Hes superfamily or Hes/Hey-related genes encompass a variety of metazoan-specific bHLH genes, with somewhat fuzzy phylogenetic relationships. Hes superfamily members are involved in a variety of major developmental mechanisms in metazoans, notably in neurogenesis and segmentation processes, in which they often act as direct effector genes of the Notch signaling pathway. RESULTS: We have investigated the molecular and functional evolution of the Hes superfamily in metazoans using the lophotrochozoan Platynereis dumerilii as model. Our phylogenetic analyses of more than 200 Metazoan Hes/Hey-related genes revealed the presence of five families, three of them (Hes, Hey and Helt) being pan-metazoan. Those families were likely composed of a unique representative in the last common metazoan ancestor. The evolution of the Hes family was shaped by many independent lineage specific tandem duplication events. The expression patterns of 13 of the 15 Hes/Hey-related genes in Platynereis indicate a broad functional diversification. Nevertheless, a majority of these genes are involved in two crucial developmental processes in annelids: neurogenesis and segmentation, resembling functions highlighted in other animal models. CONCLUSIONS: Combining phylogenetic and expression data, our study suggests an unusual evolutionary history for the Hes superfamily. An ancestral multifunctional annelid Hes gene may have undergone multiples rounds of duplication-degeneration-complementation processes in the lineage leading to Platynereis, each gene copies ensuring their maintenance in the genome by subfunctionalisation. Similar but independent waves of duplications are at the origin of the multiplicity of Hes genes in other metazoan lineages.

Posterior elongation in the annelid Platynereis dumerilii involves stem cells molecularly related to primordial germ cells.

Like most bilaterian animals, the annelid Platynereis dumerilii generates the majority of its body axis in an anterior to posterior temporal progression with new segments added sequentially. This process relies on a posterior subterminal proliferative body region, known as the "segment addition zone" (SAZ). We explored some of the molecular and cellular aspects of posterior elongation in Platynereis, in particular to test the hypothesis that the SAZ contains a specific set of stem cells dedicated to posterior elongation. We cloned and characterized the developmental expression patterns of orthologs of 17 genes known to be involved in the formation, behavior, or maintenance of stem cells in other metazoan models. These genes encode RNA-binding proteins (e.g., tudor, musashi, pumilio) or transcription factors (e.g., myc, id, runx) widely conserved in eumetazoans. Most of these genes are expressed both in the migrating primordial germ cells and in overlapping ring-like patterns in the SAZ, similar to some previously analyzed genes (piwi, vasa). The SAZ patterns are coincident with the expression of proliferation markers cyclin B and PCNA. EdU pulse and chase experiments suggest that new segments are produced through many rounds of divisions from small populations of teloblast-like posterior stem cells. The shared molecular signature between primordial germ cells and posterior stem cells in Platynereis thus corresponds to an ancestral "stemness" program.

Systematics and molecular phylogeny of the family oscarellidae (homoscleromorpha) with description of two new oscarella species.

The family Oscarellidae is one of the two families in the class Homoscleromorpha (phylum Porifera) and is characterized by the absence of a skeleton and the presence of a specific mitochondrial gene, tatC. This family currently encompasses sponges in two genera: Oscarella with 17 described species and Pseudocorticium with one described species. Although sponges in this group are relatively well-studied, phylogenetic relationships among members of Oscarellidae and the validity of genus Pseudocorticium remain open questions. Here we present a phylogenetic analysis of Oscarellidae using four markers (18S rDNA, 28S rDNA, atp6, tatC), and argue that it should become a mono-generic family, with Pseudocorticium being synonymized with Oscarella, and with the transfer of Pseudocorticium jarrei to Oscarella jarrei. We show that the genus Oscarella can be subdivided into four clades, each of which is supported by either a small number of morphological characters or by molecular synapomorphies. In addition, we describe two new species of Oscarella from Norwegian fjords: O. bergenensis sp. nov. and O. nicolae sp. nov., and we compare their morphology, anatomy, and cytology with other species in this genus. Internal anatomical characters are similar in both species, but details of external morphology and particularly of cytological characters provide diagnostic features. Our study also confirms that O. lobularis and O. tuberculata are two distinct polychromic sibling species. This study highlights the difficulties of species identification in skeleton-less sponges and, more generally, in groups where morphological characters are scarce. Adopting a multi-marker approach is thus highly suitable for these groups.

