Molecular systematics of the Acoela (Acoelomorpha,Platyhelminthes) and its concordance with morphology

The phylogenetic relationships of the lower worm group Acoela were investigated using newly obtained nuclear 18S rDNA sequences from 16 acoels in combination with 16 acoel sequences available on GenBank from other laboratories. Parsimony and maximum likelihood analyses of the molecular data supported the concept that the Acoela is monophyletic; however, the gene tree produced by these analyses conflicts with the current taxonomic system for the Acoela in several family-level groupings. Most notable is the apparent polyphyly of the largest family of acoels, the Convolutidae. DNA analysis grouped together species of small-bodied convolutids in one clade, while large-bodied convolutids grouped in a separate clade with other large-bodied acoels. Despite such conflicts, the branching pattern in the gene tree is well supported by morphological characters of sperm and body-wall musculature.     

How the worm got its pharynx: phylogeny, classification and Bayesian assessment of character evolution in Acoela

Acoela are marine microscopic worms currently thought to be the sister taxon of all other bilaterians. Acoels have long been used as models in evolutionary scenarios, and generalized conclusions about acoel and bilaterian ancestral features are frequently drawn from studies of single acoel species. There is no extensive phylogenetic study of Acoela and the taxonomy of the 380 species is chaotic. Here we use two nuclear ribosomal genes and one mitochondrial gene in combination with 37 morphological characters in an analysis of 126 acoel terminals (about one-third of the described species) to estimate the phylogeny and character evolution of Acoela. We present an estimate of posterior probabilities for ancestral character states at 31 control nodes in the phylogeny. The overall reconstruction signal based on the shape of the posterior distribution of character states was computed for all morphological characters and control nodes to assess how well these were reconstructed. The body-wall musculature appears more clearly reconstructed than the reproductive organs. Posterior similarity to the root was calculated by averaging the divergence between the posterior distributions at the nodes and the root over all morphological characters. Diopisthoporidae is the sister group to all other acoels and has the highest posterior similarity to the root. Convolutidae, including several "model" acoels, is most divergent. Finally, we present a phylogenetic classification of Acoela down to the family level where six previous family level taxa are synonymized.     

Comparative morphology of the bursal nozzles in acoels (Acoela, Acoelomorpha)

Systematics of the Acoela is particularly difficult because of the paucity of readily discernible morphological features. In other soft-bodied worms, sclerotized structures, such as copulatory stylets, provide important characters that can be seen in whole mounts, but acoels generally lack such features. Among the few sclerotized structures in acoels are bursal nozzles-tubiform outlets on the seminal bursae that are believed to be conduits (spermatic ducts) through which allosperm are transported to the oocytes. Early classifications of the Acoela used features of the female reproductive system, including bursal nozzles, for distinguishing major groups, but the current system essentially ignores them as too plastic to provide higher-level distinctions. We used confocal and electron microscopy to further characterize bursal nozzles in five acoel species, and found all composed of actin-reinforced extensions of stacked, flat mesenchymal cells. In Notocelis gullmarensis, Aphanostoma bruscai, and Daku woorimensis, the nozzle is a stiffened region of the same cells forming the wall of the bursa. By contrast, in Wulguru cuspidata cells forming the nozzle are distinct from those of the bursa. The so-called bursal cap of A. bruscai and D. woorimensis has small sclerotized disjunct units within it, also composed of stacked, flat, actin-reinforced cells. The nozzle of W. cuspidata, prominent like that of other convolutid acoels, is relatively complex, its actin-reinforced cells sandwiched with secretory cells and its base bearing a "sorting apparatus" of egg-shaped cells that send narrow processes inside the spermatic duct. Cases of sperm inside the nozzle corroborate its assumed role in reproduction. Whereas most nozzles sit at the end of the bursa facing the ovary, in species of Pseudmecynostomum and purportedly in a few other acoels, they sit between the female pore and the bursa, constituting what we call a vaginal nozzle. All bursal nozzles of acoels show a common ground pattern indicating common ancestry, but certain features discerned through electron and confocal microscopy show promise of providing synapomorphies for grouping some species.     

