Early Branching Clade Research Articles

Phylogenetic position of Zygogonium ericetorum (zygnematophyceae, charophyta) from a high alpine habitat and ultrastructural characterization of unusual aplanospores.

Zygogonium ericetorum, the type species of the genus, was studied from a natural population collected in Mt. Schönwieskopf, Tyrol, Austria. Generic concepts of Zygogonium and Zygnema were tested with atpB, psbC, and rbcL gene sequence analysis, which showed a sister relationship between Z. ericetorum and Mesotaenium, in an early branching clade sister to a grouping of Zygnema and several other filamentous and unicellular zygnematalean taxa. A variety of light, confocal, transmission electron microscopy, and cytochemical techniques provided new data on the variable chloroplast shape of Z. ericetorum, and its aplanospore structure and development, which has been previously considered taxonomically important but has been ambiguously interpreted. Zygogonium can be distinguished from other zygnematophytes (particularly Zygnema), based on the combination of two characters: (i) irregular, compressed plate-like chloroplasts and (ii) residual cytoplasmic content left in sporangia outside of the fully developed aplanospores or zygospores. The presence of a sporangial wall that separates the spores from the parent cell should be excluded from the definition of Zygogonium, because it is also observed in Zygnema. Similarly, the ecological characterization of Zygogonium as acidophilic is not unique to the genus. The names of 18 species currently belonging to Zygogonium are here changed to Zygnema, because of incompatibility with this new proposed Zygogonium concept. In the species transferred to Zygnema, chloroplasts are typically stellate in three-dimensions, and the entire content of fertile cells is transformed into the spore, so there is no cytoplasmic residue.

<i>Moraea orthrosantha</i> (Iridaceae: Irideae), a new species from Namaqualand, South Africa

Background: Recent fieldwork in Namaqualand, Northern Cape Province, South Africa,indicated the existence of an undescribed member of Moraea subgenus (subg.) Umbellatae Goldblatt J.C.Manning, a small, early branching clade of the genus.Objectives: To describe the new species of Moraea subg. Umbellatae.Method: Recent collections were compared with existing material and published literature.Results: Moraea orthrosantha is described as a new species, differing from Moraea margaretae inthe well-developed nodes between the cauline leaves. Two earlier collections of the species weremisidentified as M. margaretae.Conclusion: The new species increases our understanding of the diversity of Moraea in southernAfrica and assists in conservation assessments of both M. margaretae and M. orthrosantha.

Put a tiger in your tank: the polyclad flatworm Maritigrella crozieri as a proposed model for evo-devo

Polyclad flatworms are an early branching clade within the rhabditophoran Platyhelminthes. They provide an interesting system with which to explore the evolution of development within Platyhelminthes and amongst Spiralia (Lophotrochozoa). Unlike most other flatworms, polyclads undergo spiral cleavage (similar to that seen in some other spiralian taxa), they are the only free-living flatworms where development via a larval stage occurs, and they are the only flatworms in which embryos can be reared outside of their protective egg case, enabling embryonic manipulations. Past work has focused on comparing early cleavage patterns and larval anatomy between polyclads and other spiralians. We have selected Maritigrella crozieri, the tiger flatworm, as a suitable polyclad species for developmental studies, because it is abundant and large in size compared to other species. These characteristics have facilitated the generation of a transcriptome from embryonic and larval material and are enabling us to develop methods for gene expression analysis and immunofluorescence techniques. Here we give an overview of M. crozieri and its development, we highlight the advantages and current limitations of this animal as a potential evo-devo model and discuss current lines of research.

