Malin Strand

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.

DNA barcoding should accompany taxonomy – the case of Cerebratulus spp (Nemertea)

Many issues in DNA barcoding need to be solved before it can reach its goal to become a general database for species identification. While species delimitations are more or less well established in several taxa, there are still many groups where this is not the case. Without the proper taxonomic background/knowledge and corroboration with other kinds of data, the DNA barcoding approach may fail to identify species accurately. The classification and taxonomy of phylum Nemertea (nemerteans, ribbon worms) are traditionally based on morphology, but are not corroborated by an increasing amount of genetic data when it comes to classification either into species or into higher taxa. The taxonomy of the phylum needs to be improved before the full potential of DNA barcoding can be utilized to make sure that valid Linnean names accompany the barcode sequences. We illustrate the problematic situation in the phylum Nemertea by a case study from the genus Cerebratulus.

A DNA-based description of a new nemertean (phylum Nemertea) species

Abstract Nemerteans (Nemertea) are traditionally described from internal characters obtained from histological sections of serially sectioned animals. The procedure is time-consuming and requires careful preparation of specimens. The preparations often suffer from artefacts caused by the handling of the animals, since they contract extensively when fixed. It is commonly stated that nemerteans can only be reliably identified using internal characters and the taxon is therefore viewed as ‘difficult’ and, as such, often overlooked in e.g. marine inventories because nobody has the time, facilities or skills to do the sectioning. Contrary to this often-stated ‘fact’, our experience is that many nemerteans can be identified from external characters, although there of course are exceptions. The risk for fixation artefacts in combination with an ascertained high (natural?) intraspecific morphological variation make us pose the question whether anatomy is in fact the best and most efficient basis for phylogeny and classification. There is undoubtedly a large number of undescribed/unnamed nemerteans. To cope with this, we pragmatically propose that nemerteans can be described by a combination of external characters and DNA sequences. We apply this concept on one recently found species new to science, Pseudomicrura afzelii gen. et sp. nov.

Genetics do not reflect habitat differences in Riseriellus occultus (Heteronemertea, Nemertea) from Spain and Wales

Abstract The description of the heteronemertean species Riseriellus occultus was based on specimens from Spain, where they live imbedded in sand, and Wales, where the specimens were collected intertidally under stones and rocks. Considering these habitat differences, which are not known from any other nemertean conspecifics, we asked if this instead is an example of two sibling species that could not be separated by morphology. We sequenced specimens for the two mitochondrial genes cytochrome oxidase I (COI) and 16S and estimated an intraspecific phylogenetic network using statistical parsimony. This analysis did not disclose any differences congruent with geography/habitat; neither did the phylogenetic analysis using maximum likelihood. The phylogenetic analysis also included new sequences of Lineus longissimus, a species with many external characters in common with Riseriellus occultus.

Genus Baseodiscus (Nemertea: Heteronemertea): Molecular identification of a new species in a phylogenetic context

A new heteronemertean, Baseodiscus jonasii sp. nov., is described from Guadalcanal, Solomon Islands. It resembles B. delineatus in inner morphology but can be distinguished from this species by its different colour pattern and differences in the nucleotide sequence of the mitochondrial 16S rRNA gene. The monophyletic status of the genus is investigated by reconstructing the phylogeny of six specimens from four species assigned to this genus, together with 22 specimens from nine other heteronemertean genera, using parsimony and Bayesian analysis. The results imply that Baseodiscus is a monophyletic group while several other heteronemertean genera are non‐monophyletic.

A new nemertean species: what are the useful characters for ribbon worm descriptions?

In recent years the Norwegian Taxonomy Initiative started thorough investigations of poorly known organism groups. In this context, several marine inventories have rendered a number of marine invertebrate species new to science. Within the phylum Nemertea (ribbon worms) a characteristic hoplonemertean was encountered on two different occasions. We describe the new species Amphiporus rectangulus sp. nov. with a combination of histology and DNA data (COI). For the morphological description we use a previously proposed character matrix and, in a context given by the results, also provide a brief discussion on benefits and drawbacks with both methods. We argue that for small animals with soft bodies external characters can be more informative than hitherto expected.

Evaluating the Utility of Single-Locus DNA Barcoding for the Identification of Ribbon Worms (Phylum Nemertea)

Whereas many nemerteans (ribbon worms; phylum Nemertea) can be identified from external characters if observed alive, many are still problematic. When it comes to preserved specimens (as in e.g. marine inventories), there is a particular need for specimen identifier alternatives. Here, we evaluate the utility of COI (cytochrome c oxidase subunit I) as a single-locus barcoding gene. We sequenced, data mined, and compared gene fragments of COI for 915 individuals representing 161 unique taxonomic labels for 71 genera, and subjected different constellations of these to both distance-based and character-based DNA barcoding approaches, as well as species delimitation analyses. We searched for the presence or absence of a barcoding gap at different taxonomic levels (phylum, subclass, family and genus) in an attempt to understand at what level a putative barcoding gap presents itself. This was performed both using the taxonomic labels as species predictors and using objectively inferred species boundaries recovered from our species delimitation analyses. Our data suggest that COI works as a species identifier for most groups within the phylum, but also that COI data are obscured by misidentifications in sequence databases. Further, our results suggest that the number of predicted species within the dataset is (in some cases substantially) higher than the number of unique taxonomic labels—this highlights the presence of several cryptic lineages within well-established taxa and underscores the urgency of an updated taxonomic backbone for the phylum.

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.