Ultrastructural investigations on the differentiation of genital hard structures in free-living platyhelminths and their phylogenetic significance

Ultrastructure and differentiation of penis stylets and stylet needles have been investigated in representatives of various groups of free-living platyhelminths, viz. the Acoela, Macrostomida, Typhloplanoida, Kalyptorhynchia, and Dalyellioida. In all these groups, the differentiation of such hard parts occurs intracellularly but in different ways in the different groups. The ultrastructure of the bursal mouth piece in an acoel platyhelminth is not comparable to the hard structures in male copulatory organs. The presence of penial copulatory organs having intracellular hard structures appears to be an autapomorphy of the Euplatyhelminthes. Several characters in the ultrastructure and development of these structures can be used as autapomorphies for various platyhelminth groups.     

A new viviparous acoel Childia vivipara sp. nov. with observations on the developing embryos, sperm ultrastructure, body wall and stylet musculatures

A free‐living viviparous acoel, Childia vivipara sp. nov., from the Gullmar fjord of the Swedish coast is described. The new species is assigned to the taxon Childia based on histological, ultrastructural and molecular sequence similarities. All available molecular markers (18S rRNA, 28S rRNA and histone H3) and several morphological characters, obtained using transmission electron microscopy and confocal scanning laser microscopy of whole mount specimen stained with TRITC‐labelled phalloidin, support the placement of C. vivipara in the taxon Childia. Childia vivipara and other Childia species share the following morphological synapomorphies: well‐developed copulatory organs built of tightly packed stylet needles, proximal part of the stylet inserted into the seminal vesicle, reversed body‐wall musculature, absence of ventral diagonal muscles, presence of dorsal diagonal muscles, and presence of ventral straight longitudinal muscles between frontal pore and mouth, 9 + 1 sperm axoneme structure, six distal sperm cytoplasmic microtubules, and extensive overlap of axonemes and nucleus. The new species can be easily distinguished from other Childia species by its viviparous mode of reproduction and single curved stylet. Observations on late embryonic development based on the oldest developing embryos are discussed.     

Distinctive features of cilia in metazoans and their significance for systematics.

A comparative study of epidermal cilia in the Turbellaria and Nemertea has revealed features in these organelles that are specific to certain taxonomic groups. Turbellarians of the order Acoela, in particular, have a characteristic pattern of axonemal filament termination in the distal tips of their cilia and a characteristic ciliary rootlet system that is not seen in other turbellarian orders nor in other metazoans. Each epidermal cilium in acoels has a typical 9 + 2 axonemal pattern through the main part of its length, but near its distal tip there is an abrupt shelf-life narrowing at which filaments 4-7 terminate; filaments 1, 2, 8 and 9 continue into the thinner distal-most part of the shaft along with singlet microtubules from the axonemal center. The rootlet system in acoel cilia involves an interconnecting pattern with lateral connectives. The unique structure of these cilia has systematic and phylogenetic significance for the Acoela, and it is argued that ultrastructural characters in general, including characters of organelles, can be validly applied to the phylogeny and systematics of the Metazoa.     

Monograph of the solenofilomorphidae (Turbellaria: Acoela)

Morphological studies of eleven new species in the family permitted reconstruction of progressive evolutionary lines, and this strongly implies progressive evolutionary lines for other acoela. Solenofilomorpha funilis n. sp., Myopea crassula n. g. n. sp., M. latafaucium n. sp., Fusantrum rhammiphorum n. g. n. sp. and Endocincta punctata n. g. n. sp. are described from the coast of South Carolina, U.S.A., and S. guaymensis n. sp. is described from the Gulf of California. Five additional unnamed species from the U. S. Pacific Coast, Sweden and Tunisia are briefly described. Two species groupings were based on correlation between two different arrangements of pharynx muscles and trio arrangements of antrum muscles and further correlations with lesser characters. The membership of the two groupings in a single family make it clear that evolution proceeded by addition of structures to a less complex common ancestor. Functional and behavioral considerations support the evolutionary path drawn from morphology. Distinctive statocyst structure, ciliary interconnections and sperm morphology shared by acoels show them to have a common ancestry, but clearly different pharynges and male structures cannot be consistent with evolution by reductions. A common ancestor of low complexity has probably given rise to several partly parallel evolutionary lines which together form the structurally diverse Acoela. The history of acoel systematics is also briefly summarized.     