Revisiting the phylogeny of papilionoid legumes: New insights from comprehensively sampled early‐branching lineages

Phylogenetic relationships of the papilionoid legumes (Papilionoideae) reveal that the early branches are more highly diverse in floral morphology than are other clades of Papilionoideae. This study attempts for the first time to comprehensively sample the early-branching clades of this economically and ecologically important legume subfamily and thus to resolve relationships among them. • Parsimony and Bayesian phylogenetic analyses of the plastid matK and trnL intron sequences included 29 genera not yet sampled in matK phylogenies of the Papilionoideae, 11 of which were sampled for DNA sequence data for the first time. • The comprehensively sampled matK phylogeny better resolved the deep-branching relationships and increased support for many clades within Papilionoideae. The potentially earliest-branching papilionoid clade does not include any genus traditionally assigned to tribe Swartzieae. Dipterygeae is monophyletic with the inclusion of Monopteryx. The genera Aldina and Amphimas represent two of the nine main but as yet unresolved lineages comprising the large 50-kb inversion clade within papilionoids. The quinolizidine-alkaloid-accumulating genistoid clade is expanded to include a strongly supported subclade containing Ormosia and the previously unplaced Clathrotropis s.s., Panurea, and Spirotropis. Camoensia is the first-branching genus of the core genistoids. • The well-resolved phylogeny of the earliest-branching papilionoids generated in this study will greatly facilitate the efforts to redefine and stabilize the classification of this legume subfamily. Many key floral traits did not often predict phylogenetic relationships, so comparative studies on floral evolution and plant-animal interactions, for example, should also benefit from this study.

VincetoxicumandTylophora(Apocynaceae: Asclepiadoideae: Asclepiadeae)—two sides of the same medal: Independent shifts from tropical to temperate habitats

AbstractA revised phylogeny of Tylophorinae, based on 77 species of all genera presently attributed to the subtribe(Biondia, Blyttia, Diplosstigma, Goydera, Pentatropiss, Pleurostelma, Rhyncharrhena, Tylophora, Vincetoxicum)is presented. Five chloroplast markers(trnT–L,trnL–F,andpssbA–trnHintergenic spacers as well as thetrnLandtrnGintrons) and two nuclear markers (ITS, ETS) were used, totalling in 3809 base pairs. Tylophorinae are strongly monophyletic and consist of several well–defined clades. Backbone resolution between these clades is low, indicating a rapid early radiation.Pentatropissis sister to all remaining taxa, albeit with weak support; all other taxa form one big clade not corresponding to previous generic delimitations. Therefore, all genera except forPentatropissare subsumed underVincetoxicum.The early branching clades ofVincetoxicums.l. all grow in Africa, where the group originated ca. 18 Ma ago, correlating with the closure of the Tethys Ocean. The first round of differentiation took place between 12 and 15 Ma ago, resulting in Tylophorinae being distributed over almost all of their present range except for South Africa, Arabia and Europe. Two unrelated lineages of temperate, mostly erect plants hitherto named‘“Vincetoxicum”have arisen from more tropical lineages, hitherto named“Tylophora”.One clade of African species is nested inside the Australasian assemblage; this clade contains exclusively polyploid species and its re–immigration to Africa took place in the Messinian, at ca. 6.8 Ma. The EuropeanVincetoxicumspecies split from erect steppe plants of temperate Asia at ca. 4.5 Ma, coinciding with the uplift of the Tibetan plateau. This group contains two species that are presently aggressively spreading in North America after chance introductions.

Age and historical biogeography of the pantropically distributed Spathelioideae (Rutaceae, Sapindales)

AbstractAim The family Rutaceae (rue family) is the largest within the eudicot order Sapindales and is distributed mainly in the tropical and subtropical regions of both the New World and the Old World, with a few genera in temperate zones. The main objective of this study is to present molecular dating and biogeographical analyses of the subfamily Spathelioideae, the earliest branching clade (which includes eight extant genera), to interpret the temporal and spatial origins of this group, ascertaining possible vicariant patterns and dispersal routes and inferring diversification rates through time.Location Pantropics.Methods A dataset comprising a complete taxon sampling at generic level (83.3% at species level) of Spathelioideae was used for a Bayesian molecular dating analysis (beast). Four fossil calibration points and an age constraint for Sapindales were applied. An ancestral area reconstruction analysis utilizing the dispersal–extinction–cladogenesis model and diversification rate analyses was conducted.Results Dating analyses indicate that Rutaceae and Spathelioideae are probably of Late Cretaceous origin, after which Spathelioideae split into a Neotropical and a Palaeotropical lineage. The Palaeotropical taxa have their origin inferred in Africa, with postulated dispersal events to the Mediterranean, the Canary Islands, Madagascar and Southeast Asia. The lineages within Spathelioideae evolved at a relatively constant diversification rate. However, abrupt changes in diversification rates are inferred from the beginning of the Miocene and during the Pliocene/Pleistocene.Main conclusions The geographical origin of Spathelioideae probably lies in Africa. The existence of a Neotropical lineage may be the result of a dispersal event at a time in the Late Cretaceous when South America and Africa were still quite close to each other (assuming that our age estimates are close to the actual ages), or by Gondwanan vicariance (assuming that our age estimates provide minimal ages only). Separation of land masses caused by sea level changes during the Pliocene and Pleistocene may have been triggers for speciation in the Caribbean genus Spathelia.

