Jörn von Döhren

Disentangling Ribbon Worm Relationships: Multi-Locus Analysis Supports Traditional Classification of the Phylum Nemertea

The phylogenetic relationships of selected members of the phylum Nemertea are explored by means of six markers amplified from the genomic DNA of freshly collected specimens (the nuclear 18S rRNA and 28S rRNA genes, histones H3 and H4, and the mitochondrial genes 16S rRNA and cytochrome c oxidase subunit I). These include all previous markers and regions used in earlier phylogenetic analyses of nemerteans, therefore acting as a scaffold to which one could pinpoint any previously published study. Our results, based on analyses of static and dynamic homology concepts under probabilistic and parsimony frameworks, agree in the non‐monophyly of Palaeonemertea and in the monophyly of Heteronemerta and Hoplonemertea. The position of Hubrechtella and the Pilidiophora hypothesis are, however, sensitive to analytical method, as is the monophyly of the non‐hubrechtiid palaeonemerteans. Our results are, however, consistent with the main division of Hoplonemertea into Polystilifera and Monostilifera, the last named being divided into Cratenemertea and Distromatonemertea, as well as into the main division of Heteronemertea into Baseodiscus and the remaining species. The study also continues to highlight the deficient taxonomy at the family and generic level within Nemertea and sheds light on the areas of the tree that require further refinement.

Phylogeny and mitochondrial gene order variation in Lophotrochozoa in the light of new mitogenomic data from Nemertea

BackgroundThe new animal phylogeny established several taxa which were not identified by morphological analyses, most prominently the Ecdysozoa (arthropods, roundworms, priapulids and others) and Lophotrochozoa (molluscs, annelids, brachiopods and others). Lophotrochozoan interrelationships are under discussion, e.g. regarding the position of Nemertea (ribbon worms), which were discussed to be sister group to e.g. Mollusca, Brachiozoa or Platyhelminthes. Mitochondrial genomes contributed well with sequence data and gene order characters to the deep metazoan phylogeny debate.ResultsIn this study we present the first complete mitochondrial genome record for a member of the Nemertea, Lineus viridis. Except two trnP and trnT, all genes are located on the same strand. While gene order is most similar to that of the brachiopod Terebratulina retusa, sequence based analyses of mitochondrial genes place nemerteans close to molluscs, phoronids and entoprocts without clear preference for one of these taxa as sister group.ConclusionAlmost all recent analyses with large datasets show good support for a taxon comprising Annelida, Mollusca, Brachiopoda, Phoronida and Nemertea. But the relationships among these taxa vary between different studies. The analysis of gene order differences gives evidence for a multiple independent occurrence of a large inversion in the mitochondrial genome of Lophotrochozoa and a re-inversion of the same part in gastropods. We hypothesize that some regions of the genome have a higher chance for intramolecular recombination than others and gene order data have to be analysed carefully to detect convergent rearrangement events.

The fate of the larval epidermis in the Desor-larva of Lineus viridis (Pilidiophora, Nemertea) displays a historically constrained functional shift from planktotrophy to lecithotrophy

Pilidiophora constitutes a clade of nemerteans characterized by a peculiar larval type, the pilidium. A characteristic of this larva is the transitory epidermis in which the juvenile develops from imaginal discs. The primary function of this larval envelope is assumed to be feeding and dispersal. When juvenile development is complete, the larval epidermis is ruptured and swallowed by the juvenile. According to recent cladistic and molecular analyses of the Nemertea, the intracapsular Desor-larva of the sibling species Lineus viridis and L. ruber is thought to have evolved from a pelagic pilidium. The general course of development has been demonstrated to be similar to that of the pilidium, in which the juvenile forms from imaginal discs under the larval epidermis. The two Lineus species, however, differ in their mode of larval feeding: L. ruber being ootrophic and L. viridis being lecithotrophic. In order to elucidate the transition from the planktotrophic pilidum to lecithotrophic development, I studied the early cleavage and metamorphosis from intracapsular Desor-larva to juvenile stages in L. viridis from the island of Sylt, using light microscopical, electron microscopical, and fluorescent staining methods. Due to the specific cleavage pattern with equally sized 1st quartet animal blastomeres and vegetal blastomeres in L. viridis, the larval epidermis later contains a considerable amount of the yolk reserve. During metamorphosis, the larval epidermis is ingested by the juvenile thus displaying behavior similar to that of the pilidium larva. In contrast to the pilidium, the function of the larval epidermis of the Desor-larva has shifted from feeding and dispersal to direct food supply. Thus, the development of L. viridis is a perfect example for strong historical constraints that prevent ancestral larval structures from being lost.

