Jon L. Norenburg

Ribbon worm relationships: a phylogeny of the phylum Nemertea

We present the most extensive phylogenetic analysis to date, to our knowledge, of higher–level nemertean relationships, based on sequence data from four different genes (the nuclear genes for nuclear large subunit rRNA (28S rRNA) and histone H3 (H3), and the mitochondrial genes for mitochondrial large subunit rRNA (16S rRNA) and cytochrome c oxidase subunit I (COI)). Well–supported clades are, in general, compatible with earlier, more limited, analyses, and current classification is largely in agreement with our results, although there are some notable exceptions. Bdellonemertea (represented by Malacobdella) is found to be a part of Monostilifera, and Polystilifera is the monophyletic sister group to Monostilifera. Cratenemertidae is the sister group to the remaining monostiliferans (including Malacobdella), a group to which we apply the new name Distromatonemertea. Heteronemertea is monophyletic and forms a clade with Hubrechtella; for this clade we introduce the name Pilidiophora. Finally, Pilidiophora and Hoplonemertea (with Malacobdella) form a monophyletic group, and we introduce the name Neonemertea to refer to this group. Palaeonemertea is found to be non–monophyletic and basal among nemerteans.

Open‐ocean barriers to dispersal: a test case with the Antarctic Polar Front and the ribbon worm Parborlasia corrugatus (Nemertea: Lineidae)

Open‐ocean environments provide few obvious barriers to the dispersal of marine organisms. Major currents and/or environmental gradients potentially impede gene flow. One system hypothesized to form an open‐ocean dispersal barrier is the Antarctic Polar Front, an area characterized by marked temperature change, deep water, and the high‐flow Antarctic Circumpolar current. Despite these potential isolating factors, several invertebrate species occur in both regions, including the broadcast‐spawning nemertean worm Parborlasia corrugatus. To empirically test for the presence of an open‐ocean dispersal barrier, we sampled P. corrugatus and other nemerteans from southern South America, Antarctica, and the sub‐Antarctic islands. Diversity was assessed by analyzing mitochondrial 16S rRNA and cytochrome c oxidase subunit I sequence data with Bayesian inference and tcs haplotype network analysis. Appropriate neutrality tests were also employed. Although our results indicate a single well‐mixed lineage in Antarctica and the sub‐Antarctic, no evidence for recent gene flow was detected between this population and South American P. corrugatus. Thus, even though P. corrugatus can disperse over large geographical distances, physical oceanographic barriers (i.e. Antarctic Polar Front and Antarctic Circumpolar Current) between continents have likely restricted dispersal over evolutionary time. Genetic distances and haplotype network analysis between South American and Antarctic/sub‐Antarctic P. corrugatus suggest that these two populations are possibly two cryptic species.

Barcoding against a paradox? Combined molecular species delineations reveal multiple cryptic lineages in elusive meiofaunal sea slugs

BackgroundMany marine meiofaunal species are reported to have wide distributions, which creates a paradox considering their hypothesized low dispersal abilities. Correlated with this paradox is an especially high taxonomic deficit for meiofauna, partly related to a lower taxonomic effort and partly to a high number of putative cryptic species. Molecular-based species delineation and barcoding approaches have been advocated for meiofaunal biodiversity assessments to speed up description processes and uncover cryptic lineages. However, these approaches show sensitivity to sampling coverage (taxonomic and geographic) and the success rate has never been explored on mesopsammic Mollusca.ResultsWe collected the meiofaunal sea-slug Pontohedyle (Acochlidia, Heterobranchia) from 28 localities worldwide. With a traditional morphological approach, all specimens fall into two morphospecies. However, with a multi-marker genetic approach, we reveal multiple lineages that are reciprocally monophyletic on single and concatenated gene trees in phylogenetic analyses. These lineages are largely concordant with geographical and oceanographic parameters, leading to our primary species hypothesis (PSH). In parallel, we apply four independent methods of molecular based species delineation: General Mixed Yule Coalescent model (GMYC), statistical parsimony, Bayesian Species Delineation (BPP) and Automatic Barcode Gap Discovery (ABGD). The secondary species hypothesis (SSH) is gained by relying only on uncontradicted results of the different approaches (‘minimum consensus approach’), resulting in the discovery of a radiation of (at least) 12 mainly cryptic species, 9 of them new to science, some sympatric and some allopatric with respect to ocean boundaries. However, the meiofaunal paradox still persists in some Pontohedyle species identified here with wide coastal and trans-archipelago distributions.ConclusionsOur study confirms extensive, morphologically cryptic diversity among meiofauna and accentuates the taxonomic deficit that characterizes meiofauna research. We observe for Pontohedyle slugs a high degree of morphological simplicity and uniformity, which we expect might be a general rule for meiofauna. To tackle cryptic diversity in little explored and hard-to-sample invertebrate taxa, at present, a combined approach seems most promising, such as multi-marker-barcoding (i.e., molecular systematics using mitochondrial and nuclear markers and the criterion of reciprocal monophyly) combined with a minimum consensus approach across independent methods of molecular species delineation to define candidate species.

