Abstract

Described in 1930–31 by Steinböck who considered it the most primitive bilaterian, the turbellarian genus Nemertoderma is known for its role in platyhelminth phylogeny as much as for its muddled taxonomy. On the basis of material collected in the Mediterranean, Atlantic and Pacific Ocean since 1964 the known four genera and seven species (Nemertoderma bathycola Steinböck, 1930–31; N. westbladi Steinböck, 1938; N. psammicola Sterrer, 1970 (syn. N. rubra Faubel, 1976); Meara stichopi Westblad, 1949; Meara sp. Smith et al. 1994; Nemertinoides elongatus Riser, 1987; and Flagellophora apelti Faubel &; Dörjes, 1978) are redescribed and one new genus with two new species (Ascoparia neglecta n. g. n. sp. and A. secunda n. sp.). Live observations on morphological variability, body size vs. reproductive state, statocyst structure and statolith variability, and sperm morphology and dimorphism are provided. The paper concludes with a discussion and diagnoses of the known taxa of Nemertodermatida, including the new family Ascopariidae.     

The Nemertodermatida are basal bilaterians and not members of the Platyhelminthes

Recent hypotheses on metazoan phylogeny have recognized three main clades of bilaterian animals: Deuterostomia, Ecdysozoa and Lophotrochozoa. The acoelomate and 'pseudocoelomate' metazoans, including the Platyhelminthes, long considered basal bilaterians, have been referred to positions within these clades by many authors. However, a recent study based on ribosomal DNA placed the flatworm group Acoela as the sister group of all other extant bilaterian lineages. Unexpectedly, the nemertodermatid flatworms, usually considered the sister group of the Acoela together forming the Acoelomorpha, were grouped separately from the Acoela with the rest of the Platyhelminthes (the Rhabditophora) within the Lophotrochozoa. To re-evaluate and clarify the phylogenetic position of the Nemertodermatida, new sequence data from 18S ribosomal DNA and mitochondrial genes of nemertodermatid and other bilaterian species were analysed with parsimony and maximum likelihood methods. The analyses strongly support a basal position within the Bilateria for the Nemertodermatida as a sister group to all other bilaterian taxa except the Acoela. Despite the basal position of both Nemertodermatida and Acoela, the clade Acoelomorpha was not retrieved. These results imply that the last common ancestor of bilaterian metazoans was a small, benthic, direct developer without segments, coelomic cavities, nephrida or a true brain. The name Nephrozoa is proposed for the ancestor of all bilaterians excluding the Nemertodermatida and the Acoela, and its descendants.     

The brain of the Nemertodermatida (Platyhelminthes) as revealed by anti-5HT and anti-FMRFamide immunostainings

The taxa Nemertodermatida and Acoela have traditionally been considered closely related and classified as sister groups within the Acoelomorpha Ehlers 1984 (Platyhelminthes). Recent molecular investigations have questioned their respective position. In this study, the 5-HT and FMRFamide immunoreactivity (IR) in the nervous system of two nemertodermatids, Nemertoderma westbladi and Meara stichopi, is described. The 5-HT immunoreactive pattern differs in the two nemertodermatids studied. In M. stichopi, two loose longitudinal bundles of 5-HT-immunoreactive fibres and an basi-epidermal nerve net were observed. In N. westbladi the 5-HT-IR shows a ring-shaped commissural structure, different from the commissural brain of acoels. In both nemertodermatids, FMRFamide immunoreactive nerve fibres followed the 5-HT-immunoreactive fibres. It is demonstrated that the Nemertodermatida have neither a 'commissural brain' structure similar to that of the Acoela, nor a 'true', ganglionic brain and orthogon, typical for other Platyhelminthes. The question of the plesiomorphic or apomorphic nature of the nervous system in Nemertodermatida cannot yet be answered. The neuroanatomy of the studied worms provides no synapomorphy supporting the taxon Acoelomorpha.     