Ultrastructure of sperm and male reproductive system in Lineus viridis (Heteronemertea, Nemertea)

Nemerteans possess serially arranged gonads that lie between the midgut pouches. In both sexes the gonads are lined with an epithelium. During maturity, they gain contact to the exterior by a ciliated duct, which is generally assumed to be a derivative of the gonad. Gonad lining and sperm ultrastructure are little known in heteronemerteans, a group of nemerteans belonging to the Anopla, one of the two large nemertean subgroups. Reproduction biology in heteronemertean Lineus viridis allows predicting a modified sperm type, so-called introsperm for this taxon. Nothing is known on the fate of the testes at the end of the reproductive period of this perennial species. In order to test the predictions and to broaden the data base, males of L. viridis were collected at different times of the year. Histological and ultrastructural data show that the gonad wall is lined with different aciliated endothelial cells and germ cells, while the gonoduct is formed by densely ciliated cells. The testes are completely filled with sperm cells during maturity; there is no hint at ongoing spermiogenesis at this time. The sperm consists of head, midpiece and tail. Externally, head and midpiece cannot be discriminated. The acrosome is cup-shaped and lies anterior to the nucleus which contains 6–8 lateral ridges. Three long mitochondria mark the midpiece. They line the posterior section of the nucleus and extend up to the level of the ciliary basal structures. The sperm morphology corroborates the predictions derived from the mode of reproduction. At the end of the reproductive period the male gonads change cellular composition, while the gonoduct degenerates. Provided that both sexes show the same growth rate, male offspring acquire sexual maturity earlier than female offspring, since L. viridis males are always smaller than the females. In contrast to the males, females keep their gonads and gonoducts during most time of the year. Since large males were never found within the studied population, these data indicate that L. viridis might be a consecutive hermaphrodite.

The future of nemertean taxonomy (phylum Nemertea) — a proposal

Submitted: 15 January 2016 Accepted: 6 March 2016 doi:10.1111/zsc.12182 Sundberg, P., Andrade, S.C.S., Bartolomaeus, T., Beckers, P., von D€ ohren, J., Kr€amer, D., Gibson, R., Giribet, G., Herrera-Bachiller, A., Juan, J., Kajihara, H., Kvist, S., K anneby, T., Sun S.-C., Thiel, M., Turbeville, J.M. , Strand, M. (2016). The future of nemertean taxonomy (phylum Nemertea) — a proposal. —Zoologica Scripta, 45: 579–582. Corresponding author: Per Sundberg, University of Gothenburg, Department of Marine Sciences, Gothenburg, Sweden. E-mail: per.sundberg@gu.se Per Sundberg, University of Gothenburg, Department of Marine Sciences, Gothenburg, Sweden.. E-mail: per.sundberg@gu.se S onia C. S. Andrade, Departamento de Gen etica e Biologia Evolutiva, IB-Universidade de, S~ao Paulo, Brazil, S~ao Paulo, Brazil. E-mail: soniacsandrade@gmail.com Thomas Bartolomaeus, Patrick Beckers, J€orn von D€ohren, and Daria Kr€amer, University of Bonn, Institute of Evolutionary Biology and Animal Ecology, Bonn, Germany. E-mails: tbartolomaeus@evolution.uni-bonn.de, pbeckers@evolution.uni-bonn.de, jdoehren@evolution.uni-bonn.de, dkraemer@evolution.uni-bonn.de Ray Gibson, 94 Queens Avenue, Meols, Wirral, CH47 0NA, U.K. E-mail: bfginc@hotmail.co.uk Gonzalo Giribet, Museum of Comparative Zoology, Department of Organismic and Evolutionary Biology, Harvard University, Cambridge, MA, USA. E-mail: ggiribet@g.harvard.edu Alfonso Herrera-Bachiller, and Juan Junoy, Departamento de Ciencias de la Vida, Universidad de Alcal a, Madrid, Spain. E-mails: alfonso@herrerabachiller.com, juan.junoy@uah.es Hiroshi Kajihara, Faculty of Science, Hokkaido University, Sapporo, Japan. E-mail: kazi@mail.sci.hokudai.ac.jp Sebastian Kvist, Department of Natural History, Royal Ontario Museum, Toronto, Canada and Department of Ecology and Evolutionary Biology, University of Toronto, Toronto, Canada. E-mail: sebastian.kvist@utoronto.ca Tobias K anneby, Swedish Museum of Natural History, Department of Zoology, Stockholm, Sweden. E-mail: Tobias.Kanneby@nrm.se, tobiaskanneby@gmail.com Shi-Chun Sun, Institute of Evolution & Marine Biodiversity, Ocean University of China, Qingdao, China. E-mail: sunsc@ouc.edu.cn Martin Thiel, Facultad Ciencias del Mar, Centro de Estudios Avanzados en Zonas Áridas (CEAZA), Millennium Nucleus Ecology and Sustainable Management of Oceanic Island (ESMOI), Universidad Cat olica del Norte, Coquimbo, Chile. E-mail: thiel@ucn.cl James M. Turbeville, Department of Biology, Virginia Commonwealth University, Richmond, VA, USA. E-mail: jmturbeville@vcu.edu Malin Strand, Swedish Species Information Centre, The Sven Lov en Centre for Marine Sciences, Str€omstad, Sweden. E-mail: malin.strand@slu.se

