Xenacoelomorpha Nervous Systems
Summary and Keywords
The emergence and diversification of bilateral animals are among the most important transitions in the history of life on our planet. A proper understanding of the evolutionary process will derive from answering such key questions as, how did complex body plans arise in evolutionary time, and how are complex body plans “encoded” in the genome? the first step is focusing on the earliest stages in bilaterian evolution, probing the most elusive organization of the genomes and microscopic anatomy in basally branching taxa, which are currently assembled in a clade named Xenacoelomorpha. This enigmatic phylum is composed of three major taxa: acoel flatworms, nemertodermatids, and xenoturbellids. Interestingly, the constituent species of this clade have an enormously varied set of morphologies; not just the obvious external features but also their tissues present a high degree of constructional variation. This interesting diversity of morphologies (a clear example being the nervous system, with animals showing different degrees of compaction) provides a unique system in which to address outstanding questions regarding the parallel evolution of genomes and the many morphological characters encoded by them. A systematic exploration of the anatomy of members of these three taxa, employing immunohistochemistry, in situ hybridization, and high-throughput transmission electron microscopy, will provide the reference framework necessary to understand the changing roles of genes and gene networks during the evolution of xenacoelomorph morphologies and, in particular, of their nervous systems.
Xenacoelomorpha and Their Constitutive Clades: General Morphology
The clade Xenacoelomorpha, which some authors assign the rank of a phylum (Brusca, Moore, & Shuster, 2016; Philippe, Brinkmann, Copley, Moroz, Nakano, Poustka et al., 2011), is composed of three major taxa: the Acoela, Nemertodermatida, and Xenoturbellida (Figures 1 and 2). They are simple worms that live almost exclusively in marine environments, at different depths, from intertidal sand beaches to hydrothermal vents (at a depth of more than 3,000 meters). Acoela and Nemertodermatida are sister groups, and together they form the Acoelomorpha. Hence, xenoturbellids are the sister group of the Acoelomorpha (Bourlat, Juliusdottir, Lowe, Freeman, Aronowicz, Kirschner et al., 2006; Bourlat, Nielsen, Lockyer, Littlewood, & Telford, 2003). Xenoturbellida is represented by only six nominal species, of which the best described are Xenoturbella bocki (Westblad, 1949) and Xenoturbella westbladi (Israelsson, 1999), though most probably these are the same species. In 2016, four newly identified species were identified in the Pacific deep sea (Rouse, Wilson, Carvajal, & Vrijenhoek, 2016) Adult Xenoturbellida are notably larger than adult Acoelomorpha, but share their relatively simple morphology. The Nemertodermatida class consists of only 18 species of marine worms (Meyer-Wachsmuth, Raikova, & Jondelius, 2013; Sterrer, 1998). The largest taxon, Acoela, is represented by more than 380 nominal species (reviewed in Achatz, Chiodin, Salvenmoser, Tyler, & Martinez, 2013).
All xenacoelomorphs are characterized by being bilaterally symmetrical, containing a ciliated epithelium and a single digestive opening (mouth); however, they lack several features common to most other bilaterians, for example, an anus, nephridia, coelom, or a circulatory system. Some representatives of this phylum are shown in Figure 1.
Xenoturbellids have a relatively simple morphology, range in size from around 1 cm (Xenoturbella bocki) to 15 cm long (Xenoturbella profunda) and live in muddy sediments (Nakano, 2015). The digestive tract of xenoturbellids is sac shaped and lined with an internal gastrodermis. The mouth opens ventrally and all specimens have a prominent anterior statocyst (of unclear function). The muscular system is organized as a grid with outer circular and inner longitudinal muscles (Ehlers & Sopott-Ehlers, 1997). Gonopores and other specialized reproductive organs are absent. Sperm and eggs are present in different positions within the body. Development is direct, with embryos being ciliated and bearing an anterior apical tuft. Gastrulation has not been observed (Nakano, Lundin, Bourlat, Telford, Funch, Nyengaard et al., 2013).
