Juan Junoy

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.

New insights into the phylogeny, systematics and DNA barcoding of Nemertea

Abstract. Although some clades of ribbon worms (phylum Nemertea) are consistently recovered with high support in molecular phylogenies, the placement and inter-relationships of some taxa have proven problematic. Herein, we performed molecular phylogenetic analyses aimed at resolving these recalcitrant splits, using six loci (nuclear 18S rRNA, 28S rRNA, histones H3 and H4, and mitochondrial 16S rRNA and COI) for 133 terminals, with particular emphasis on the problematic families Hubrechtidae and Plectonemertidae. Three different datasets were used for phylogenetic analyses and both maximum likelihood and maximum parsimony methodologies were applied. All but one of the resulting tree topologies agree on the paraphyly of the class Palaeonemertea, whereas Heteronemertea, Hoplonemertea, Polystilifera, Monostilifera and Hubrechtidae are always recovered as reciprocally monophyletic. Hubrechtidae is sister group to Heteronemertea (the Pilidiophora hypothesis) only when length variable regions of 18S rRNA and 28S rRNA are excluded. Moreover, the terrestrial and freshwater family Plectonemertidae is recovered with high support and the implications of this finding are further discussed. Finally, we evaluate the utility of DNA barcoding for specimen identification within Nemertea using an extended dataset containing 394 COI sequences. Results suggest that DNA barcoding may work for Nemertea, insofar as a distinct barcoding gap (the gap between the maximum intraspecific variation and the minimum interspecific divergence) may exist, but its recognition is regularly hampered by low accuracy in species level identifications.

Enzyme electrophoresis, genetic identity and description of a new genus and species of heteronemertean (Nemertea, Anopla) from northwestern Spain and North Wales

The anoplan order Heteronemertea, particularly the genera Cerebratulus, Lineus and Micrura, contains a very large number of nominate species, many of which are inadequately described. As a consequence, systematic difficulties are encountered with the identification of many taxa in this group, especially those originally established primarily on the basis of their external features. The present paper concerns heteronemerteans collected from two locations, the Foz Estuary (north-western Spain) and Llandudno (North Wales). The Spanish collection included specimens identified as Lineus longissimus (Gunnerus), whilst samples from Llandudno contained large numbers of Lineus viridis (Muller); samples of a third similar but apparently undescribed species were found at both locations. Starch gel electrophoresis showed that samples of the apparent third species were genetically almost identical from each of the two locations, but were clearly different from the two described Lineus species. Histological studies of the unknown specimens revealed anatomical characters, including the unique feature of a proboscis epithelium ciliated throughout its length, which exclude it from any known heteronemertean taxon; it is accordingly placed in a new genus and species, for which the name Riseriellus occultus is proposed.

Redescription of Lineus acutifrons Southern, 1913 (Nemertea: Pilidiophora) and comments on its phylogenetic position

The nemertean Lineus acutifrons Southern (1913) is a species rarely encountered in sandy beaches of Europe and is only really known from its original and short description. This species has been found almost a century later in Galician sandy beaches (northwest Spain) and is re-described here. Data detailing its internal morphology and external anatomy, and digital photographs are included. In addition, sequences of the ribosomal gene 28S and the mitochondrial gene cytochrome oxidase I (CO1) were obtained. These sequences were used in a phylogenetic analysis to test its phylogenetic position with respect to other sequences available for pilidiophorans.

Mating behavior of nemerteans: present knowledge and future directions

In most nemertean species, members of the two sexes aggregate before fertilization takes place. Few specific studies on the mating behaviour of nemerteans have been conducted but several observational reports indicate that important processes known from other organisms, such as sexual selection and sperm competition, may also be at work in nemerteans. Herein, we review some of these observations and discuss their possible implications. We produce a summary table and reproduce some important observations, placing them in an evolutionary context. Four types of gamete‐transfer mechanisms are distinguished: (1) free‐spawning, where members of both sexes release gametes freely into the water column; (2) mucus‐spawning, where gametes are released within a mucus matrix; (3) internal fertilization, where spermatozoans are transferred to the immediate vicinity of oviducts, which they penetrate; and (4) gamete transfer aided by specific structures. While little is known about the last transfer mechanism, anecdotal observations are mainly available for the first three mechanisms. Mating interactions frequently involve more than two individuals. In several species, individuals only invest part of their gametes in a single mating interaction, apparently saving additional gametes for potential future mating events. These observations suggest that males of some species are exposed to mating competition. Males might counteract sperm competition by producing large numbers of sperm, as suggested by their high fertilization potential. Previous authors inferred that sperm morphology may be an adaptation to spawning behavior and possibly also to the risk of sperm competition. Based on the results of our analysis and the above observations, we suggest that sexual selection and sperm competition are not uncommon in nemerteans.