Phylogenetic significance of chaetal arrangement and chaetogenesis in Maldanidae (Annelida)

Chaetae are important structures to facilitate locomotion in annelids. Being at the interface between the organisms and its environment, chaetae are supposed to underlie strong functional constraints to optimize the relation between structure and function. As such chaetae are potentially susceptible for convergent evolution. On the other hand, chaetae gained enormous taxonomic importance due to their conservative structure in species and supraspecific taxa which reasonably can only be explained by strong evolutionary constrains that conserve their structure. In this paper, we study the chaetation and chaetogenesis in two species of Maldanidae, Clymenura clypeata Saint-Joseph 1894 and Johnstonia clymenoides Quatrefages 1866 to unravel conservative traits in their structure and development. In a literature survey across maldanids, we address questions on the ontogenetic variation, on homology and on the phylogenetic significance especially of the bearded hooked neurochaetae. We provide evidence that functionally constraint ontogenetic variation overlies historically (phylogenetically) constraint expression of structural information and can show that within maldanids a variety of different chaetal types must be homologous due to their ontogenetic continuity. Furthermore, we use chaetation and chaetal characters to discuss the subgroup relationships within Maldanomorpha in the light of recent cladistics analyses based on morphological and molecular data. This study shows that functional considerations need to use phylogenies as backbone.

Comparative morphology and evolution of the nephridia in Nemertea

In various discussions on the phylogenetic position of the Nemertea, nephridial morphology seems to support current hypotheses for a close relationship to lophotrochozoan subtaxa. These arguments are based on isolated findings and suffer from the lack of a phylogeny-based inference. In order to fill in this gap, the structure of the nemertean nephridia is reviewed. Based on the structural data presently available for nemertean nephridia, 30 characters are described. Their ancestral states are inferred from cladograms derived from recent molecular analyses. According to these, paired protonephridia restricted to the foregut region and associated with the blood vascular system represent the primary state in nemerteans. The terminal cells are covered by the blood vessel endothelium and have no open connection to the lumen of the blood vascular system. Serially arranged protonephridia that could support the hypothesis of an annelid–nemertean–relationship are either apomorphic for Cephalothricidae, a nemertean subgroup, or a cephalothricid ingroup.