The nemertodermatids show a slightly more complex morphology than the xenoturbellids. The size of all the species described is in the millimeter range. They have an epithelial digestive tract, with a partially occluded lumen; a nervous system; and an anterior statocyst (with two statoliths, as opposed to the one seen in the acoels). They live in mud or in the interstices of sand, or are commensal (Meara lives in the foregut of a sea cucumber). Their early embryonic development follows a very similar path to that found in the acoels (Henry, Martindale, & Boyer, 2000), and is characterized by a duet cleavage pattern (Børve & Hejnol, 2014; Jondelius, Larsson, & Raikova, 2004). Nemertodermatids are hermaphroditic. Their gonads are asaccate (the germ cells are not lined and separated from the surrounding parenchyma by specialized tissue) and bear a single reproductive opening, the male gonopore. The musculature is basically organized with circular and longitudinal muscles forming the muscular sheath and diagonal fibers lying between the other two sets (though only occupying the anterior-most part of the animal body (Hooge, 2001; Meyer-Wachsmuth et al., 2013). There is no evidence of a muscular pharynx in the Nemertodermatida.
The original appraisal of the world nemertodermatid diversity was carried out by W. Sterrer (Sterrer, 1998). A first cladistics analysis of the interrelationships of the nemertodermatids was performed by K. Lundin in 2000 (Lundin, 2000). More recently the collection of many specimens from 33 locations worldwide has provided some further insights on the internal phylogeny of this poorly studied group (Meyer-Wachsmuth, Curini-Galletti, & Jondelius, 2014). Meyer-Wachsmuth et al. suggest, based on the number of cryptic species in their samples, the existence of a high diversity of nemertodermatids (and possibly acoels, too) that are present in the world marine environments but still uncharacterized (see also Arroyo, López-Escardó, de Vargas, & Ruiz-Trillo, 2016).
Acoels are bilateral marine worms with a simple unsegmented body plan, whose size is in the millimeter range. They share several morphological features with the above-mentioned nemertodermatids, such as epidermal ciliation, intestinal structure, some glandular and sensory structures, and the limited presence of an extracellular matrix (Achatz et al., 2013; Raikova, Reuter, Gustafsson, Maule, Halton, & Jondelius, 2004a). The epidermis of acoels is completely ciliated and rich with gland openings. Most cilia are required for locomotion; some specialized ciliary cells function as sensory receptors. A thick mucus layer covers the epidermis. Several other types of glands can also be distinguished between the epidermal cells along the length of the body. Acoels bear a prominent frontal organ that consists of a collection of two or more large mucus-secreting glands whose necks emerge together through a frontal pore at the apical pole of the body (Smith & Tyler, 1986). The statocyst (Ehlers, 1991) is a sensory organ informing the animal about its relative position. Most acoels have eyes (Yamasu, 1991) of a very special kind. The complexity of nervous system architecture in acoels is variable (Achatz & Martinez, 2012).
Acoela are characterized by the absence of a central cavity, as evinced by the name of this phylum (“a” = without; “coel” = cavity). Compared to the nervous system, the structure of the digestive system is poorly understood, although it is agreed that in most species, it is syncytial (see Pedersen, 1964, for an alternative opinion). The digestive system opens at the surface of the body through a mouth, a highly variable structure among species, which can be localized at, almost, any position along the main body axis of the animal. Acoels are hermaphroditic. The male copulatory apparatus is generally located posterior to the female copulatory apparatus. Both apparatuses open on the ventral side of the body. All acoel species possess three main types of muscle: longitudinal, circular, and diagonal. They differ mainly in the presence of accessory muscle types (V-shape, cross-over, parenchymal) and in the relative position of the main muscle types (outer circular and inner longitudinal). Some specialized musculature is associated with the copulatory organs (Ladurner & Rieger, 2000).
Pharynges are present in many acoel families, showing a great variety of morphologies, including musculature, the nature of the lining cells and the types of receptor present (see, for instance, Todt, 2009).
