Alexei V. Chernyshev

CLSM Analysis of the Phalloidin-Stained Muscle System of the Nemertean Proboscis and Rhynchocoel

The proboscis and rhynchocoel musculature of 56 nemertean species was studied using phalloidin labelling and confocal laser scanning microscopy. Six types of muscle layers are found in the anterior proboscis of the nemerteans: inner circular, inner diagonal, inner longitudinal, outer diagonal, outer circular, and outer longitudinal. Only the inner circular and inner longitudinal muscle layers are present in all the nemerteans studied. Ten types of arrangement of the proboscis musculature are described. Three primary types (‘palaeotype’, ‘heterotype’, and ‘hoplotype’) correspond to the three nemertean supergroups (Palaeonemertea, Heteronemertea, and Hoplonemertea). The evolutionary transformations of the initial ‘palaeotype’ proboscis proceeded in two primary ways: increasing bilateral symmetry (Callinera, Hubrechtella, and most of Heteronemertea) and increasing polyradial symmetry (Baseodiscidae, Oxypolellinae, and Hoplonemertea). The musculature of the middle portion of the proboscis differs among the three groups with armature: Palaeonemertea (genus Callinera), Polystilifera, and Monostilifera. The musculature of the stylet apparatus of the monostiliferous nemerteans is more complicated than that of the polystiliferous nemerteans, and consists of four muscle components—basal and anterior sphincters, radial and longitudinal musculature. Among the studied monostiliferans, the different components of the stylet musculature are developed to varying degrees. In addition, data on the structure of the rhynchocoel with interwoven musculature are provided. The taxonomic significance and phylogenetic interpretation of the proboscis and rhynchocoel musculature is discussed.

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.

DNA taxonomy of paranemertes (Nemertea: Hoplonemertea) with spirally fluted stylets

Of the 14 nominal species that are now or have ever been assigned to the genus Paranemertes Coe, 1901, four have been reported to have stylets with a spirally fluted or braided appearance. Although differentiation in color patterns has been documented among species/populations, these nemerteans share similar external characters. Using the sequence datasets of mitochondrial cytochrome c oxidase subunit I (COI), 16S rRNA, and nuclear 28S rRNA genes of specimens from 14 localities of Canada, USA, Russia, Japan, and China, we analyzed the genetic differentiation and reconstructed the phylogenetic trees for these nemerteans. In conjunction with the external characters, we discuss their taxonomy and species delimitation. An analysis based on COI dataset showed high genetic variations among populations and even among worms from the same geographic area. The analyzed 111 individuals were assigned into seven networks by statistical parsimony analysis. The inter-network uncorrected p-distances ranged from 0.044 to 0.172 and the mean intra-network uncorrected p-distances varied from 0.001 to 0.005. With the exception of two networks that contain specimens from the East China Sea, all networks were well-supported by the results of Bayesian and neighbor-joining analyses on the COI data. Phylogenetic trees based on 16S rRNA and 28S rRNA datasets were basically similar to the COI trees, but specimens in some networks were merged into larger clades. Present molecular analyses support the validity of P. sanjuanensis and the synonymization of P. cylindracea with P. peregrina. Nemerteans previously recorded as P. peregrina may contain several species and sympatric speciation might have been occurred in this nemertean group.

Phylogeny of Nemertea with special interest in the placement of diversity from Far East Russia and northeast Asia

Recent investigations, based mostly on molecular data, have unraveled the evolutionary history of several common ribbon worm (phylum Nemertea) species and solidified the taxonomic status of many higher taxa within the group. However, a large proportion of enigmatic species have yet to be placed in a phylogenetic framework. We investigated the phylogenetic positions of 26 novel and/or perplexing nemertean species from the Sea of Okhotsk, the Sea of Japan, the Kuril–Kamchatka Trench, and Vietnam (including the first record of a reptant nemertean from the Far East seas of Russia). We conducted both maximum likelihood and parsimony analyses, utilizing four molecular loci—mitochondrial COI and 16S rRNA, as well as nuclear 18S rRNA and 28S rRNA—finding that the current classification for most of these species is corroborated by their phylogenetic placement. We then discuss the evolution of some unique morphological traits possessed by some of these species, using the molecular phylogeny as a backbone for our general conclusions.

The structure of the proboscis musculature in Baseodiscus delineatus (Delle Chiaje, 1825) (Heteronemertea) and the comparative analysis of the proboscis musculature in heteronemerteans

The proboscis musculature was studied in the nemertean Baseodiscus delineatus using confocal laser scanning and electron transmission microscopy. Three muscle layers were differentiated in the proboscis wall: the outer-longitudinal, the diagonal, and the inner-circular layer. The endothelium consists of two cell types: apical supportive cells with rudimentary cilia and subapical myocytes making up the inner-circular musculature of the proboscis. The supportive cells have thin processes attached to the basal extracellular matrix and their perikarya are spread over the apical surfaces of myocytes. The endothelium of B. delineatus is characterized by a folded basal layer of the extracellular matrix and by different heights of myocyte processes, giving an impression that the inner-circular musculature is multilayered. Comparative analysis shows that the diagonal musculature of Baseodiscus is not homologous to that of other heteronemerteans. An assumption is made that the inner-circular muscles have endothelial origin in all heteronemerteans.