Observations and Experiments on the Biology and Life History of Riseriellus occultus (Heteronemertea: Lineidae)

Studies on the biology and life history of nemerteans are scarce, mostly because these animals are nocturnal. In order to broaden the knowledge base on the life history of nemerteans as a prerequisite for comparative analyses, we studied a population of Riseriellus occultus (Heteronemertea: Lineidae) inhabiting the rocky intertidal in southern Brittany near Concarneau (France) for more than 10 years. Our studies show that R. occultus is an iteroparous, perennial species exclusively inhabiting rocky shore crevices that result from onionskin weathering of the granite. From September through October R. occultus reproduces by external fertilization and develops via a planktonic pilidium larva, which, under laboratory conditions, metamorphoses after about six weeks. Adults of R. occultus are nocturnal macrophagous predators that preferentially feed on the gastropods Gibbula umbilicalis and Patella species, but also consume the bivalve Mytilus edulis. Since R. occultus devours the snail inside the shell, we fixed individuals while feeding, and serially sectioned them. Reconstruction of the sections shows that R. occultus swallows the entire soft body and finally detaches the columellar muscle from the shell. Estimates on the density of R. occultus inside the rock crevices provide evidence for clustered distribution and locally high abundance on the rocky shore. These data strongly suggest that R. occultus affects the structure of the rocky shore gastropod community. Although our data are still fragmentary with respect to the ecology of this species and its role in the local food web, our knowledge has grown to such extent that R. occultus can now be regarded as one of the few well characterized nemertean species.

Unravelling the Lineus ruber/viridis species complex (Nemertea, Heteronemertea)

Lineus ruber (Müller, ) and Lineus viridis (Müller, ) are among the longest known and most abundant intertidal nemertean species found on both sides of the North Atlantic. Due to easy maintenance in captivity, both species have been well studied concerning morphology, reproduction, development and ecology. Originally described as two separate species in the 18th century, they were subsequently synonymised and considered colour varieties of a single species. It was not until the mid‐20th century that complementary redescriptions, including information on reproduction and development, re‐established the specific identities of L. ruber and L. viridis. With the advent of molecular markers in nemertean systematics, however, doubt was again cast on their specific identities. To solve one of the longest standing problems in nemertean systematics, we assembled a comprehensive data set combining external morphology and three genetic markers (mitochondrial cytochrome c oxidase subunit I, 16S rRNA and nuclear internal transcribed spacer region) from 160 specimens of L. ruber and L. viridis collected at six sampling sites along the continental coast of Western Europe. The data set was analysed with tree‐based and non‐tree‐based species delimitation methods. The results from all methods used confidently delimit three separate clades. A distinct barcoding gap was detected in our data set which thus provides a framework to unequivocally identify specimens as members of any of the three species. Comparison of our findings with published data enables us to assign one lineage to L. ruber and one to L. viridis. We designated neotypes for both species. The third clade is very similar to L. viridis, only distinguishable by a conspicuous, iridescent ventral fold in some male specimens This lineage shows a syntopic distribution along western European coasts and represents a species new to science and is described as Lineus clandestinus sp. n. based on its external characters and the analyses of the molecular data provided in this study.

Development of the Nervous System of Carinina ochracea (Palaeonemer-tea, Nemertea)

The various clades of Lophotrochozoa possess highly disparate adult morphologies. Most of them, including Nemertea (ribbon worms), are postulated to develop via a pelagic larva of the trochophora type, which is regarded as plesiomorphic in Lophotrochozoa. With respect to the nervous system, the trochophora larva displays a set of stereotypic features, including an apical organ and trochal neurites, both of which are lost at the onset of metamorphosis. In the investigated larvae of Nemertea, the nervous system is somewhat divergent from the postulated hypothetical trochophore-like pattern. Moreover, no detailed data is available for the “hidden” trochophore larva, the hypothetical ancestral larval type of palaeonemertean species. Therefore, the development of the nervous system in the larva of Carinina ochracea, a basally branching palaeonemertean species, was studied by means of immunofluorescence and confocal laserscanning microscopy. Like in the other investigated nemertean larvae, the prospective adult central nervous system in C. ochracea develops in an anterior to posterior direction, as an anterior brain with paired longitudinal nerve cords. Thus, development of the adult nervous system in Nemertea is largely congruent with currently accepted hypotheses of nervous system development in Spiralia. In early development, transitory apical, serotonin-like immunoreactive flask-shaped cells are initially present, but the trochal neurites that have been considered as pivotal to lophotrochozoan development, are absent. In the light of the above stated hypothesis, trochal neurites have to be interpreted as reduced in Nemertea. On the other hand, due to the unsettled systematic status of Palaeonemertea, more comparative data are desirable to answer the remaining questions regarding the evolution of nervous system development in Nemertea.