Vestigial Prototroch in a Basal Nemertean, Carinoma tremaphoros (Nemertea; Palaeonemertea)

Summary Nemerteans have been alleged to belong to a protostome clade called the Trochozoa that includes mollusks, annelids, sipunculids, echiurids, and kamptozoans and is characterized by, among other things, the trochophore larva. The trochophore possesses a prototroch, a preoral belt of specialized ciliary cells, derived from the trochoblast cells. Nemertea is the only trochozoan phylum for which presence of the trochophore larva possessing a prototroch had never been shown. However, so little is known about nemertean larval development that comparing it with development of other trochozoans is difficult. Development in the nemertean clade Pilidiophora is via a highly specialized planktonic larva, the pilidium, and most of the larval body is lost during a drastic metamorphosis. Other nemerteans (hoplonemerteans and palaeonemerteans) lack a pilidium, and their development is direct, forming either an encapsulated or planktonic “planuliform” larva, producing a juvenile without a dramatic change in body plan. We show that early in the development of a member of a basal nemertean assemblage, the palaeonemertean Carinoma tremaphoros, large squamous cells cover the entire larval surface except for the apical and posterior regions. Although apical and posterior cells continue to divide, the large surface cells cleavage arrest and form a contorted preoral belt. Based on its position, cell lineage, and fate, we suggest that this belt corresponds to the prototroch of other trochozoans. Lack of differential ciliation obscures the presence of the prototroch in Carinoma, but differentiation of the trochoblasts is clearly manifested in their permanent cleavage arrest and ultimate degenerative fate. Our results allow a meaningful comparison between the development of nemerteans and other trochozoans. We review previous hypotheses of the evolution of nemertean development and suggest that a trochophore‐like larva is plesiomorphic for nemerteans while a pilidium type of development with drastic metamorphosis is derived.

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.

Phylogenetics of Trachylina (Cnidaria: Hydrozoa) with new insights on the evolution of some problematical taxa

Some of the most interesting and enigmatic cnidarians are classified within the hydrozoan subclass Trachylina. Despite being relatively depauperate in species richness, the clade contains four taxa typically accorded ordinal status: Actinulida, Limnomedusae, Narcomedusae and Trachymedusae. We bring molecular data (mitochondrial 16S and nuclear small and large subunit ribosomal genes) to bear on the question of phylogenetic relationships within Trachylina. Surprisingly, we find that a diminutive polyp form, Microhydrula limopsicola (classified within Limnomedusae) is actually a previously unknown life stage of a species of Stauromedusae. Our data confirm that the interstitial form Halammohydra sp. (Actinulida) is derived from holopelagic direct developing ancestors, likely within the trachymedusan family Rhopalonematidae. Trachymedusae is shown to be diphyletic, suggesting that the polyp stage has been lost independently at least two times within trachyline evolution. Narcomedusae is supported as a monophyletic group likely also arising from trachymedusan ancestors. Finally, some data, albeit limited, suggest that some trachyline species names refer to cryptic species that have yet to be sorted taxonomically.

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.

The Necessity of DNA Taxonomy to Reveal Cryptic Diversity and Spatial Distribution of Meiofauna, with a Focus on Nemertea

Meiofauna represent one of the most abundant and diverse communities in marine benthic ecosystems. However, an accurate assessment of diversity at the level of species has been and remains challenging for these microscopic organisms. Therefore, for many taxa, especially the soft body forms such as nemerteans, which often lack clear diagnostic morphological traits, DNA taxonomy is an effective means to assess species diversity. Morphological taxonomy of Nemertea is well documented as complicated by scarcity of unambiguous character states and compromised by diagnoses of a majority of species (and higher clades) being inadequate or based on ambiguous characters and character states. Therefore, recent studies have advocated for the primacy of molecular tools to solve the taxonomy of this group. DNA taxonomy uncovers possible hidden cryptic species, provides a coherent means to systematize taxa in definite clades, and also reveals possible biogeographic patterns. Here, we analyze diversity of nemertean species by considering the barcode region of the mitochondrial gene Cytochrome Oxidase subunit I (COI) and different species delineation approaches in order to infer evolutionarily significant units. In the aim to uncover actual diversity of meiofaunal nemerteans across different sites in Central America, COI sequences were obtained for specimens assigned here to the genera Cephalothrix, Ototyphlonemertes, and Tetrastemma-like worms, each commonly encountered in our sampling. Additional genetic, taxonomic, and geographic data of other specimens belonging to these genera were added from GenBank. Results are consistent across different DNA taxonomy approaches, and revealed (i) the presence of several hidden cryptic species and (ii) numerous potential misidentifications due to traditional taxonomy. (iii) We additionally test a possible biogeographic pattern of taxonomic units revealed by this study, and, except for a few cases, the putative species seem not to be widely distributed, in contrast to what traditional taxonomy would suggest for the recognized morphotypes.

Redescription of a brooding nemertine, Cyanophthalma obscura (Schultze) gen. et comb.n., with observations on its biology and discussion of the species of Prostomatella and related taxa*†

A new genus, Cyanophthalma, is erected with the type species Tetrastemma obscura Schultze, 1851. Cyanophthalma obscura is transferred from Prostomatella and is redescribed from observations of more than 300 living specimens and serial sections of 59 specimens from the northeast coast of North America. Comparison is made with serial sections of C. obscura from the Baltic and with serially sectioned specimens of three other described species of Prostomatella. Two of these, P. enteroplecta and P. merula, do not fit either genus and are provisionally placed in the aggregate genus Tetrastemma, whose present diagnosis they do fit. Prostomatella remains with only its type species, P. arenicola. Amphiporus cordiceps is also transferred to Cyanophthalma. Some of the characters that form the basis for classifying Monostilifera are assessed for phylogenetic significance. Cyanophthalma obscura in Nova Scotia lives in salt marshes. Young are brooded through the winter while ice covers most of the marsh.