Ancestral state reconstruction reveals multiple independent evolution of diagnostic morphological characters in the "Higher Oribatida" (Acari), conflicting with current classification schemes

Background  

The use of molecular genetic data in phylogenetic systematics has revolutionized this field of research in that several taxonomic groupings defined by traditional taxonomic approaches have been rejected by molecular data. The taxonomic classification of the oribatid mite group Circumdehiscentiae ("Higher Oribatida") is largely based on morphological characters and several different classification schemes, all based upon the validity of diagnostic morphological characters, have been proposed by various authors. The aims of this study were to test the appropriateness of the current taxonomic classification schemes for the Circumdehiscentiae and to trace the evolution of the main diagnostic traits (the four nymphal traits scalps, centrodorsal setae, sclerits and wrinkled cuticle plus octotaxic system and pteromorphs both in adults) on the basis of a molecular phylogenetic hypothesis by means of parsimony, likelihood and Bayesian approaches.     

Evolution of body-wall musculature in the Platyhelminthes (Acoelomorpha, Catenulida, Rhabditophora)

In an effort to understand the phylogeny of the Platyhelminthes, the patterns of body-wall musculature of flatworms were studied using fluorescence microscopy and Alexa-488-labeled phalloidin. Species of the Catenulida have a simple orthogonal gridwork of longitudinal and circular muscles. Members of the Rhabditophora have the same gridwork of musculature, but also have diagonal muscles over their entire body. Although a few species of Acoelomorpha possessed a simple orthogonal grid of musculature, most species typically have distinctly different patterns of dorsal and ventral body-wall musculature that include sets of longitudinal, circular, U-shaped, and several kinds of diagonal muscles. Several distinct patterns of musculature were identified, including 8 patterns in 11 families of acoels. These patterns have proven to be useful in clarifying the phylogeny of the Acoelomorpha, particularly with regard to the higher acoels. Patterns of musculature as well as other morphological characters are used here for revisions of acoel systematics, including the return of Eumecynostomum sanguineum (Mecynostomidae) to the genus Aphanostoma (Convolutidae), the revision of the family Childiidae, and the formation of a new family, Actinoposthiidae.     

New tools for resolving phylogenies: a systematic revision of the Convolutidae (Acoelomorpha, Acoela)

Molecular sequence data, morphological characters of spermatozoa, and newly obtained morphological characters of penis musculature are used here to revise the systematics of the family Convolutidae (Acoela). Species having isodiametric penes with non-anastomosing longitudinal muscles are transferred to the family Isodiametridae fam. nov. Species with longitudinal penis muscle fibres that anastomose or cross-over each other remain in the Convolutidae. Some species of the genera Convoluta and Conaperta (Convolutidae) are transferred to the genus Isodiametra gen. nov. (Isodiametridae fam. nov.). The genus Stomatricha (Otocelididae) is transferred to the family Convolutidae. Convoluta opisthandropora (Convolutidae) is transferred to the genus Pseudohaplogonaria (Haploposthiidae). Aphanostoma sanguineum (Convolutidae) is transferred to the genus Pseudactinoposthia (Actinoposthiidae).     

Morphology and ultrastructure of the pharynx in Solenofilomorphidae (Acoela)

The homology of pharynges within the mostly pharynx-less Acoela has been a matter of discussion for decades. Here, we analyze the pharynges of three members of the Solenofilomorphidae, Myopea sp. and two species of the genus Solenofilomorpha, by means of light and transmission electron microscopy. Special focus is placed on the ultrastructure of the pharyngeal musculature, epidermis surrounding the mouth, pharyngeal epithelium, and junction with the digestive parenchyma. The main goal of this study was to evaluate the usefulness of certain characters for broader comparisons within the Acoela. Among the three species, characters relating to position of the mouth, presence and elaboration of sphincter muscles, presence of pharyngeal glands, and ultrastructure of epitheliosomes proved to be variously species- and genus-specific. The arrangement of pharyngeal muscles and their connection with body wall musculature, ultrastructure of receptor cells, and morphology of a nonciliated glandular region in the posterior pharynx, in contrast, appear to be characteristic of the family Solenofilomorphidae and thus of predominant interest for comparisons with other acoel families.     

