Currently, bilaterian animals are subdivided into four major groups: the supposedly basal Acoelomorpha, the Ecdysozoa (combining all molting animals such as arthropods and nematodes), the Lophotrochozoa with a trochophore-like ciliated larva (e.g., Annelida, Entoprocta, Mollusca, Platyhelminthes), and Deuterostomia (including chordates, hemichordates and echinoderms) [1–4]. Despite ongoing efforts, the interrelationships of the phyla that nest within the Lophotrochozoa remain unresolved [5, 6]. Entoprocta is a phylum that has been proposed to belong to a clade of spirally cleaving animals, the so-called Spiralia, which together with its suggested sister group, the Lophophorata (Ectoprocta, Brachiopoda, and Phoronida), forms the Lophotrochozoa . Typically, entoprocts are microscopic, mostly marine, sessile metazoan animals. Its approximately 150 hitherto described species are divided into four subgroups, the solitary (and supposedly basal) Loxosomatidae and the colonial Barentsiidae, Pedicellinidae, and Loxocalypodidae . Their adult gross morphology is characterized by a ciliated tentacle crown, which surrounds both the mouth and the anus. The calyx houses the reproductive organs, mostly one pair of protonephridia, and the cerebral ganglion. Entoprocts reproduce asexually by budding, as well as sexually, whereby two major larval types can be recognized, namely the creeping, supposedly basal, lecithotrophic and the more common swimming, planktotrophic larval type . Metamorphosis is very complex and often involves settlement and adhesion with the frontal body region to the substrate as well as rotation of the gut .
Morphological and molecular analyses have proposed several phylogenetic hypotheses concerning entoproct interphyletic relationships. Traditionally, Entoprocta and Ectoprocta have been comprised to form the monophyletic Bryozoa (Bryozoa-concept), based on a metamorphosing larval stage with a completely retracted and cavity-enclosed prototroch as well as additional common features during metamorphosis [8, 9]. This hypothesis has been revived by a recent molecular study , although subsequent analyses of partly the same authors are far less clear . The cryptic Cycliophora, one of the most recently erected phyla , have also argued to be associated with Entoprocta and Ectoprocta, notably as a monophyletic assemblage termed "Polyzoa" [13, 14], while other authors suggest a sister group relationship of Cycliophora and Entoprocta alone [15, 16]. On the contrary, the recently proposed Tetraneuralia-concept has strengthened the so-called Lacunifera- or Sinusoida- hypothesis, suggesting a monophyletic assemblage of Entoprocta and Mollusca based on numerous larval and adult autapomorphies [4, 17–19]. Resembling a mosaic of larval and adult molluscan characters, the entoproct creeping-type larva shares a number of morphological traits with the polyplacophoran trochophore, including a highly complex apical organ with eight centrally located flask-shaped and several peripheral cells, as well as a typical molluscan-like tetraneurous condition of longitudinal nerve cords [4, 19–21]. Additional shared characters are, among a total set of nine, the distinct creeping foot, a large pedal gland, frontal cirri, and a ventrally intercrossing dorsoventral musculature [4, 19, 20]. Despite the spiral cleavage pattern, which has traditionally been used to unite polyclad flatworms, nemerteans, annelids, and molluscs as "Spiralia" , other developmental characters, such as the cellular arrangement into an "apical cross pattern" during early embryogenesis, have been used to infer protostome interrelationships. For a long time, only two cross patterns had been clearly defined, namely the molluscan and the annelid cross, respectively. Since a seemingly "molluscan-type" cross pattern had also been reported for sipunculans, a close relationship to molluscs was suggested . Recently, additional cross patterns, such as a nemertean cross, have been described . For entoprocts, a spiral cleavage pattern has been mentioned in the literature and is often referred to in textbooks, but its documentation is restricted to only a few sketch drawings [25–27]. Apical cross patterns, which would be expected for a spirally cleaving taxon, have not been reported by these studies.
In order to fill the significant gaps in knowledge concerning entoproct early embryology, we herein describe the development of a representative of the supposedly basal entoproct genus Loxosomella by applying immunochemistry and confocal microscopy. Using our detailed description of the early cleavage pattern, we also clarify whether or not a distinct "cross pattern" is present in this species. These data are discussed with those available for other lophotrochozoans in order to assess their suitability for phylogenetic inferences.