Structure of the proboscis endothelium in Nemertea

We studied the ultrastructure of the proboscis endothelium of 14 nemertean species. In all nemerteans examined, the endothelium is organized as a pseudostratified myoepithelium consisting of two types of cells resting on the basal extracellular matrix: apically situated supportive cells and subapical myocytes covered by cytoplasmic sheets of the supportive cells. Myocytes form the inner circular musculature of the proboscis; the endothelium in the bulb of monostiliferous nemerteans lacks myocytes. The endothelium of the studied species differs in the number of rows of muscle fibres (one vs. several rows), the number of myofibrils in myocytes (one vs. two to five), the number of processes of myocytes covered by one supportive cell (one vs. two to 23), and in the number of processes in supportive cells (one vs. two to five). In some of the species, rudimentary cilia of supportive cells were revealed by using cLSM and an antibody against tubulin. The data obtained indicate that the proboscis endothelium in nemerteans is in fact a coelothelium, but recognition of the ancestral state of the coelomic lining in Nemertea is problematic, as the rhynchocoel peritoneum lacks myocytes.

Taxonomy and phylogeny of Lineus torquatus and allies (Nemertea, Lineidae) with descriptions of a new genus and a new cryptic species

The heteronemertean genus Lineus Sowerby, 1806 has been badly in need of revision because of its apparent non-monophyly. In this paper, we focus on Lineus torquatus Coe, 1901, one of the heteronemertean species that occur commonly in waters around the North Pacific, as well as a few other allied species distributed in the western North Pacific, including Lineus alborostratus Takakura, 1898 and Cerebratulus montgomeryi Coe, 1901. Based on phylogenetic analyses using 16S, COI, 28S, 18S, and H3 gene and ITS sequences, we detected a well-supported clade comprised of heteronemerteans with a frontal white band on the head, to which we add Kulikovia gen. nov. This genus is nested within a more comprehensive, highly supported clade, here named the Siphonenteron-clade, which contains Tenuilineus bicolour (Verrill, 1892), Lineus flavescens Coe, 1905, Siphonenteron bilineatum (Renier, 1804), S. cf. bilineatum, Lineus cf. caputornatus, and Lineus sp. from Guam. Our analyses confirmed the presence of a cryptic species of what was formerly known as the cherry-red and reddish forms of Lineus torquatus, herein described as Kulikovia manchenkoi sp. nov. based on some external characters, internal morphology, and the four genetic markers (COI, 16S, H3, and ITS). In contrast to the species pair K. torquata–K. manchenkoi, the reddish form of K. alborostrata does not differ genetically from the typical form of this species. The significance of the external and internal characters for distinguishing cryptic species is discussed. http://zoobank.org/urn:lsid:zoobank.org:pub:9BECBC60-9C82-48EC-AD36-FC564D82A5BChttp://zoobank.org/urn:lsid:zoobank.org:act:D02B2339-4F65-4517-9B13-DD4AAB0C55C5http://zoobank.org/urn:lsid:zoobank.org:act:8D239B62-E655-4721-90F0-A4944DD8A3C7

Special Issue: Proceedings of the 8th International Conference on Nemertean Biology

1Institute of Evolution & Marine Biodiversity, Ocean University of China, 5 Yushan Road, Qingdao 266003, China 2Faculty of Science, Hokkaido University, Sapporo 060-0810, Japan 3A.V. Zhirmunsky Institute of Marine Biology, 17 Palchevsky St, 690059, Vladivostok, Russia 4Far Eastern Federal University 29 Oktyabrskaya St, 690600, Vladivostok, Russia 5Organizers of the 8th International Conference on Nemertean Biology; Guest Editors, Zoological Science

Genetic diversity and phylogeny of limpets of the genus Nipponacmea (Patellogastropoda: Lottiidae) based on mitochondrial DNA sequences

Abstract Species of the genus Nipponacmea inhabit only the Pacific coast of Asia, including the Russian Far East. Their external morphological characters are highly variable and often lead to misidentifications of species. So far, little research has been conducted using molecular markers. We used sequences from three mitochondrial genes (fragments of cytochrome c oxidase subunit I gene (COI), 12S and 16S rDNA). For comparison, additional genetic and taxonomic data on other species belonging to this genus were derived from GenBank. The molecular phylogenetic trees suggest that the species N. fuscoviridis and N. nigrans are species complexes. N. fuscoviridis is divided into three subgroups with high support and relatively large distances between them (N. fuscoviridis A, B and C); N. nigrans fall into two subgroups and one of them (N. nigrans A) is more closely related to N. moskalevi than to the other subgroup of N. nigrans (B).

First records of Hubrechtella ijimai (Nemertea, Hubrechtiiformes) from Korea and China

Hubrechtella ijimai is reported for the first time from South Korea (East China Sea) and China (Yellow Sea), about 260 and 930 km from the nearest locality in Japan. Additional morphological data, confocal laser scanning microphotographs, and DNA data (COI sequences) are provided. This species possesses high intraspecific genetic COI p-distances for nemerteans (1.6–6.3%).