Revision of the Childiidae (Acoela), a total evidence approach in reconstructing the phylogeny of acoels with reversed muscle layers

The Childiidae sensu Dörjes 1968 comprises the acoel worms characterized by a cone‐shaped penis with muscular or sclerotized elements. Based on differences in body‐wall musculature arrangement, Hooge (2001) recently restricted the family to the genus Childia Graff, 1910 and placed the remaining genera to his new family Actinoposthiidae Hooge 2001 . This rearrangement has been questioned ( Raikova et al. 2004 ). We reconstructed the phylogeny of the Childiidae sensu Dörjes 1968 by means of a total evidence analysis including Histone H3, 28S rDNA and new 18S rDNA sequences, as well as 50 morphological characters. New characters of the muscular system and copulatory organs discovered through confocal laser scanning microscopy of phalloidin‐stained specimens are included in the phylogenetic analysis. A total of 12 taxa (nine ingroup and three outgroup) were used in the parsimony analysis of the 18S data set, which was aligned with different parameters for a sensitivity analysis, and the combined data set (18S + 28S + H3 + morphology). Incongruence in the node support of the groups among the four partitions was very low in the total evidence tree; except for the H3 partition. The conflict observed in the H3 partition is likely due to large homoplasy observed in the synonymous alternatives at both first and third codon positions. All data partitions demonstrated that Actinoposthia beklemischevi Mamkaev 1965 , and the newly defined taxon Childiidae (comprising Childia and Paraphanostoma Westblad 1942 ) are not close relatives. The monophyly of Childia and Paraphanostoma is strongly supported by both the 18S and 28S data partitions. Our study also reveals additional apomorphies uniting Childia with Paraphanostoma from body‐wall musculature, statocyst muscles and male copulatory organ. Muscular system, statocyst muscles, male copulatory organ and nervous system characters proved to be the best characters for taxonomic delimitations of subtaxa within the Childiidae, whereas the seminal bursa (a frequently used character in the taxonomy of Acoela) was highly homoplastic. We also described the body‐wall musculature of six Paraphanostoma species, which is characterized by the reversed arrangement of the longitudinal and circular muscle layers, and by the absence of diagonal muscles on the ventral side of the body and the presence of two types of diagonal muscles on the dorsal side. Childia groenlandica (Levinsen, 1879) is nested among the Paraphanostoma species in our total evidence tree, so we synonymize Paraphanostoma with Childia; all former members of Paraphanostoma are transferred to Childia.     

Elongation factor 1-alpha sequences do not support an early divergence of the Acoela

The phylogenetic position of the Acoela is a key problem in the understanding of metazoan evolution. Recent studies based on 18S ribosomal DNA (rDNA) sequences have placed the Acoela in an extremely basal position as the sister group to all other extant triploblastic animals, suggesting that the phylum Platyhelminthes is polyphyletic. In order to test the results obtained with 18S rDNA, we sequenced elongation factor 1-alpha (EF1a) for the acoel Convoluta roscoffensis and five species of Turbellaria (two polyclads, Leptoplana tremellaris, and Prostheceraeus vittatus, and three triclads, Crenobia alpina, Schmidtea polychroa, and Girardia tigrina). Phylogenetic analyses of EF1a sequences show that the acoel sequences branch within the Platyhelminthes, in opposition to the 18S rDNA data. Moreover, comparison of the central variable region of EF1a shows similar sequence signatures between C. roscoffensis and the three triclad species. Although EF1a sequences fail to prove the monophyly of the phylum Platyhelminthes, they do not confirm the early divergence of the Acoela.     

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.     

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.     

New model for the formation and function of sagittocysts: Symsagittifera corsicae n. sp. (Acoela)

Abstract. This study is focused on the formation and function of sagittocysts, which are secretions typical of members of the acoel family Sagittiferidae. The needle-shaped sagittocysts are produced in specialized gland cells (sagittocytes) whose distal necks are often surrounded by muscle mantles. Contraction of the muscle mantle ejects the sagittocyst. We establish a model for the development of sagittocytes and muscle mantles out of the stem cell pool of the new acoel species Symsagittifera corsicae . We used various techniques, especially interference and phase-contrast microscopy of living specimens as well as labeling of the body-wall musculature, for species characterization. In addition to the morphological features, we provide the third complete sequence of the 18S rDNA gene in the family Sagittiferidae.     