Flatworm phylogeny from partial 18S rDNA sequences

Partial 18S rDNA sequences from 29 flatworms and 2 outgroup taxa were used in a cladistic analysis of the Platyhelminthes. Support for the clades in the resulting single most parsimonious tree was estimated through bootstrap analysis, jack-knife analysis and decay indices. The Acoelomorpha (Acoela and Nemertodermatida) were absent from the most parsimonious tree. The Acoela and the Fecampiidae form a strongly supported clade, the sister group of which may be the Tricladida. There is some support for monophyly of the rhabdocoel taxon Dalyellioida, previously regarded as paraphyletic. The sister group of the Neodermata remains unresolved. This revised version was published online in July 2006 with corrections to the Cover Date.     

Cleavage in<Emphasis Type="Italic"> Nemertoderma westbladi</Emphasis> (Nemertodermatida) and its phylogenetic significance

Recent phylogenetic analyses of ribosomal and protein coding nuclear genes place the marine worms within the Nemertodermatida as one of the oldest lineages among the bilaterian animals. We studied the early embryonic cleavage in Nemertoderma westbladi to provide the first account of nemertodermatid early development. Live embryos were studied with interference microscopy and fixed embryos were either sectioned or studied with confocal laser scanning microscopy. Initially the divisions in the embryo are radial, but then micromeres are shifted clockwise generating a spiral pattern. The four-cell stage is characterized by duets of macromeres and micromeres and thus resembles the duet cleavage reported from members of the Acoela. However, subsequent stages differ from the acoel duet pattern and also from quartet spiral cleavage. The optimization of the cleavage pattern on current phylogenetic hypotheses with Nemertodermatida and Acoela as early bilaterian branches is discussed.     

Are the Platyhelminthes a monophyletic primitive group? An assessment using 18S rDNA sequences

In most zoological textbooks, Platyhelminthes are depicted as an early- emerging clade forming the likely sister group of all the other Bilateria. Other phylogenetic proposals see them either as the sister group of most of the Protostomia or as a group derived from protostome coelomate ancestors by progenesis. The main difficulty in their correct phylogenetic placing is the lack of convincing synapomorphies for all Platyhelminthes, which may indicate that they are polyphyletic. Moreover, their internal phylogenetic relationships are still uncertain. To test these hypotheses, new complete 18S rDNA sequences from 13 species of "Turbellaria" have been obtained and compared to published sequences of 2 other "Turbellaria," 3 species of parasitic Platyhelminthes, and several diploblastic and deuterostome and protostome triploblastics. Maximum-parsimony, maximum-likelihood, and neighbor-joining methods were used to infer their phylogeny. The results show the order Catenulida to form an independent early- branching clade and emerge as a potential sister group of the rest of the Bilateria, while the rest of Platyhelminthes (Rhabditophora), which includes the parasites, form a clear monophyletic group closely related to the protostomes. The order Acoela, morphologically considered as candidates to be ancestral, are shown to be fast-clock organisms for the 18S rDNA gene. Hence, long-branching of acoels and insufficient sampling of catenulids and acoels leave their position still unresolved and call for further studies. Within the Rhabditophora, our analyses suggest (1) a close relationship between orders Macrostomida and Polycladida, forming a clear sister group to the rest of orders; (2) that parasitic platyhelminthes appeared early in the evolution of the group and form a sister group to a still-unresolved clade made by Nemertodermatida, Lecithoepitheliata, Prolecithophora, Proseriata, Tricladida, and Rhabdocoela; and (3) that Seriata is paraphyletic.      

Acoel flatworms are not platyhelminthes: evidence from phylogenomics

Acoel flatworms are small marine worms traditionally considered to belong to the phylum Platyhelminthes. However, molecular phylogenetic analyses suggest that acoels are not members of Platyhelminthes, but are rather extant members of the earliest diverging Bilateria. This result has been called into question, under suspicions of a long branch attraction (LBA) artefact. Here we re-examine this problem through a phylogenomic approach using 68 different protein-coding genes from the acoel Convoluta pulchra and 51 metazoan species belonging to 15 different phyla. We employ a mixture model, named CAT, previously found to overcome LBA artefacts where classical models fail. Our results unequivocally show that acoels are not part of the classically defined Platyhelminthes, making the latter polyphyletic. Moreover, they indicate a deuterostome affinity for acoels, potentially as a sister group to all deuterostomes, to Xenoturbellida, to Ambulacraria, or even to chordates. However, the weak support found for most deuterostome nodes, together with the very fast evolutionary rate of the acoel Convoluta pulchra, call for more data from slowly evolving acoels (or from its sister-group, the Nemertodermatida) to solve this challenging phylogenetic problem.     

