Genus Tetrastemma Ehrenberg, 1831 (Phylum Nemertea)—A natural group? Phylogenetic relationships inferred from partial 18S rRNA sequences

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Abstract

We investigated the monophyletic status of the hoplonemertean taxon Tetrastemma by reconstructing the phylogeny for 22 specimens assigned to this genus, together with another 25 specimens from closely related hoplonemertean genera. The phylogeny was based on partial 18S rRNA sequences using Bayesian and maximum likelihood analyses. The included Tetrastemma-species formed a well-supported clade, although the within-taxon relationships were unsettled. We conclude that the name Tetrastemma refers to a monophyletic taxon, but that it cannot be defined by morphological synapomorphies, and our results do not imply that all the over 100 species assigned to this genus belong to it. The results furthermore indicate that the genera Amphiporus and Emplectonema are non-monophyletic.

Introduction

Members of the phylum Nemertea possess an eversible proboscis surrounded by a hydrostatic cavity called the rhynchocoel; this apparatus is unique to nemerteans and represents a novel synapomorphy that identifies Nemertea as a monophyletic taxon. The nemerteans has been recognized as a distinct taxon for more than 150 years and as a phylum for 60 years, but the relationships and systematics of the included species are still basically unresolved. The first published species descriptions were obviously inadequate by modern standards and contained only external characters. Authors tended to put new findings in already established species, and species in known higher taxa, simply because they did not have the information to do otherwise. For a time species described were therefore assigned to just a few major genera (Amphiporus, Cerebratulus, Lineus, Prostoma Tetrastemma), and new species were kept being placed into these genera instead of creating new ones on rather unstable grounds. As a consequence, more than 40% (Gibson, 1985) of the currently known over 1200 species of nemerteans are placed in these four genera alone. The standard of descriptions has obviously improved over the years and most descriptions today contain detailed observations of both exterior and anatomical characters. The increased amount of information has transformed the systematics of nemerteans and the number of genera has increased to currently around 250 in the phylum. However, improved descriptions have not really changed the actual systematic thinking and nemertean classification is still largely based on an approach where combinations of similarities/dissimilarities are used to erect taxa. Sundberg (1993) criticized this approach and argued that it was non-phylogenetic and thus not likely to identify monophyletic groups. Even though the situation is slowly changing, it still means that many of the nemertean taxon names we know today, especially families and genera, do not refer to monophyletic groups.

However, the reason for this is not entirely the lack of a phylogenetic approach, but is also caused by problems inherited in morphological characters. First, the number of available morphological characters is essentially rather low, and fewer the tinier the species is which in itself causes problems in systematizing nemerteans. Older descriptions are furthermore so poor when it comes to morphological details that in most cases they cannot be used for any systematic work. Second, since there are very few external characters, systematic conclusions must be based on observations of anatomical features which require time-consuming histology. Third, most nemertean species are extremely contractile which means that the animal will contract during fixation unless careful measures are taken when fixing for histology, and this will further add to the genetically induced intraspecific variation always present. Our conclusion is that although species descriptions should be based on morphology, it may be that their systematic position cannot be deduced from this information alone and we strongly believe that systematic conclusions must be based on nucleotide information. See also Strand and Sundberg (in press) who showed that there is little concordance between morphological “species” and evolutionary lineages when they analysed the intrageneric variation in a group of Tetrastemma species. Their conclusion was that speciation can take place without apparent morphological diversification.