Involvement of the Wnt/ß-catenin pathway in neurectoderm architecture in Platynereis dumerilii.

Signalling pathways are essential for the correct development of the central nervous system (CNS) in bilaterian animals. Here we show that in the CNS of the annelid Platynereis dumerilii, neural progenitor cells (NPCs) are located close to the ventral midline and express axin, a negative regulator of the Wnt/ß-catenin pathway. Using pharmacological inhibitors, we observe that Wnt/ß-catenin is required for the transition between proliferating NPCs and differentiating neurons. We also show that the Rho-associated kinase (Rok) is necessary for neurectoderm morphogenesis and ventral midline formation, and indirectly affects the distribution of the NPCs and the development of axonal scaffolds. Moreover, seven genes belonging to the planar cell polarity (PCP) pathway are expressed in the developing Platynereis neurectoderm, suggesting an involvement in its morphogenesis. When compared with previous studies in vertebrates, our data suggest that the involvement of the Wnt/ß-catenin pathway in the control of neural cell proliferation/differentiation is ancestral to bilaterians.

Molecular phylogeny restores the supra-generic subdivision of homoscleromorph sponges (Porifera, Homoscleromorpha).

BACKGROUND: Homoscleromorpha is the fourth major sponge lineage, recently recognized to be distinct from the Demospongiae. It contains <100 described species of exclusively marine sponges that have been traditionally subdivided into 7 genera based on morphological characters. Because some of the morphological features of the homoscleromorphs are shared with eumetazoans and are absent in other sponges, the phylogenetic position of the group has been investigated in several recent studies. However, the phylogenetic relationships within the group remain unexplored by modern methods. METHODOLOGY/PRINCIPAL FINDINGS: Here we describe the first molecular phylogeny of Homoscleromorpha based on nuclear (18S and 28S rDNA) and complete mitochondrial DNA sequence data that focuses on inter-generic relationships. Our results revealed two robust clades within this group, one containing the spiculate species (genera Plakina, Plakortis, Plakinastrella and Corticium) and the other containing aspiculate species (genera Oscarella and Pseudocorticium), thus rejecting a close relationship between Pseudocorticium and Corticium. Among the spiculate species, we found affinities between the Plakortis and Plakinastrella genera, and between the Plakina and Corticium. The validity of these clades is furthermore supported by specific morphological characters, notably the type of spicules. Furthermore, the monophyly of the Corticium genus is supported while the monophyly of Plakina is not. CONCLUSIONS/SIGNIFICANCE: As the result of our study we propose to restore the pre-1995 subdivision of Homoscleromorpha into two families: Plakinidae Schulze, 1880 for spiculate species and Oscarellidae Lendenfeld, 1887 for aspiculate species that had been rejected after the description of the genus Pseudocorticium. We also note that the two families of homoscleromorphs exhibit evolutionary stable, but have drastically distinct mitochondrial genome organizations that differ in gene content and gene order.

Polyphyly of the genus Axinella and of the family Axinellidae (Porifera: Demospongiaep).

The genus Axinella is difficult to define on the basis of morphological characters and includes a heterogeneous assemblage of species. Several previous authors have suspected the polyphyly of both this genus and the family Axinellidae. To clarify the phylogeny of Axinellidae and Axinella, we propose a new hypothesis based on two molecular markers. In our analyses, Axinellidae and Axinella are polyphyletic assemblages. The 15 species of Axinellidae in our dataset belong to five clades and the nine species of Axinella to three clades. One Axinella clade, named Axinella(p), contains the type-species of the genus: A. polypoides (plus A. aruensis, A. dissimilis, A. infundibuliformis and A. vaceleti). A new clade, Cymbaxinella(p), is proposed, following the PhyloCode, it includes C. damicornis, C. verrucosa, C. corrugata and C. cantharella. The species Axinella cannabina is reallocated to a clade named Acanthella(p). The clades Agelas(p) and Cymbaxinella(p) constitute a new clade: Agelasida(p). Few morphological, biochemical and secondary structures characters support these groupings, highlighting the need for new characters for such problematic sponge groups. This work is an attempt to build a framework for the phylogeny of taxa allocated to Axinella and Axinellidae in the traditional classification.