Phylogenetic and Ecological Signals in the Wood of Spathelioideae (Rutaceae)

Subfamily Spathelioideae of Rutaceae constitutes a well-supported early branching clade of eight small woody genera that were formerly assigned to five different Sapindalean/Rutalean families. This study brings together detailed wood anatomical information on all eight genera (for four the wood anatomy is described for the first time in detail). Wood anatomy strongly supports the inclusion of all Spathelioid genera in Rutaceae and underpins the molecular phylogeny with a set of interesting apomorphies at different nodes of the cladogram. The wood anatomy of Cneorum tricoccon with its semi-ring porosity, dendritic vessel pattern, vascular tracheids and helical vessel wall thickenings stands out in Spathelioideae. This wood anatomical syndrome is hypothesized to be due to adaptive evolution for hydraulic safety and efficiency of this species in a typical Mediterranean climate, where similar syndromes have evolved in many unrelated clades of woody dicots. In at least six unrelated genera of Rutaceae outside Spathelioideae from Mediterranean or cool temperate and montane climates, the syndrome has also evolved in presumably parallel, adaptive evolution.

Evolution of MIR159/319 microRNA genes and their post-transcriptional regulatory link to siRNA pathways

BackgroundMicroRNAs (miRNAs) are prevalent and important endogenous gene regulators in eukaryotes. MiR159 and miR319 are highly conserved miRNAs essential for plant development and fertility. Despite high similarity in conservation pattern and mature miRNA sequences, miR159 and miR319 have distinct expression patterns, targets and functions. In addition, both MIR319 and MIR159 precursors produce multiple miRNAs in a phased loop-to-base manner. Thus, MIR159 and MIR319 appear to be related in origin and considerably diverged. However the phylogeny of MIR159 and MIR319 genes and why such unusual style of miRNA production has been conserved during evolution is not well understood.ResultsWe reconstructed the phylogeny of MIR159/319 genes and analyzed their mature miRNA expression. The inferred phylogeny suggests that the MIR159/319 genes may have formed at least ten extant early-branching clades through gene duplication and loss. A series of duplications occurred in the common ancestor of seed plants leading to the original split of flowering plant MIR159 and MIR319. The results also indicate that the expression of MIR159/319 is regulated at post-transcriptional level to switch on the expression of alternative miRNAs during development in a highly spatio-temporal specific manner, and to selectively respond to the disruption of defensive siRNA pathways. Such intra-stem-loop regulation appears diverged across the early-branching clades of MIR159/319 genes.ConclusionsOur results support that the MIR159 and MIR319 genes evolve from a common ancestor, which is likely to be a phased stem-loop small RNA. Through duplication and loss of genes this miRNA gene family formed clades specific to moss, lycopods, gymnosperms and angiosperms including the two major clades of flowering plants containing the founding members of MIR319 and MIR159 genes in A.thaliana. Our analyses also suggest that some MIR159/319 have evolved into unusual miRNA genes that are regulated at post-transcriptional level to express multiple mature products with variable proportions under different circumstances. Moreover, our analyses reveal conserved regulatory link of MIR159/319 genes to siRNA pathway through post-transcriptional regulation.