Embryonic development of Acoela was recorded by several authors (Apelt, 1969; Bresslau, 1909; Georgévitvch, 1899) but was better described in the later studies of Childia groenlandica (Boyer, 1971, who also manipulated the embryos), and Neochildia fusca performed by Henry and colleagues (2000) and Ramachandra, Gates, Ladurner, Jacobs, and Hartenstein (2002). It follows a specific duet spiral cleavage, where the second cleavage occurs in a leiotropically oblique plane relative to the animal-vegetal axis. At the four-cell stage, the plane of first cleavage corresponds to the plane of bilateral symmetry. Following cleavage, the embryo forms a solid mass of cells that differentiate “in situ,” whereby the outermost layer of cells becomes the ciliated epidermis. Cells more interiorly differentiate as muscle, neurons, glands, and neoblasts. Yolk-rich cells in the inner core of the embryo fuse into the digestive syncytium. Many acoels are able to regenerate body parts (Hanson, 1967; Perea-Atienza, Botta, Salvenmoser, Gschwentner, Egger, Kristof et al., 2013; Sprecher, Bernardo-Garcia, van Giesen, Hartenstein, Reichert, Neves et al., 2015), with some species showing dramatic examples in the context of their natural asexual reproduction (Sikes & Bely, 2008).
The Phylogenetic Context
Classification of Xenacoelomorpha and their constituent taxa has long been a matter of continuous controversy (see Hejnol & Pang, 2016, for a critical assessment). Since the 2000s, and primarily because of the introduction of phylogenomic analysis, Xenacoelomorpha are considered a monophyletic group, a phylum, in which the Xenoturbellida and the Acoelomorpha (Acoela + Nemertodermatida) are united as sister taxa (Figure 2). Originally, however (see, for instance, Hyman, 1951), the acoelomorphs and the enigmatic Xenoturbella were classified within the Platyhelminthes (Turbellaria) because of the similarities of their body plans (all were “flat-worms”). This classification also relied on an assumed “progressive” evolution of animal groups within the Metazoa, with increasing degrees of complexity being used as a major classificatory criterion, from the “primitive” acoelomate taxa to the “advanced” coelomates, passing through all pseudo-coelomate intermediates. Since then, the history of classification of the different xenacoelomorph taxa has followed an interesting path.
The taxon Xenoturbellida comprises a few nominal species, but for a long time, the only species described was Xenoturbella bocki (Westblad, 1949). This was originally described as a turbellarian. Its “simple” morphology has prompted the classification of Xenoturbella as either a basal metazoan (Jagersten, 1959) or a basal bilaterian (Sopott-Ehlers & Ehlers, 1997). Based on the structure of the epithelia, the statocyst, and the musculature (myocytes), Reisinger (1960) also suggested affinities with the Deuterostomia, and specifically with the clade today known as Ambulacraria (Hemicordata + Echinodermata), a proposal also supported by Obst, Nakano, Bourlat, Thorndyke, Telford, Nyengaard et al. (2011) based on the ultrastructure of the spermatozoon. However, some authors dispute this position and maintain that there is no clear, specific similarity between xenacoelomorphs and the deuterostome larvae, making this affinity difficult to accept (see Haszprunar, 2015). For a period of time, it was proposed that Xenoturbella was a Mollusca group, but this was shown to be based on the presence of contaminant DNA sequences in the Xenoturbella extracts used to infer the molecular phylogenies (see Noren & Jondelius, 1997). The affinities of Xenoturbella with the Acoelomorpha was observed early on (Westblad, 1949; see also Nielsen, 2010, for comments) and later emphasized by Franzen, Afzelius, and Franzkn (1987) and Lundin (1998). The affinity of all these groups is now supported by various phylogenomic studies (Cannon, Vellutini, Smith, Ronquist, Jondelius, & Hejnol, 2016; Hejnol, Obst, Stamatakis, Ott, Rouse, Edgecombe et al., 2009; Philippe et al., 2011; Srivastava, Mazza-Curll, van Wolfswinkel, & Reddien, 2014). Based on these affinities, Philippe and collaborators (2011) have suggested assigning this group the rank of a phylum (others, however, have elevated each of the three constitutive taxa to the level of phylum; Nielsen, 2012). The alternative, phylogenetic, scenarios will be explained below.