Ultrastructure of sperms in Acoela (Acoelomorpha) and its concordance with molecular systematics

Abstract. The sperms of the Acoela, a group of lower worms, are filiform cells with 2 flagella incorporated into the cell body. Their axonemes can variously have 9+2, 9+1, or 9+0 patterns of microtubules; and singlet microtubules in the cell body can be arranged in axial or cortical positions. An analysis of phylogenetic relationships of acoels based on molecular characters (18S rDNA sequence data) showed that these patterns of microtubules, where known, fell into discrete monophyletic groups. To test this hypothesis, we have expanded the database of sperm characters by examining the ultrastructure of a further 10 species representing 4 acoel families. As expected, the Convolutidae fell into 2 unrelated groups: “small‐bodied convolutids”(Convoluta pulchra, Praeconvoluta tigrina, Pseudaphanostoma smithrii) having 9+2 axonemes and cortical microtubules, and “large‐bodied convolutids” (including Wulguru cuspidata) having 9+0 axonemes and axial microtubules. Also, as expected, a member of the Mecynostomidae (Paedomecynostomum bruneum) has 9+1 axonemes and axial microtubules. Members of a family that appears intermediate by molecular characters, the Otocelididae, significantly have a variety of patterns: axonemes with both 9+2 and 9+0 patterns (Notocelis gullmarensis) or just 9+2 (the other species), and either axial (Philocelis brueggemanni), both axial and cortical (N. gullmarensis) microtubules, or microtubules that bend between axial and cortical positions along the length of the sperm (Otocelis sandara). Members of the Dakuidae (Daku woorimensis) also belong to this intermediate group, having 9+2 axonemes and axial microtubules, while in a fifth otocelidid (Stomatricha hochbergi), sperm characters are like those of the “large‐bodied convolutids” (9+0 axonemes and axial microtubules). Characters of sperm morphology generally support the molecular hypothesis of relationships and confirm a suspected polyphyly of the families Convolutidae, Otocelididae, and Actinoposthiidae.     

Phylogenetics and evolution of Capitata (Cnidaria: Hydrozoa), and the systematics of Corynidae

Nawrocki, A. M., Schuchert, P. & Cartwright, P. (2009). Phylogenetics and evolution of Capitata (Cnidaria: Hydrozoa), and the systematics of Corynidae.—Zoologica Scripta, 39, 290–304. Generic‐ and family level classifications in Hydrozoa have been historically problematic due to limited morphological characters for phylogenetic analyses and thus taxonomy, as well as disagreement over the relative importance of polyp vs. medusa characters. Within the recently redefined suborder Capitata (Cnidaria: Hydrozoa: Hydroidolina), which includes 15 families and almost 200 valid species, family level relationships based on morphology alone have proven elusive, and there exist numerous conflicting proposals for the relationships of component species. Relationships within the speciose capitate family Corynidae also remain uncertain, for similar reasons. Here, we combine mitochondrial 16S, and nuclear 18S and 28S sequences from capitate hydrozoans representing 12 of the 15 valid capitate families, to examine family level relationships within Capitata. We further sample densely within Corynidae to investigate the validity of several generic‐level classification schemes that rely heavily on the presence/absence of a medusa, a character that has been questioned for its utility in generic‐level classification. We recover largely congruent tree topologies from all three markers, with 28S and the combined dataset providing the most resolution. Our study confirms the monophyly of the redefined Capitata, and provides resolution for family level relationships of most sampled families within the suborder. These analyses reveal Corynidae as paraphyletic and suggest that the limits of the family have been underestimated. Our results contradict all available generic‐level classification schemes for Corynidae. As classification schemes for this family have been largely based on reproductive characters such as the presence/absence of a medusa, our results suggest that these are not valid generic‐level characters for the clade. We suggest a new taxonomic structure for the lineage that includes all members of the newly redefined Corynidae, based on molecular and morphological synapomorphies for recovered clades within the group.