Hiroshi Kajihara

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.

Statistical Parsimony Networks and Species Assemblages in Cephalotrichid Nemerteans (Nemertea)

Background It has been suggested that statistical parsimony network analysis could be used to get an indication of species represented in a set of nucleotide data, and the approach has been used to discuss species boundaries in some taxa. Methodology/Principal Findings Based on 635 base pairs of the mitochondrial protein-coding gene cytochrome c oxidase I (COI), we analyzed 152 nemertean specimens using statistical parsimony network analysis with the connection probability set to 95%. The analysis revealed 15 distinct networks together with seven singletons. Statistical parsimony yielded three networks supporting the species status of Cephalothrix rufifrons, C. major and C. spiralis as they currently have been delineated by morphological characters and geographical location. Many other networks contained haplotypes from nearby geographical locations. Cladistic structure by maximum likelihood analysis overall supported the network analysis, but indicated a false positive result where subnetworks should have been connected into one network/species. This probably is caused by undersampling of the intraspecific haplotype diversity. Conclusions/Significance Statistical parsimony network analysis provides a rapid and useful tool for detecting possible undescribed/cryptic species among cephalotrichid nemerteans based on COI gene. It should be combined with phylogenetic analysis to get indications of false positive results, i.e., subnetworks that would have been connected with more extensive haplotype sampling.

A Transcriptomic Approach to Ribbon Worm Systematics (Nemertea): Resolving the Pilidiophora Problem

Resolving the deep relationships of ancient animal lineages has proven difficult using standard Sanger-sequencing approaches with a handful of markers. We thus reassess the relatively well-studied phylogeny of the phylum Nemertea (ribbon worms)-for which the targeted gene approaches had resolved many clades but had left key phylogenetic gaps-by using a phylogenomic approach using Illumina-based de novo assembled transcriptomes and automatic orthology prediction methods. The analysis of a concatenated data set of 2,779 genes (411,138 amino acids) with about 78% gene occupancy and a reduced version with 95% gene occupancy, under evolutionary models accounting or not for site-specific amino acid replacement patterns results in a well-supported phylogeny that recovers all major accepted nemertean clades with the monophyly of Heteronemertea, Hoplonemertea, Monostilifera, being well supported. Significantly, all the ambiguous patterns inferred from Sanger-based approaches were resolved, namely the monophyly of Palaeonemertea and Pilidiophora. By testing for possible conflict in the analyzed supermatrix, we observed that concatenation was the best solution, and the results of the analyses should settle prior debates on nemertean phylogeny. The study highlights the importance, feasibility, and completeness of Illumina-based phylogenomic data matrices.

Molecular phylogeny of kinorhynchs.

We reconstructed kinorhynch phylogeny using maximum-likelihood and Bayesian analyses of nuclear 18S and 28S rRNA gene sequences from 30 species in 13 genera (18S) and 23 species in 12 genera (28S), representing eight families and both orders (Cyclorhagida and Homalorhagida) currently recognized in the phylum. We analyzed the two genes individually (18S and 28S datasets) and in combination (18S+28S dataset). We detected four main clades (I-IV). Clade I consisted of family Echinoderidae. Clade II contained representatives of Zelinkaderidae, Antygomonidae, Semnoderidae, Centroderes, and Condyloderes, the latter two currently classified in Centroderidae; within Clade II, Zelinkaderidae, Antygomonidae, and Semnoderidae comprised a clade with strong nodal support. Clade III contained only two species in Campyloderes, also currently classified in the Centroderidae, indicating polyphyly for this family. Clades I-III, containing all representatives of Cyclorhagida included in the analysis except for Dracoderes abei, formed a clade with high nodal support in the 28S and 18S+28S trees. Clade IV, resolved in the 18S and 18S+28S trees with high nodal support, contained only species in order Homalorhagida, with the exception of the cyclorhagid Dracoderes abei. Order Cyclorhagida as it currently stands is thus polyphyletic, and order Homalorhagida paraphyletic. Our results indicate that Dracoderidae has been misplaced in Cyclorhagida based on homoplasious characters. Our analyses did not resolve the relationships among Clades I-III within Cyclorhagida. Neither gene alone nor the combined dataset resolved all nodes in trees, indicating that additional markers will be needed to reconstruct kinorhynch phylogeny.

Highly Toxic Ribbon Worm Cephalothrix simula Containing Tetrodotoxin in Hiroshima Bay, Hiroshima Prefecture, Japan