The classification of Acoelomorpha has had a more complex history. Ever since the phylum Platyhelminthes was defined, it has included the Acoelomorpha (Gegenbaur, 1859; Ruiz-Trillo & Paps, 2015). The phylum contained three major constituents: the Acoelomorpha, the Catenulida, and the Rhabditophora (Ehlers, 1985). The monophyly of the group was questioned soon after, because of the ambiguity of the characters used in the analysis (Smith & Tyler, 1986). In fact, the first extensive molecular phylogeny of Platyhelminthes, done by Carranza Baguna, and Riutort (1997), showed that they formed a paraphyletic group. However, there are some characters that are still shared by the Acoela and the Rabditophora: the stem-cell system (expressing the marker gene piwi), the specific form of replacement of the epidermal cells (from mesodermally located precursors), including the presence of one gut opening, a multiciliated epidermis and the orthogon-like (a regular pattern of longitudinal nerve cords connected by transverse commissures) structure of the central nervous system, with a brain and multiple radially arranged longitudinal nerve cords. Most molecular phylogenies do not support the grouping of Acoelomorpha within the Platyhelminthes, leaving the puzzle of how these previous characters have originated over evolutionary time (Egger, Steinke, Tarui, De Mulder, Arendt, Borgonie et al., 2009). The Acoela and Nemertodermatida were united in the clade Acoelomorpha based on some shared anatomical characters, such as the ciliary rootlet system and the embryo cleavage form, called duet-spiral. It was the use of acoel sequences in the phylogenetic analysis conducted by Ruiz-Trillo, Riutort, Littlewood, Herniou, and Baguña (1999) that showed for the first time that the Acoela formed the sister group of the remaining Bilateria, separate from the rest of the Platyhelminthes that were now classified as protostomes (in fact, Carranza and collaborators had shown already the affinities of Platyhelminthes with Protostomia). This placement of Acoela as basal bilaterians was confirmed in several studies, using ribosomal genes (see, for instance, Jondelius, Ruiz-Trillo, Baguñà, & Riutort, 2002; Telford, Lockyer, Cartwright-Finch, & Littlewood, 2003) and other multigene datasets (nuclear or mitochondrial; for instance, Mwinyi, Bailly, Bourlat, Jondelius, Littlewood, & Podsiadlowski, 2010; Paps, Baguña, & Riutort, 2009). The position of the nemertodermatids was either as sister to the acoels (Acoelomorpha) or sister to the remaining Bilateria, branching after the Acoela (Wallberg, Curini-Galletti, Ahmadzadeh, & Jondelius, 2007).
The differences between all these different phylogenetic/phylogenomic studies suggest that the “uncertain” placement of the Xenacoelomorpha (and its constitutive clades) is highly dependent on taxon sampling, the evolutionary model used, and, most probably, the intrinsic uncertainty imposed by a rapid cladogenesis occurring at the origin of bilaterian groups (Telford, Budd, & Philippe, 2015). A critical assessment of the biological implications derived from those alternative placements is given by G. Haszprunar (2015). Independently of the placement of the Xenacoelomorpha within the Metazoan tree, there is a consensus that they form a monophyletic group.
The internal phylogeny of the Acoela was most recently being studied in great detail by Jondelius and collaborators, using a combination of morphological and molecular (two nuclear ribosomal and one mitochondrial) characters on a sample of 126 acoel species (Jondelius, Wallberg, Hooge, & Raikova, 2011; Figure 3). Older phylogenies were built based on the character distribution of some anatomical structures, such as pharynx (Todt, 2009), spermatozoa (Achatz, Hooge, Wallberg, Jondelius, & Tyler, 2010; Hooge, Haye, Tyler, Litvaitis, & Kornfield, 2002) and body-wall musculature (Hooge, 2001). In all cases, the sampling represented a relatively low diversity of acoel species. The study of Jondelius and collaborators has challenged the conclusions of these previous studies through the exhaustive sampling of the world acoel diversity (using data from one third of all known species). The main conclusions derived from their study are (a) Diopisthoporidae is the sister taxon to all other acoels; (b) Paratomellidae is the sister taxon of the Bursalia and both constitute the taxon Bitesticulata; (c) there is a clade uniting Hallangia, Hofsteniidae, and Solenofilomorphidae; and (d) the most divergent clade is Convolutidae, which includes most acoel “model” species.
In this context, it is of special interest that by using the Bayesian approach to the reconstruction of ancestral states Jondelius and collaborators (2011) have been able to model the putative anatomical traits that characterized an ancestral acoel. This animal would have had a cylindrical shape and a frontal gland complex, also a pharynx, a hermaphrodite gonad, a posterior gonopore, a penis stylet and sperm with a 9 + 2 axoneme structure. Needless to say, and in the context of the putative basal bilaterian position of the Xenacoelomorpha, the reconstruction of an ancestral acoel has enormous importance, providing us with key insights on the origin of the bilaterian body plans (see, for instance, Baguñà & Riutort, 2004).