Amylocorticiales ord. nov. and Jaapiales ord. nov.: Early diverging clades of Agaricomycetidae dominated by corticioid forms

The Agaricomycetidae is one of the most morphologically diverse clades of Basidiomycota that includes the well known Agaricales and Boletales, which are dominated by pileate-stipitate forms, and the more obscure Atheliales, which is a relatively small group of resupinate taxa. This study focused taxon sampling on resupinate forms that may be related to these groups, aimed at resolving the early branching clades in the major groups of Agaricomycetidae. A specific goal was to resolve with confidence sister group relationships among Agaricales, Boletales and Atheliales, a difficult task based on conflicting results concerning the placement of the Atheliales. To this end we developed a six-locus nuclear dataset (nuc-ssu, nuc-lsu, 5.8S, rpb1, rpb2 and tef1) for 191 species, which was analyzed with maximum parsimony, maximum likelihood and Bayesian methods. Our analyses of these data corroborated the view that the Boletales are closely related to athelioid forms. We also identified an additional early branching clade within the Agaricomycetidae that is composed primarily of resupinate forms, as well as a few morphologically more elaborate forms including Plicaturopsis and Podoserpula. This clade, which we describe here as the new order Amylocorticiales, is the sister group of the Agaricales. We introduce a second order, the Jaapiales, for the lone resupinate genus Jaapia consisting of two species only. The Jaapiales is supported as the sister group of the remainder of the Agaricomycetidae, suggesting that the greatest radiation of pileate-stipitate mushrooms resulted from the elaboration of resupinate ancestors.

A neglected lineage of North American turtles fills a major gap in the fossil record

Abstract: The fossil record of the two primary subclades of softshell turtles (Trionychidae) is exceedingly asymmetric, as a result of a ghost range of total clade Cyclanorbinae that is estimated at 80 Ma. Herein, we present the first phylogenetic analysis of Trionychidae that includes a representative of the poorly studied taxon Plastomenidae, which is known from the Campanian to Eocene of North America. The analysis reveals that plastomenids are stem cyclanorbines, thus significantly reducing the apparent ghost range of total group Cyclanorbinae to approximately 30 Ma. Plastomenids are either an early branching clade of stem Cyclanorbinae, or they represent a paraphyletic grade that gave rise to modern cyclanorbines. Although abundant, the fossil record is still too poorly understood to distinguish between these two primary hypotheses. The previously persistent extremely long ghost range of total clade Cyclanorbinae appears to have been the result of a research bias.

Moving towards a complete molecular framework of the Nematoda: a focus on the Enoplida and early-branching clades

BackgroundThe subclass Enoplia (Phylum Nematoda) is purported to be the earliest branching clade amongst all nematode taxa, yet the deep phylogeny of this important lineage remains elusive. Free-living marine species within the order Enoplida play prominent roles in marine ecosystems, but previous molecular phylogenies have provided only the briefest evolutionary insights; this study aimed to firmly resolve internal relationships within the hyper-diverse but poorly understood Enoplida. In addition, we revisited the molecular framework of the Nematoda using a rigorous phylogenetic approach in order to investigate patterns of early splits amongst the oldest lineages (Dorylaimia and Enoplia).ResultsMorphological identifications, nuclear gene sequences (18S and 28S rRNA), and mitochondrial gene sequences (cox1) were obtained from marine Enoplid specimens representing 37 genera. The 18S gene was used to resolve deep splits within the Enoplia and evaluate the branching order of major clades in the nematode tree; multiple phylogenetic methods and rigorous empirical tests were carried out to assess tree topologies under different parameters and combinations of taxa. Significantly increased taxon sampling within the Enoplida resulted in a well-supported, robust phylogenetic topology of this group, although the placement of certain clades was not fully resolved. Our analysis could not unequivocally confirm the earliest splits in the nematode tree, and outgroup choice significantly affected the observed branching order of the Dorylaimia and Enoplia. Both 28S and cox1 were too variable to infer deep phylogeny, but provided additional insight at lower taxonomic levels.ConclusionsAnalysis of internal relationships reveals that the Enoplia is split into two main clades, with groups consisting of terrestrial (Triplonchida) and primarily marine fauna (Enoplida). Five independent lineages were recovered within the Enoplida, containing a mixture of marine and terrestrial species; clade structure suggests that habitat transitions have occurred at least four times within this group. Unfortunately, we were unable to obtain a consistent or well-supported topology amongst early-branching nematode lineages. It appears unlikely that single-gene phylogenies using the conserved 18S gene will be useful for confirming the branching order at the base of the nematode tree-future efforts will require multi-gene analyses or phylogenomic methods.