In 1998, during a toxicological surveillance of various marine fouling organisms in Hiroshima Bay, Japan, specimens of the ribbon worm, Cephalothrix simula (Nemertea: Palaeonemertea) were found. These ribbon worms contained toxins with extremely strong paralytic activity. The maximum toxicity in terms of tetrodotoxin (TTX) was 25,590 mouse units (MU) per gram for the whole worm throughout the monitoring period. The main toxic component was isolated and recrystallized from an acidified methanolic solution. The crystalline with a specific toxicity of 3520 MU/mg was obtained and identified as TTX by high performance liquid chromatography (HPLC)-fluorescent detection (FLD) (HPLC-FLD), electrospray ionization-mass spectrometry (ESI-MS), infrared (IR), nuclear magnetic resonance (NMR) and gas chromatography–mass spectrometry (GC-MS). The highest toxicity of C. simula exceeded the human lethal dose per a single worm. A toxicological surveillance of C. simula from 1998 to 2005 indicated approximately 80% of the individuals were ranked as “strongly toxic” (≥1000 MU/g). Forty-eight percent of the specimens possessed toxicity scores of more than 2000 MU/g. Seasonal variations were observed in the lethal potency of C. simula. Specimens collected on January 13, 2000 to December 26, 2000 showed mean toxicities of 665–5300 MU/g (n = 10). These data prompted a toxicological surveillance of ribbon worms from other localities with different habitats in Japan, including Akkeshi Bay (Hokkaido) under stones on rocky intertidal beaches, as well as Otsuchi (Iwate) among calcareous tubes of serpulid polychaetes on rocky shores. Within twelve species of ribbon worms examined, only C. simula possessed extremely high toxicity. Therefore, C. simula appears to show generally high toxicity irrespective of their locality and habitat.

Molecular Systematics of Tanaidacea (Crustacea: Peracarida) Based on 18S Sequence Data, with an Amendment of Suborder/Superfamily-Level Classification

Phylogenetic relationships within Tanaidacea were analyzed based on sequence data for the 18S rRNA gene. Our results strongly supported a monophyletic group composed of Neotanaidae, Tanaoidea, and Paratanaoidea, with the first two taxa forming a clade. These results contradict three previously suggested hypotheses of relationships. Based on the molecular results, and considering morphological similarities/differences between Neotanaidomorpha and Tanaidomorpha, we demoted Suborder Neotanaidomorpha to Superfamily Neotanaoidea within Tanaidomorpha; with this change, the classification of extant tanaidaceans becomes a two-suborder, four-superfamily system. This revision required revision of the diagnoses for Tanaidomorpha and its three super-families. The results for Apseudomorpha were ambiguous: this taxon was monophyletic in the maximum likelihood and Bayesian analyses, but paraphyletic in the maximum parsimony and minimum evolution analyses.

A new genus and species of monostiliferous hoplonemertean (Nemertea: Enopla: Monostilifera) from Japan

A new genus and species of monostiliferous hoplonemertean, Diopsonemertes acanthocephala gen. et sp. nov., is described from Otsuchi Bay, Japan. Significant anatomical features of the new form include a body wall longitudinal musculature anteriorly divided into inner and outer layers by connective tissue, no pre-cerebral septum, the presence of a thin coat of diagonal muscle fibres between the body wall longitudinal and circular muscle layers in the foregut body region, cephalic retractor muscles derived only from the inner portion of the divided longitudinal muscles and a rhynchocoel more than half the body length.

Taxonomic Identity of a Tetrodotoxin-Accumulating Ribbon-worm Cephalothrix simula (Nemertea: Palaeonemertea) : A Species Artificially Introduced from the Pacific to Europe

We compared the anatomy of the holotype of the palaeonemertean Cephalothrix simula (Iwata, 1952) with that of the holotypes of Cephalothrix hongkongiensis Sundberg, Gibson and Olsson, 2003 and Cephalothrix fasciculus (Iwata, 1952), as well as additional specimens from Fukue (type locality of C. simula) and Hiroshima, Japan. While there was no major morphological discordance between these specimens, we found discrepancies between the actual morphology and some statements in the original description of C. simula with respect to supposedly species-specific characters. Our observation indicates that these three species cannot be discriminated by the anatomical characters so far used to distinguish congeners. For objectivity of scientific names, topogenetypes of the mitochondrial cytochrome c oxidase subunit I (COI) sequences are designated for C. simula, C. hongkongiensis, and C. fasciculus. Analysis of COI sequence showed that the Hiroshima population can be identified as C. simula, which has been found in previous studies from Trieste, Italy, and also from both the Mediterranean and Atlantic coasts of the Iberian Peninsula, indicating an artificial introduction via (1) ballast water, (2) ship-fouling communities, or (3) the commercially cultured oyster Crassostrea gigas (Thunberg, 1793) brought from Japan to France in 1970s. Cephalothrix simula is known to be toxic, as it contains large amounts of tetrodotoxin (TTX). We report here that the grass puffer Takifugu niphobles (Jordan and Snyder, 1901)—also known to contain TTX— consumes C. simula. We suggest that the puffer may be able to accumulate TTX by eating C. simula.

Potamostoma shizunaiense gen. et sp. nov. (Nemertea: Hoplonemertea: Monostilifera): a New Brackish-Water Nemertean from Japan

Abstract Potamostoma shizunaiense gen. et sp. nov. (Nemertea: Hoplonemertea: Monostilifera) is described from the mouth of the River Shizunai, Hokkaido, Japan. This genus is readily distinguished from other monostiliferans by an oesophagus opening far anteriorly into the rhynchodaeum, a well developed excretory system extending the whole body length, terminals of the excretory collecting tubules situated between the body wall circular muscle layer and the dermis, and bilobed testes in males.

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