Neural Architecture of the Xenacoelomorpha
Clarifying the Terminology
The overview of the structure of the nervous system in Xenacoelomorpha that follows uses a few terms employed in the literature to describe specific elements of neural architecture. These include the terms brain, ganglion, nerve ring, nerve tract, nerve cord, nerve net, basiepithelial, and subepithelial. Definitions of these terms as they are used in this article follow:
The term “brain” refers to a conglomeration of nerve cells, typically associated with sensory receptors, in the anterior (relative to direction of movement) part of the body. Most depictions of aceolomorph architecture include an anterior brain. It is likely that, in cases that emphasize the absence of a “true brain” (e.g., Reuter, Raikova, & Gustafsson, 1998), only specific, sparsely distributed neuron types were labeled, which does not allow one to draw conclusions about the extent and structure of the nervous system as a whole. In some papers, the anterior brain is called “cerebral ganglion” or “nerve ring” (nerve ring understood in the literature as a “circular neuropil”). A distinction between “brain” and these other terms lacks clear (= quantifiable) criteria. All three terms refer to conglomerations of neuronal cells, associated with a more or less voluminous neuropil formed by neuronal processes (neurites) and synapses, thus the term “brain” is used in the following.
“Nerve tracts” (or fiber tracts) and “nerve cords” are bundles of long neurites distinguished by the absence and presence of neuronal cell bodies and neuropil, respectively (Richter, Loesel, Purschke, Schmidt-Rhaesa, Scholtz, Stach et al., 2010). In case of a tract, neuronal cell bodies forming the bundle are located together at one end; individual fibers project for long distances without branching and synapses. To put it more specifically, any specific bundles that are embedded within a (larger) neuropil can be called a tract. In a cord, cell bodies flank the bundle and send branched, interconnected neurites into it. The number and packing density of neuronal somata in a chord may vary; however, and as for the brain, there is no generally agreed upon threshold number of somata that has to be surpassed in order to call the association of nerve fibers a cord, rather than a tract or a nerve. Following this rationale, the nervous system of aceolomorphs that have been analyzed at a sufficient level of resolution possesses a set of bilaterally symmetric nerve cords, with cell bodies and neuropil, which emanate from the anteriorly located brain. Typically, the density of cell bodies decreases from anterior to posterior along the cords, which has prompted several authors to define the fiber systems of acoels as “neurite bundles” (e.g., Achatz & Martinez, 2012; Hejnol, 2016). The term “nerve chord” is used here for consistency.
A “nerve net” consists of scattered, more or less evenly distributed neurons, interconnected by single neurites (rather than bundles). A nerve net, in addition to a deeper brain and nerve cords, has been described for several invertebrate taxa, including mollusks, platyhelminths, and hemichordates. It also exists in Xenoturbella and many, if not all, acoelomorph species (e.g., Bedini & Lanfranchi, 1991; Bery et al., 2010).
In regard to their basic histological structure, invertebrate nervous systems fall into two main types, subepithelial and basiepithelial. In a basiepithelial nervous system, neuronal cell bodies and their processes are located within the epidermis. They are separated from the body cavity and deeper tissue layers, such as muscle, by a basement membrane. By contrast, in subepithelial nervous systems, neurons, and their fibers are located within the body cavity, separated from the epidermis by a basement membrane. The distinction between these two architectures is often not very clear-cut. Thus, even for animals that possess a basement membrane, it has been demonstrated that in numerous instances, both types of neural architecture occur in the same species at sequential developmental stages. For example, in the polychaete Capitella sp., neural progenitors, postmitotic neurons, and fiber tracts are observed at a basiepithelial position in embryos, whereas they move deeper, beyond the basement membrane, toward later stages (Meyer & Seaver, 2009).