Characterization of the stem cell system of the acoel Isodiametra pulchra

BackgroundTissue plasticity and a substantial regeneration capacity based on stem cells are the hallmark of several invertebrate groups such as sponges, cnidarians and Platyhelminthes. Traditionally, Acoela were seen as an early branching clade within the Platyhelminthes, but became recently positioned at the base of the Bilateria. However, little is known on how the stem cell system in this new phylum is organized. In this study, we wanted to examine if Acoela possess a neoblast-like stem cell system that is responsible for development, growth, homeostasis and regeneration.ResultsWe established enduring laboratory cultures of the acoel Isodiametra pulchra (Acoela, Acoelomorpha) and implemented in situ hybridization and RNA interference (RNAi) for this species. We used BrdU labelling, morphology, ultrastructure and molecular tools to illuminate the morphology, distribution and plasticity of acoel stem cells under different developmental conditions. We demonstrate that neoblasts are the only proliferating cells which are solely mesodermally located within the organism. By means of in situ hybridisation and protein localisation we could demonstrate that the piwi-like gene ipiwi1 is expressed in testes, ovaries as well as in a subpopulation of somatic stem cells. In addition, we show that germ cell progenitors are present in freshly hatched worms, suggesting an embryonic formation of the germline. We identified a potent stem cell system that is responsible for development, homeostasis, regeneration and regrowth upon starvation.ConclusionsWe introduce the acoel Isodiametra pulchra as potential new model organism, suitable to address developmental questions in this understudied phylum. We show that neoblasts in I. pulchra are crucial for tissue homeostasis, development and regeneration. Notably, epidermal cells were found to be renewed exclusively from parenchymally located stem cells, a situation known only from rhabditophoran flatworms so far. For further comparison, it will be important to analyse the stem cell systems of other key-positioned understudied taxa.

THE IMPORTANCE OF PREADAPTED GENOMES IN THE ORIGIN OF THE ANIMAL BODYPLANS AND THE CAMBRIAN EXPLOSION

The genomes of taxa whose stem lineages branched early in metazoan history, and of allied protistan groups, provide a tantalizing outline of the morphological and genomic changes that accompanied the origin and early diversifications of animals. Genome comparisons show that the early clades increasingly contain genes that mediate development of complex features only seen in later metazoan branches. Peak additions of protein-coding regulatory genes occurred deep in the metazoan tree, evidently within stem groups of metazoans and eumetazoans. However, the bodyplans of these early-branching clades are relatively simple. The existence of major elements of the bilaterian developmental toolkit in these simpler organisms implies that these components evolved for functions other than the production of complex morphology, preadapting the genome for the morphological differentiation that occurred higher in metazoan phylogeny. Stem lineages of the bilaterian phyla apparently required few additional genes beyond their diploblastic ancestors. As disparate bodyplans appeared and diversified during the Cambrian explosion, increasing complexity was accommodated largely through changes in cis-regulatory networks, accompanied by some additional gene novelties. Subsequently, protein-coding genic richness appears to have essentially plateaued. Some genomic evidence suggests that similar stages of genomic evolution may have accompanied the rise of land plants.

The diploid nature of the Chilean Epipetrum and a new base number in the Dioscoreaceae

Chromosomal analyses were conducted for the first time in all three species of the Chilean endemic Epipetrum Phil. (Dioscoreaceae; the yam family). Mitotic chromosome counts showed that all studied individuals of the rare Epipetrum bilobum and E. polyanthes, and most studied individuals of the more widespread E. humile, had 2n = 14 chromosomes. However, two populations of E. humile had 2n = 28 chromosomes. The ideograms shared a similar karyotype constitution for the three species but with a duplicated chromosome number in some populations of E. humile. Our results suggest that the Epipetrum taxa are mainly diploids, with a base number of x = 7, and the tetraploid cytotypes have arisen only in few cases. After that of Borderea (x = 6), this chromosome base number is the second lowest one found in the Dioscoreaceae. Epipetrum and Borderea are two small, relict, satellite genera of the Dioscoreaceae, endemic to Chile (South America) and the Pyrenees (Southwestern Europe), respectively. The two taxa share several morphological attributes and are adapted to rocky and mountain habitats. However, whereas Borderea is an allotetra‐ploid genus of relatively recent hybrid origin, Epipetrum is diploid in common with the species found in the early branching clades of Dioscorea.