The muscular system of Nemertoderma westbladi and Meara stichopi (Nemertodermatida, Acoelomorpha)

Nemertodermatida is a small taxon of marine worm-like animals; its position in the tree of life is highly contested. The musculature of Nemertoderma westbladi and Meara stichopi is studied here in detail using fluorescent phalloidin and confocal microscopy. In both species, the musculature is composed of an outer layer of circular and an inner layer of longitudinal musculature, diagonal muscles form a distinct layer in N. westbladi, but in M. stichopi these fibres connect to both other layers. The supraterminally opening male pore and antrum are formed by invagination of the whole body-wall in both species, and the seminal vesicle is lined by a thin net of musculature only in full male maturity. Modifications of the ventral body-wall adjacent to the mouth are small and transient in N. westbladi including no extra musculature, whereas it consists of additional strong U-shaped musculature in M. stichopi. Myogenesis in N. westbladi is not finished in hatchlings and will be completed dorsally in juvenile specimens and ventrally in male mature ones, after the loss of the mouth. Musculature between the two species differs considerably and might give insights into the internal relationships of Nemertodermatida and might prove to be useful in studies investigating their phylogenetic position. More data of other species and developmental changes are needed.     

Combined large and small subunit ribosomal RNA phylogenies support a basal position of the acoelomorph flatworms

The phylogenetic position of the phylum Platyhelminthes has been re-evaluated in the past decade by analysis of diverse molecular datasets. The consensus is that the Rhabditophora + Catenulida, which includes most of the flatworm taxa, are not primitively simple basal bilaterians but are related to coelomate phyla such as molluscs. The status of two other groups of acoelomate worms, Acoela and Nemertodermatida, is less clear. Although many characteristics unite these two groups, initial molecular phylogenetic studies placed the Nemertodermatida within the Rhabditophora, but placed the Acoela at the base of the Bilateria, distant from other flatworms. This contradiction resulted in scepticism about the basal position of acoels and led to calls for further data. We have sequenced large subunit ribosomal RNA genes from 13 rhabditophorans + catenulids, three acoels and one nemertodermatid, tripling the available data. Our analyses strongly support a basal position of both acoels and nemertodermatids. Alternative hypotheses are significantly less well supported by the data. We conclude that the Nemertodermatida and Acoela are basal bilaterians and, owing to their unique body plan and embryogenesis, should be recognized as a separate phylum, the Acoelomorpha.     

Mitochondrial genome data support the basal position of Acoelomorpha and the polyphyly of the Platyhelminthes

We determined 9.7, 5.2, and 6.8 kb, respectively, of the mitochondrial genomes of the acoel Paratomella rubra, the nemertodermatid Nemertoderma westbladi, and the free-living rhabditophoran platyhelminth Microstomum lineare. The identified gene arrangements are unique among metazoans, including each other, sharing no more than one or two single gene boundaries with a few distantly related taxa. Phylogenetic analysis of the amino acid sequences inferred from the sequenced genes confirms that the acoelomorph flatworms (acoels+nemertodermatids) do not belong to the Platyhelminthes, but are, instead, the most basal extant bilaterian group. Therefore, the Platyhelminthes, as traditionally constituted, is a polyphyletic phylum.     

Dismissal of Acoelomorpha: Acoela and Nemertodermatida are separate early bilaterian clades

We used new 18S and 28S rRNA sequences analysed with parsimony, maximum likelihood and Bayesian methods of phylogenetic reconstruction to show that Nemertodermatida, generally classified as the sister group of Acoela within the recently proposed Phylum Acoelomorpha, are a separate basal bilaterian lineage. We used several analytical approaches to control for possible long branch attraction (LBA) artefacts in our results. Parsimony and the model based phylogenetic reconstruction methods that incorporate 'corrections' for substitution rate heterogenities yielded concordant results. When putative long branch taxa were experimentally removed the resulting topologies were consistent with our total evidence analysis. Deletion of fast-evolving nucleotide sites decreased resolution and clade support, but did not support a topology conflicting with the total evidence analysis. Establishment of Acoela and Nemertodermatida as two early lineages facilitates reconstruction of ancestral bilaterian features. The ancestor of extant Bilateria was a small, benthic direct developer without coelom or a planktonic larval stage. The previously proposed Phylum Acoelomorpha is dismissed as paraphyletic.     