On the identity of two Antarctic brooding nemerteans: redescription of Antarctonemertes valida (Burger, 1893) and description of a new species in the genus Antarctonemertes Friedrich, 1955 (Nemertea, Hoplonemertea)

Nemerteans (ribbon worms) constitute an abundant and occasionally conspicuous group of benthic invertebrates in the Southern Ocean. Although recent work has confirmed that this group is far more diverse than previously recognized, the Antarctic nemertean fauna remains poorly understood when compared to other geographic regions. In most cases, the taxonomic information on the known nemertean fauna is incomplete for this region and/or has been inappropriately documented. As a consequence, many of the species described are considered species inquirendae. Among the nearly 50 species described so far for the Southern Ocean, two hoplonemerteans are known to brood eggs in cocoons: Amphiporus incubator Joubin, 1914 and Amphiporus michaelseni Bürger, 1895a. Here, we redescribe Antarctonemertes valida (Bürger, 1893), a senior synonym of A. michaelseni, and describe a new congeneric species, Antarctonemertes riesgoae sp. nov. Both species show a similar reproductive strategy by brooding their cocoons, and similar external appearance, but clearly differ in other aspects of their morphology, such as the cephalic coloration pattern and the number of proboscidial nerves. We provide novel information about their life habitus, reproductive behaviour, internal anatomy, and their phylogenetic placement within hoplonemerteans using one nuclear (28S rRNA) and two mitochondrial [cytochrome c oxidase subunit I (COI) and 16S rRNA] markers. We also provide a parsimony haplotype network using 16S rRNA, COI, and the internal transcribed spacer region 2 (ITS-2) showing a clear distinction between individuals of both species. Our results stress the need of combining molecular and morphological information when dealing with closely related species of nemerteans.

Ultrastructural study of two glandular systems in the proboscidial glandular epithelium of Riseriellus occultus (Nemertea, Heteronemertea)

Abstract Two different types of glandular system in the proboscidial epithelium of Riseriellus occultus have been investigated by transmission electron microscopy. As expected, most of the epithelial cells are glandular in nature. With regard to differences in the ultrastructure of these gland cells and in the formation and morphology of their secretory granules, we have categorized and described four types of gland cell, indicated as G1, G2, G3, and G4. Each gland cell has a completely intraepithelial body characterized by a prominent nucleus, developed rough endoplasmic reticulum, Golgi complexes, and numerous secretory granules at different stages of maturation. These four types of gland cell appear associated in pairs forming numerous glandular systems of two types (A, B). These glandular systems are restricted to the ventral surface of the proboscis and are scattered irregularly throughout its length. Each glandular system consists of two gland cells of different types. The gland cell necks in each glandular system extend together to the epithelial surface; they protrude onto this and form a papilla where they open in a common area. The epithelial supportive cells adjacent to the glandular systems have long, stout microvilli which have a core of tonofilaments. These tonofilaments gather into dense bundles which pass vertically through the supportive cells and attach to the extracellular matrix underlaying the cells by hemidesmosomes. Moreover, a single sensory process stands close to each papilla. The ultrastructural morphology of the type A glandular systems suggests that they have an adhesive function operating in a similar way to that of the duo-gland adhesive systems in other invertebrate groups, although they are not homologous with these. The spatial arrangement of the secreted products of the type B glandular systems suggests that these may contribute to increasing the grip of the proboscis on the prey. The secretory granules (=pseudocnids) of the type G3 gland cells are very likely an autapomorphy of the Anopla, providing a character by which the relationships within the Nemertea can be evaluated.

Ultrastructural study of sensory cells of the proboscidial glandular epithelium of Riseriellus occultus (Nemertea, Heteronemertea)

Only one sensory cell type has been observed within the glandular epithelium of the proboscis in the heteronemertine Riseriellus occultus. These bipolar cells are abundant and scattered singly throughout the proboscis length. The apical surface of each dendrite bears a single cilium enclosed by a ring of six to eight prominent microvilli. The cilium has the typical 9×2 + 2 axoneme arrangement and is equipped with a cross‐striated vertical rootlet extending from the basal body. No accessory centriole or horizontal rootlet was observed. Large, modified microvilli (stereovilli) surrounding the cilium are joined together by a system of fine filaments derived from the glycocalyx. Each microvillus contains a bundle of actin‐like filaments which anchor on the indented inner surface of a dense, apical ring situated beneath the level of the ciliary basal body. The tip of the cilium is expanded and modified to form a bulb‐like structure which lies above the level where the surrounding microvilli terminate. In the region where the cilium emerges from the microvillar cone, the membrane of the microvillar apices makes contact with a corresponding portion of the ciliary membrane. At this level microvilli and cilium are apparently firmly linked by junctional systems resembling adherens junctions. The results suggest that these sensory cells may be mechanoreceptors.

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

Ultrastructural study of the proboscis endothelium of Riseriellus occultus (Nemertea, Heteronemertea)

We have examined with transmission electron microscopythe epithelial layer exposed to the rhynchocoel fluidof the proboscis in the heteronemertine Riseriellus occultus. This epithelium is organized asa monociliated, pseudostratified myoepitheliumconsisting of two cell types: apically situatedmonociliated supportive cells and subapical myocyteslacking cilia. The low supportive cells form acontinuous adluminal sheet and reach with numerouscytoplasmic processes into the extracellular matrix;these cells are characterized by numerous, irregularlyshaped, apical folds projecting into the rhynchocoelfluid, delimiting broad extracellular spaces. Theauthors suppose that both apical and basal folds couldaccommodate stretching of the endothelium when theproboscis is everted. The apical folds of thesupportive cells increase the interface of these withthe rhynchocoel fluid; this feature, together with thepresence of pinocytotic vesicles in such cells,suggest that they could be involved in the exchange ofsubstances between the rhynchocoel fluid and theproboscis. The myocytes are scattered singly withinthe monociliated pseudostratified myoepithelium. Theyare situated between the supportive cells and thesubjacent extracellular matrix. Basement membraneseparating both cells types is lacking. Myofibrillarparts protrude basally from the myocyte somata. Themyofibrillar parts lie in direct apposition to theextracellular matrix, and are oriented circular to thelongitudinal axis of the proboscis. We consider themyocytes to be intra-epithelial, myoepithelial cells.