18S rDNA phylogeny and evolution of cap development in Polyphysaceae (formerly Acetabulariaceae; Dasycladales, Chlorophyta)

Cells of the members of the Dasycladales have a unique body plan well known from fossils. They persist today in 38 recognized species. This study investigates in detail the development of reproductive structures in 17 Polyphysaceae (= Acetabulariaceae) species and provides a molecular phylogenetic analysis of 18S ribosomal DNA sequence data of 23 species of the order Dasycladales, including 17 of the 19 extant members of the family Polyphysaceae. Reproductive cap development is documented by scanning electron microscopy in 17 species, by histological sections in five species, and by growth measurements. Other morphometric data are also provided for most species. Bayesian analysis of DNA data reveals three well-supported clades for which morphological synapomorphies exist, but which are not completely in accordance with previous generic concepts. An early-branching clade comprising the monotypic Chalmasia and two species of Halicoryne is characterized by the formation of buds by the pointed cap rays. The second clade, comprising five species of Polyphysa (except P. peniculus), is characterized by corrugation of the cell stalk. It forms a sister group to the third clade, which comprises all eight Acetabularia species, plus P. peniculus and Acicularia schenckii. This clade is united by the lack of a velum covering the developing caps and by the relative timing of cap ray initiation. The extended Acetabularia clade is also characterized by the presence of a corona inferior, a structure that apparently was secondarily reduced in P. peniculus. Acetabularia acetabulum, a model species for cell biology, is sister to all other species of this clade. Its unique, congenitally fused cap rays may have evolved by a simple shift of growth zones during cap initiation. We propose to include both the monotypic Acicularia (as Acetabularia schenckii) and Polyphysa peniculus (as Acetabularia peniculus) in the genus Acetabularia. For the remaining Polyphysa species of clade 2, a new genus name, Parvocaulis S. Berger et al., is proposed.

Molecular phylogeny of conjugating green algae (Zygnemophyceae, Streptophyta) inferred from SSU rDNA sequence comparisons.

Nuclear-encoded SSU rDNA sequences have been obtained from 64 strains of conjugating green algae (Zygnemophyceae, Streptophyta, Viridiplantae). Molecular phylogenetic analyses of 90 SSU rDNA sequences of Viridiplantae (inciuding 78 from the Zygnemophyceae) were performed using complex evolutionary models and maximum likelihood, distance, and maximum parsimony methods. The significance of the results was tested by bootstrap analyses, deletion of long-branch taxa, relative rate tests, and Kishino-Hasegawa tests with user-defined trees. All results support the monophyly of the class Zygnemophyceae and of the order Desmidiales. The second order, Zygnematales, forms a series of early-branching clades in paraphyletic succession, with the two traditional families Mesotaeniaceae and Zygnemataceae not recovered as lineages. Instead, a long-branch Spirogyra/Sirogonium clade and the later-diverging Netrium and Roya clades represent independent clades. Within the order Desmidiales, the families Gonatozygaceae and Closteriaceae are monophyletic, whereas the Peniaceae (represented only by Penium margaritaceum) and the Desmidiaceae represent a single weakly supported lineage. Within the Desmidiaceae short internal branches and varying rates of sequence evolution among taxa reduce the phylogenetic resolution significantly. The SSU rDNA-based phylogeny is largely congruent with a published analysis of the rbcL phylogeny of the Zygnemophyceae (McCourt et al. 2000) and is also in general agreement with classification schemes based on cell wall ultrastructure. The extended taxon sampling at the subgenus level provides solid evidence that many genera in the Zygnemophyceae are not monophyletic and that the genus concept in the group needs to be revised.

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.