In acoels and nemertodermatids, which lack a basement membrane, the defining criterion to distinguish between basiepithelial versus subepithelial nervous system is lacking. As discussed in more detail in the section “The Cytology of Xenacoelomorph Neural Components” cell bodies of epidermal cells, muscle cells, neurons, and other cell types occur intermingled. However, terms like subepidermal and basiepithelial have been used in the literature, and merely refer to the perceived distance between the neural elements and the apical surface of the epidermis. In cases where one or more layers of relatively densely packed cell bodies separate the neuronal fiber masses (neuropil) of the brain or nerve cords or both from the body surface, the term “subepidermal” is usually preferred. On the other hand, where cell bodies are sparse, and fiber masses are in closer contact to the muscle fibers or apical epidermal processes, authors tend to speak of a “basiepithelial” structure. Similar as for brain or ganglion, such a distinction is relatively inconsequential. In the same animal, the nerve cords in the anterior part of the body (where they emerge from the brain) are surrounded by relatively large numbers of cell bodies and would be characterized as subepidermal, whereas in the posterior tail, where cell bodies are scarce, cords are closer to the surface and appear “basiepithelial.”
The Nervous System of Xenaturbellida
Xenaturbellidae are the most basal clade among the Xenacoelomorpha and until recently consisted only of two described Xenoturbella species. The first described species is X. bocki (Westblad, 1949), and only in 1999 was a second species, X. westbaldi, identified (Israelsson, 1999). However, an analysis from 2016 has shown that these two species are the same (Rouse et al., 2016). More recently, four new deep-sea Xenoturbella species X. monstrosa, X. churro, X. profunda, X. hollandorum were described, which were collected at different depths in the Monterey Canyon, Gulf of Mexico, and Gulf of California.
The body of Xenoturbella is subdivided into an external epidermis, a layer containing muscle fibers, and an internal gastrodermis. A thick, multilayered basement membrane termed “subepidermal membrane complex” separates the epidermis from the gastrodermis. The nervous system of Xenoturbella (Figure 4A) consists of a large basiepidermal nerve net (Bock, 1923; Raikova, 2004; Stach, 2016; Stach, Dupont, Israelson, Fauville, Nakano, Kånneby et al., 2005; Westblad, 1949). Labeling with anti-serotonin and FRMFamide antibodies showed non-overlapping parts of the nerve net, suggesting that both neurotransmitter systems are used in Xenoturbella and this in diverse sets of neurons (Raikova, 2004). Cells expressing, FMRFamide and SALMFamide have a bipolar or multipolar shape (Stach et al., 2005). Since some of these neurons have apical, ciliated processes, they may correspond to sensory cells. The basiepidermal neurons do not appear to cross the basement membrane, nor do they innervate the gastrodermis. Similarly to acoels there are also neurites connecting from the nerve net toward the peripheral part of the epidermis likely innervating peripheral muscles and ciliary cells.
While it has been observed that the density of nerve cells along the anterior-posterior body axis seems to be variable, no anterior agglomeration of nerve cells has been observed, giving rise to the notion that Xenoturbella lacks a brain. Because in past analyses it was only possible to investigate a small subset of neurons by light microscopy, it is challenging to extract a complete picture. Similarly, not much is known regarding the development of the Xenoturbella nervous system. The lack of detailed developmental studies is a further obstacle to inferring commonalities or differences among the xenacoelomorph nervous system. If at any developmental stage a more concentrated brain-like structure exists, this remains to be shown. The side furrows have been suggested as a sensory organ (Westbald, 1949), though there is as yet no direct evidence of this role.
In terms of sensory receptors, Xenoturbella possesses a variety of not further characterized monociliated sensory neurons (see section “The Cytology of Xenacoelomorph Neural Components”), as well as an unusually structured statocyst with putative sensory function (Israelsson, 2007; Raikova, Reuter, Jondelius, & Gustafsson, 2000). In acoels, as in most other animal phyla containing such an organ, the statocyst is required for gravity sensing and often for geotaxis behavior. The behavioral repertoire of Xenoturbella has not yet been investigated, but the presence of a statocyst suggests that they may be sense gravity, which in turn may be relevant for movement on and in the muddy habitat.
The Nervous System of Nemertodermatida
The structure of the nervous system of adult Nemertodermatida is characterized by a relatively low number and density of neurons and a tenuous neuropil, similar to that in xenoturbellids (Nemertoderma westbladi, Meara stichopi, Flagellophora sp. and Sterreria sp. were described (Børve & Hejnol, 2014; Raikova, Meyer-Wachsmuth, & Jondelius, 2016; Raikova et al., 2004a). The nervous system includes a brain and nerve cords, as well as a peripheral nerve net.
