The interrelationships of the flatworms (phylum Platyhelminthes) are poorly resolved despite decades of morphological and molecular phylogenetic studies. The earliest-branching clades (Catenulida, Macrostomorpha, and Polycladida) share spiral cleavage and entolecithal eggs with other lophotrochozoans. Lecithoepitheliata have primitive spiral cleavage but derived ectolecithal eggs. Other orders (Rhabdocoela, Proseriata, Tricladida and relatives, and Bothrioplanida) all have derived ectolecithal eggs but have uncertain affinities to one another. The orders of parasitic Neodermata emerge from an uncertain position from within these ectolecithal classes. To tackle these problems, we have sequenced transcriptomes from 18 flatworms and 5 other metazoan groups. The addition of published data produces an alignment of >107,000 amino acids with less than 28% missing data from 27 flatworm taxa in 11 orders covering all major clades. Our phylogenetic analyses show that Platyhelminthes consist of the two clades Catenulida and Rhabditophora. Within Rhabditophora, we show the earliest-emerging branch is Macrostomorpha, not Polycladida. We show Lecithoepitheliata are not members of Neoophora but are sister group of Polycladida, implying independent origins of the ectolecithal eggs found in Lecithoepitheliata and Neoophora. We resolve Rhabdocoela as the most basally branching euneoophoran taxon. Tricladida, Bothrioplanida, and Neodermata constitute a group that appears to have lost both spiral cleavage and centrosomes. We identify Bothrioplanida as the long-sought closest free-living sister group of the parasitic Neodermata. Among parasitic orders, we show that Cestoda are closer to Trematoda than to Monogenea, rejecting the concept of the Cercomeromorpha. Our results have important implications for understanding the evolution of this major phylum.

The lion’s share of studies on regeneration in Plathelminthes (flatworms) has been so far carried out on a derived taxon of rhabditophorans, the freshwater planarians (Tricladida), and has shown this group’s outstanding regeneration capabilities in detail. Sharing a likely totipotent stem cell system, many other flatworm taxa are capable of regeneration as well. In this paper, we present the regeneration capacity of Macrostomum lignano, a representative of the Macrostomorpha, the basal-most taxon of rhabditophoran flatworms and one of the most basal extant bilaterian protostomes. Amputated or incised transversally, obliquely, and longitudinally at various cutting levels, M. lignano is able to regenerate the anterior-most body part (the rostrum) and any part posterior of the pharynx, but cannot regenerate a head. Repeated regeneration was observed for 29 successive amputations over a period of almost 12 months. Besides adults, also first-day hatchlings and older juveniles were shown to regenerate after transversal cutting. The minimum number of cells required for regeneration in adults (with a total of 25,000 cells) is 4,000, including 160 neoblasts. In hatchlings only 1,500 cells, including 50 neoblasts, are needed for regeneration. The life span of untreated M. lignano was determined to be about 10 months.

Traditionally, regeneration research has been closely tied to flatworm research, as flatworms (Plathelminthes) were among the first animals where the phenomenon of regeneration was discovered. Since then, the main focus of flatworm regeneration research was on triclads, for which various phenomena were observed and a number of theories developed. However, free-living flatworms encompass a number of other taxa where regeneration was found to be possible. This review aims to display and to compare regeneration in all major free-living flatworm taxa, with special focus on a new player in the field of regeneration, Macrostomum lignano (Macrostomorpha). Findings on the regeneration capacity of this organism provide clues for links between regeneration and (post-)embryonic development, starvation, and asexual reproduction. The role of the nervous system and especially the brain for regeneration is discussed, and similarities as well as particularities in regeneration among free-living flatworms are pointed out.

Background: Tissue 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. Results: We 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. Conclusions: We 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.

Flatworm embryology has attracted attention since the early beginnings of comparative evolutionary biology. Considered for a long time the most basal bilaterians, the Platyhelminthes (excluding Acoelomorpha) are now robustly placed within the Spiralia. Despite having lost their relevance to explain the transition from radially to bilaterally symmetrical animals, the study of flatworm embryology is still of great importance to understand the diversification of bilaterians and of developmental mechanisms. Flatworms are acoelomate organisms generally with a simple centralized nervous system, a blind gut, and lacking a circulatory organ, a skeleton and a respiratory system other than the epidermis. Regeneration and asexual reproduction, based on a totipotent neoblast stem cell system, are broadly present among different groups of flatworms. While some more basally branching groups - such as polyclad flatworms - retain the ancestral quartet spiral cleavage pattern, most flatworms have significantly diverged from this pattern and exhibit unique strategies to specify the common adult body plan. Most free-living flatworms (i.e. Platyhelminthes excluding the parasitic Neodermata) are directly developing, whereas in polyclads, also indirect developers with an intermediate free-living larval stage and subsequent metamorphosis are found. A comparative study of developmental diversity may help understanding major questions in evolutionary biology, such as the evolution of cleavage patterns, gastrulation and axial specification, the evolution of larval types, and the diversification and specialization of organ systems. In this review, we present a thorough overview of the embryonic development of the different groups of free-living (turbellarian) platyhelminths, including the Catenulida, Macrostomorpha, Polycladida, Lecithoepitheliata, Proseriata, Bothrioplanida, Rhabdocoela, Fecampiida, Prolecithophora and Tricladida, and discuss their main features under a consensus phylogeny of the phylum.