The interrelationships of all major groups of Platyhelminthes: phylogenetic evidence from morphology and molecules

We used a data matrix of 65 morphological characters from 25 ingroup and 6 outgroup taxa, and an alignment comprising complete 18S rDNA sequences from 82 species of parasitic and free-living Platyhelminthes and from 19 species of lower invertebrates to analyse phylogenetic relationships of various platyhelminth taxa. Of the 1358 unambiguously alignablc molecular positions, 995 were variable and 757 were phylogenctically informative (parsimony criterion); complete 18S rDNA sequences ranged in length from 1755 to 2873 bp. Main conclusions are: Ncodermata are monophylctic, and the Trematoda, Monogenca and Cestoda within them are monophylctic as well. The sister group of the Ncodermata is all the other Ncoophora; the Kalyptorhynchia, Typhloplanida, Dalyelliida and Tcmnocephalida form one clade, and the last three another. Monophyly of the Seriata is rejected, but Polycladida/ Macrostomida/Haplopharyngida are monophylctic, as arc the last two taxa. As a consequence, validity of the taxon Trepaxonemata is rejected. Further studies must show the correct position of the Acocla and Nemertodermatida. It is stressed that morphological and molecular data in some respects lead to contradictory results, for instance concerning the position of the Fecampiidac/ Urastoma/Ichthyophaga and the relative position of the Lccithoepitheliata. Denser sampling of taxa for molecular data, complementary sequences from independent genes, and inclusion of additional morphological data are necessary to resolve these contradictions.     

Hox and ParaHox genes in Nemertodermatida, a basal bilaterian clade

Molecular evidence suggests that Acoelomorpha, a proposed phylum composed of acoel and Nemertodermatida flatworms, are the most basal bilaterian animals. Hox and ParaHox gene complements characterised so far in acoels consist of a small set of genes, comprising representatives of anterior, central and posterior genes, altogether Hox and ParaHox, but no PG3-Xlox representatives have been reported. It has been proposed that this might be the ancestral Hox repertoire in basal bilaterians. However, no studies of the other members of the group, the Nemertodermatida, have been done. In order to get a more complete picture of the basal bilaterian Hox and ParaHox complement, we have analysed the Hox/ParaHox complement of the nemertodermatid Nemertoderma westbladi. We have found representatives of two central and one posterior Hox genes, as well as an Xlox and a Caudal ParaHox gene. From our data we conclude that a PG3-Xlox gene was present in the ancestor of bilaterians. These findings support the speculation that basal bilaterians already had the beginnings of the extended central Hox set, driving back gene duplications in the central part of the Hox cluster deeper in phylogeny than previously suggested.     

Evolution of ITS1 rDNA in the Digenea (Platyhelminthes: Trematoda): 3′ End Sequence Conservation and Its Phylogenetic Utility

A comparison of ribosomal internal transcribed spacer 1 (ITS1) elements of digenetic trematodes (Platyhelminthes) including unidentified digeneans isolated from Cyathura carinata (Crustacea: Isopoda) revealed DNA sequence similarities at more than half of the spacer at its 3′ end. Primary sequence similarity was shown to be associated with secondary structure conservation, which suggested that similarity is due to identity by descent and not chance. Using an analysis of apomorphies, the sequence data were shown to produce a distinct phylogenetic signal. This was confirmed by the consistency of results of different tree reconstruction methods such as distance approaches, maximum parsimony, and maximum likelihood. Morphological evidence additionally supported the phylogenetic tree based on ITS1 data and the inferred phylogenetic position of the unidentified digeneans of C. carinata met the expectations from known trematode life-cycle patterns. Although ribosomal ITS1 elements are generally believed to be too variable for phylogenetic analysis above the species or genus level, the overall consistency of the results of this study strongly suggests that this is not the case in digenetic trematodes. Here, 3′ end ITS1 sequence data seem to provide a valuable tool for elucidating phylogenetic relationships of a broad range of phylogenetically distinct taxa. Received: 20 October 1997 / Accepted: 24 March 1998     