The brain of Flagelophora contains bipolar and unipolar neurons, some of them expressing sand FMRFamide. Sensory neurons with peripheral dendrites are located in the brain. In N. westbladi (Figure 4B) the brain is formed by two ring-like neuropils surrounding the statocyst and interconnecting commissures. Serotonin and FMRFamide-immunoreactive neurites are found in the brain and in the nerve net. From the brain, two cords extend posteriorly. The brain of Sterreria includes an elaborate neuropil consisting of a pair of longitudinal cords interconnected by seven to eight commissures. By contrast, in the parasitic species M. stichopi, no anterior condensation of neurons and neuropil appears to exist. Two loose nerve cords run laterally along the anteroposterior body axis. Similarly to Xenoturbella bocki, no apparent innervation of the gut was described among serotonin and FRMFamide-immunoreactive neurons (no neurites are sent to the gut).
Unlike Xenoturbella, developmental studies are lacking for most Nemertodermatida. The exception is the parasitic species M. stichopi, for which aspects of neural structure have been followed from hatching onward (Børve & Hejnol, 2014). Juveniles show a wide distribution of serotonin-immunoreactive neurons along the anterior-posterior body axis. Comparable to the adult animal, the dominating structure is two lateral cords. Interestingly, these bundles are FMRFamide positive and dense in the anterior part of the animal with an elaborate commissural bundle (“brain”) connecting the cords. It is possible that additional elements, expressing transmitters other than serotonin or FMRFamide, contribute to the brain of the juvenile Meara. Thus, as described for many parasitic species, features of the adult animal may be secondarily reduced during postembryonic development due to its lifestyle.
The Acoel Nervous System
The central nervous system of acoels consists of a brain and three to five pairs of nerve cords, and a peripheral nerve plexus. Nerve cords are generally distributed evenly around the circumference of the body and are of a similar diameter; however, the dorsal and ventrolateral cords tend to be more pronounced (Raikova, Reuter, Kotikova, & Gustafsson, 1998). The nerve net displays a well-developed serotonin-immunoreactive pattern in most acoels. A pharyngeal (stomatogastric) nervous system associated with the mouth, pharynx, or digestive syncytium has not been detected in any acoel. This general feature aside, there is a considerable variability in neural architecture when comparing different acoel clades, in terms of the number of cords, their dorsoventral position, and the presence/absence of some sensory structures (i.e., the eyes).
The following paragraphs describe the specifics of the nervous system architecture in all major acoel clades, starting with the older lineages and using the phylogenetic framework proposed by Jondelius and collaborators (2011).
Diopisthoporidae is the sister taxon to all other acoels (Figure 3). Westblad (1940) and Dörjes (1968) described the architecture of the nervous system in the species Diophisthoporus psammophilus and Diophisthoporus longitubus as consisting of an anterior neural mass surrounding the statocyst from which emanate six nerve cords located at different dorsoventral positions. A similar architecture was seen with serotonin immunostaining in D. longitubus by Raikova and colleagues (Kotikova & Raikova, 2008; Raikova, 2004). These authors describe the presence of an extensive peripheral nerve plexus. According to the description of Smith and Tyler (1985), the nervous system of the species Diopisthoporus gymnopharyngeus consists of paired dorsolateral ganglionic lobes located immediately posterior to the statocyst. The neuropil are described as taking the form of two ring-shaped commissures flanking the statocyst, as well as a two longitudinal tracts connecting these “nerve rings” and extending posteriorly as the nerve cords. Taking into account the phylogenetic position of the Diopisthoporidae, the basic pattern of the acoel nervous system would consist of a small brain associated with the statocyst, one or two ring commissures and two to six nerve cords. A stomatogastric nervous system appears to be absent.
The family Paratomellidae, a member of the class Bitesticulata, constitutes the sister group to all members of Bursalia (Figure 3). The best-described species within this clade is Paratomella rubra, which possesses a prominent neuropil surrounding the statocyst. The neuropil features longitudinal fiber tracts that extend both anteriorly and posteriorly, and two circular neuropiles. A similar structure is described by Crezée and Tyler (1976) for the genus Hesiolicium, where they detect a brain located dorsal to the statocyst and a pair of, slightly insunk, nerve cords that run posteriorly. Among the nerve cords extending from the brain of paratomellids, the dorsolateral cords are the best developed (Crezée, 1978; Crezée & Tyler, 1976).