The higher classification of nemerteans is still based on Stiasny-Wijnhoff’s (1936) arrangement of the nemerteans into the subclasses Palaeonemertea, Heteronemertea, Hoplonemertea, and Bdellonemertea. Iwata’s (1960) later division of some palaeonemerteans into Archinemertea has not gained wide acceptance and has been refuted by later phylogenetic analyses (Sundberg and Hylbom, 1994, Thollesson and Norenburg, 2003). Phylogenetic analyses based on molecular data (Sundberg et al., 2001, Thollesson and Norenburg, 2003), have established the monophyly of the clade Hoplonemertea when Bdellonemertea is included (it is interpreted as an apomorphic group within the hoplonemerteans). This means that Enopla and Hoplonemertea are identical, but although the name Enopla should have priority, Thollesson and Norenburg argue for keeping the name Hoplonemertea for this clade. At present there are approximately 650 hoplonemertean species, placed in 155 genera. Of these, two genera contain together more than one third of these species; Amphiporus with 117 species and Tetrastemma with 113 (Gibson, 1995). The genus Tetrastemma was erected by Ehrenberg (1828) with illustrations of the type-species (T. flavidum), while the genus was not diagnosed until later (Ehrenberg, 1831) by the feature “four eyes.” The definition of the genus Tetrastemma has expanded over the years and has become more detailed, but distinct morphological features of the genus are still few and sometimes dubious. The generic diagnoses of both Amphiporus and Tetrastemma (like most old generic diagnoses in fact) do not point to any synapomorphy(-ies) that unambiguously would identify the taxon as monophyletic. These diagnoses are lists of observed character states in species assigned to each genus respectively, instead of being shared similarities, and these characters “define” the genus. These diagnoses may be useful for species identifications but do not (in the case of nemerteans) normally point to monophyletic groups; the difference between characters good for defining a monophyletic taxon and those good for identifying species (for other reasons) placed in a taxon is muddled in many cases. When the diagnoses of Tetrastemma and Amphiporus are listed character by character (Table 1) it is clear that the differences between the two diagnoses are minor and often ambiguous, and furthermore that there are no apparent synapomorphies for either of the genera. Thus, there are reasons to question if these genera are monophyletic taxa. We used 18S rRNA sequences from 22 specimens of Tetrastemma, together with 25 other hoplonemerteans (Table 2) to test the monophyletic status of the former genus. We reconstructed the phylogeny for these species using Bayesian and maximum likelihood analyses and found a well corroborated clade of Tetrastemma-species. Our conclusion from the species examined is that the taxon name refers to a monophyletic group, although not implying that all species hitherto assigned to this genus actually belong there. We will also argue that the taxon cannot be morphologically synapomorphy-defined with our present knowledge of the morphology.

Section snippets

Specimens and DNA extraction

Sequenced specimens and collection sites are listed in Table 2. The study includes a total of 34 species, and in the case of three putative species we have several samples from different geographic areas. Specimens were placed in 70–95% ethanol and stored until the DNA was extracted using either a Chelex (5–10%) protocol or the QIAamp DNA Mini Kit for tissue (QIAgen) following the protocol supplied by the manufacturer.

Amplification and sequencing

Amplification of the 18S rRNA gene was carried out by PCR using a thermal

Results

The aligned data set contained 1349 basepairs (bp) after excluding sites that could not be reliably aligned. Of the used data set, 95 (7%) bp were parsimony informative. Fig. 1 shows the resulting phylogram from the Bayesian analysis. All species in the taxon Tetrastemma form a monophyletic group with posterior probability 0.97, but we do not see any significant grouping of the presumed same species in cases where there are multiple representatives of a species. Other well supported (posterior

Discussion

There is today a growing number of papers showing that Bayesian posterior probabilities are inflated while the bootstrap values are underestimates (see e.g., Taylor and Piel, 2004). Still, we consider the Bayesian probability 0.97 to be sufficient for our conclusion that there is a taxon Tetrastemma. Although both analyses support the monophyletic status of Tetrastemma, this does not imply that all species currently assigned to this genus refer to this natural group, as only 13 of approximately

Acknowledgments

We thank N.N. for critical comments on this manuscript, and Drs. J. Moore, M. Envall, and A.V. Chernyshev for providing us with specimens. The study was financially supported by the Swedish Research Council (to P.S.), and by the Nilsson-Ehle, Ax:son Johnson, Colliander funds (to M.S.). P.S. is grateful for Dr. Duncan’s support during his sabbatical stay in Liverpool.

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