Characters of the female gonad and the phylogeny of Platyhelminthes

The female gonad of Platyhelminthes shows a wide variability in its anatomical and cellular organization and submicroscopic structure, and some characteristics may be useful for elucidating the questioned monophyly of some taxa and reconstructing the phylogenetic tree of the group. The morphological characters of the female gonad have been subdivided into three levels: macroscopic, microscopic and ultrastructural, and some examples with potential phylogenetic value are presented for each level. This revised version was published online in July 2006 with corrections to the Cover Date.     

To Be or Not to Be a Flatworm: The Acoel Controversy

Since first described, acoels were considered members of the flatworms (Platyhelminthes). However, no clear synapomorphies among the three large flatworm taxa - the Catenulida, the Acoelomorpha and the Rhabditophora - have been characterized to date. Molecular phylogenies, on the other hand, commonly positioned acoels separate from other flatworms. Accordingly, our own multi-locus phylogenetic analysis using 43 genes and 23 animal species places the acoel flatworm Isodiametra pulchra at the base of all Bilateria, distant from other flatworms. By contrast, novel data on the distribution and proliferation of stem cells and the specific mode of epidermal replacement constitute a strong synapomorphy for the Acoela plus the major group of flatworms, the Rhabditophora. The expression of a piwi-like gene not only in gonadal, but also in adult somatic stem cells is another unique feature among bilaterians. These two independent stem-cell-related characters put the Acoela into the Platyhelminthes-Lophotrochozoa clade and account for the most parsimonious evolutionary explanation of epidermal cell renewal in the Bilateria. Most available multigene analyses produce conflicting results regarding the position of the acoels in the tree of life. Given these phylogenomic conflicts and the contradiction of developmental and morphological data with phylogenomic results, the monophyly of the phylum Platyhelminthes and the position of the Acoela remain unresolved. By these data, both the inclusion of Acoela within Platyhelminthes, and their separation from flatworms as basal bilaterians are well-supported alternatives.     

Is the sperm type of the Nemertodermatida close to that of the ancestral Platyhelminthes?

Results from a transmission electron microscope study of the spermiogenesis and spermatozoon of Meara stichopi (Nemertodermatida, Platyhelminthes) indicate that the sperm type of the Nemertodermatida has evolved from the primitive metazoan sperm type rather than from an aberrant biflagellar sperm type as found in many other flatworms. The spirally coiled mitochondrial derivative in the mature spermatozoon develops from two large oval mitochondria in the early spermatid stages. A single flagellum grows out from a peripheral basal body adjacent to a perpendicularly placed accessory centriole. The basal body moves to a distal depression of the nucleus, and becomes equipped with an anchoring fibre apparatus. Most of the flagellum becomes axially incorporated into the developing spermatid. No trace of a second flagellum was found in any stage of the spermiogenesis. Rounded vesicles appear around the proximal, tapering end of the elongating nucleus. Most probably these vesicles form a thin acrosomal structure in the mature spermatozoon. No dense bodies, characteristic of many other ‘turbellarian’ flatworm sperm types, were found. This revised version was published online in July 2006 with corrections to the Cover Date.     

Molecular phylogeny of Gastrotricha on the basis of a comparison of the 18S rRNA genes: Rejection of the hypothesis of a relationship between Gastrotricha and Nematoda

Gastrotricha are the small meiobenthic acoelomate worms whose phylogenetic relationships between themselves and other invertebrates remain unclear, despite all attempts to clarify them on the basis of both morphological and molecular analyses. The complete sequences of the 18S rRNA genes (8 new and 7 known) were analyzed in 15 Gastrotricha species to test different hypotheses on the phylogeny of this taxon and to determine the reasons for the contradictions in earlier results. The data were analyzed using both maximum likelihood and Bayesian methods. Based on the results, it was assumed that gastrotrichs form a monophyletic group within the Spiralia clade, which also includes Gnathostomulida, Plathelminthes, Syndermata (Rotifera + Acanthocephala), Nemertea, and Lophotrochozoa. Statistical tests rejected a phylogenetic hypotheses considering Gastrotricha to be closely related to Nematoda and other Ecdysozoa or placing them at the base of the Bilateria tree, close to Acoela or Nemertodermatida. Among gastrotrichs, species belonging to the orders Chaetonotida and Macrodasyida form two well-supported clades. The analysis confirmed monophyly of the families Chaetonotidae and Xenotrichulidae from the order Chaetonida, as well as the families Turbanellidae and Thaumastodermatidae from the order Macrodasyida. Lepidodasyidae is a polyphyletic family, because the genus Mesodasys forms a sister group for Turbanellidae; genus Cephalodasys forms a separate branch at the base of Macrodasyida; and Lepidodasys groups with Neodasys between Thaumastodermatidae and Turbanellidae. To confirm these conclusions and to get an authentic view of the phylogeny of Gastrotricha, it is necessary to study more Gastrotricha species and to analyze some other genes.     

Phylogenetic position of Nemertea derived from phylogenomic data

Nemertea and Platyhelminthes have traditionally been grouped together because they possess a so-called acoelomate organization, but lateral vessels and rhynchocoel of nemerteans have been regarded as coelomic cavities. Additionally, both taxa show spiral cleavage patterns prompting the placement of Nemertea as sister to coelomate Protostomia, that is, either to Neotrochozoa (Mollusca and Annelida) or to Teloblastica (Neotrochozoa plus Arthropoda). Some workers maintain a sister group relationship of Nemertea and Platyhelminthes as Parenchymia because of an assumed homology of G?tte's and Müller's larvae of polyclad Platyhelminthes and the pilidium larvae of heteronemerteans. So far, molecular data were only able to significantly reject a sister group relationship to Teloblastica. Although phylogenomic data are available for Platyhelminthes, Annelida, Mollusca, and Arthropoda, they are lacking for Nemertea. Herein, we present the first analysis specifically addressing nemertean phylogenetic position using phylogenomic data. More specifically, we collected expressed sequence tag data from Lineus viridis (O.F. Müller, 1774) and combined it with available data to produce a data set of 9,377 amino acid positions from 60 ribosomal proteins. Maximum likelihood analyses and Bayesian inferences place Nemertea in a clade together with Annelida and Mollusca. Furthermore, hypothesis testing significantly rejected a sister group relationship to either Platyhelminthes or Teloblastica. The Coelomata hypothesis, which groups coelomate taxa together to the exclusion of acoelomate and pseudocoelomate taxa, is not congruent with our results. Thus, the supposed acoelomate organization evolved independently in Nemertea and Platyhelminthes. In Nemertea, evolution of acoely is most likely due to a secondary reduction of the coelom as it is found in certain species of Mollusca and Annelida. Though looking very similar, the G?tte's and Müller's larvae of polyclad Platyhelminthes are not homologous to the pilidium larvae of heteronemerteans. Finally, the convergent evolution of segmentation in Annelida and Arthropoda is further substantiated.     

衣藻属的系统发育分析——基于形态形状和nrDNA ITS序列

通过实验分析莱茵衣藻 ( Chlamydomonas reinhardtii) 1个种和互连网获得衣藻属 1 5个种及丝藻属 1个种 ( Ulothrix zonata) ,共 1 7个种的 nr DNA ITS序列 ,并以 U.zonata为外类群 ,采用计算机分析软件包对其进行分析及构建分子系统发育树图。同时以 1 2个传统分类性状 ,对此 1 6种衣藻构建数据矩阵 ;以 U.zonata动孢子的相应性状为外类群原始性状 ,用Wagner法在计算机上对其进行分枝分析 ;然后比较并分析分子系统树和表征性状分支分析树的异同。初步尝试以 ITS分子序列系统发育分析作为传统性状分析的补充来研究衣藻种间的亲缘关系。