Within Bursalia, the Hofsteniidae and Solenofilomorphidae form a monophyletic group, which is sister to the small group Hallangiidae (there are no data on the nervous system of this group). The three clades compose the Prosopharyngida (Figure 3).
For Hofstenia atroviridis and Hofstenia miamia, the brain has the shape of a subepidermal cylinder completely encircling the body in the area of the statocyst (Bock, 1923; Corrêa, 1960; Steinböck, 1966). The cylinder is the thickest on the dorsal side, gradually becoming thinner toward the ventral side. Recent analyses, employing in situ hybridization with neural markers, show a similar pattern, without revealing the cellular arrangement of the diverse neurons (Srivastava et al., 2014). For H. arabiensis, from the Red Sea, the brain was described as a submuscular “nerve plexus” that occupies the one sixth most anterior part of the animal (Beltagi & Mandura, 1991). This plexus issues thin processes with possible sensory function into the epidermis
It is interesting to note that the relationship between the epidermis and the nervous system appears to vary in different members of the Hofsteniidae, similar to what has been described in “higher” acoels The H. pardii nervous system consists of a basiepidermal plexus, whereas the Marcusiola tinga nervous system lies subepidermally, but the dorsal nerve cords are positioned basiepidermally (Steinböck, 1966).
The taxon Solenofilomorphidae has been mostly described by Michael Crezée (1975), using serial sections and squeezed specimens. Solenofilomorphidae have a minuscule brain whose neuropil forms a set of one to three ring-shaped commissures, connected by longitudinal tracts, in the vicinity of the statocyst. Paired dorsolateral “ganglia” or “thickenings” described for Diopisthoporidae or Hofsteniidae are absent. Longitudinal nerve tracts or cords (the difference cannot be made out in the histological material used) extending from the brain include an unpaired dorsal and ventral cord, and paired dorsolateral, lateral, and ventrolateral cords, which tend to appear at various stages of fusion. There is a common trend within Solenofilomorphidae of an enlargement of dorsolateral and ventrolateral cords and their separation from the epidermis. The other, smaller cords remain in close association with the epidermis and are frequently difficult to find, particularly the ventral nerve cord.
Crucimusculata (“Higher” Acoels)
Moving on to Crucimusculata (acoels with ventral crossover muscles and wrapping cells (Jondelius et al., 2011), this class contains most of the so-called acoel “model organisms” (Symsagittifera roscoffensis, Isodiametra pulchra, and Convolutriloba longifissura), all belonging to the families Isodiametridae and Convolutidae. This discussion, however, will begin with descriptions of other members of these families that are less well known.
Several species belonging to the family Proporidae (Figure 4C) have been studied, two (Haploposthia viridis and Haploposthia. lactomaculata) by immunochemical methods, and two (H. brunea and Kuma albiventer) by histological sections. In all of these species, the nervous system is composed of a well-developed peripheral neural plexus and a “statocyst ganglion” (brain; Kotikova & Raikova, 2008).
The thorough analysis of the acoel phylogeny by Jondelius and collaborators (2011) revealed a mixed clade of actinoposthiids and isodiametrids, which have an unstable phylogenetic position. For several species of this clade, including Faerlia glomerata, Actinoposthia beklemishevi, and Avagina incola, we have only very rudimentary information about the nervous system. F. glomerata has been described as having a small superficial brain with several ring-shaped commissures (“commissural brain”; Raikova et al., 1998). Nerve cords exiting the brain are quickly lost in the superficial nervous plexus. A. beklemishevi has a commissural brain with one or two ring-shaped commissures and eight longitudinal connectives that posteriorly become longitudinal nervous trunks (Kotikova & Raikova, 2008). By contrast, A. incola is characterized by a clearly pronounced bilaterally symmetrical dorsal brain that is deeply immersed in the parenchyma (Reuter, Raikova, Jondelius, Gustafsson, Maule, & Halton, 2001).
Within the Isodiametridae, we find one of the best-characterized nervous systems of the Acoela, that of Isodiametra pulchra, a “model” species that is amenable to gene knockdown experiments investigating gene activities (De Mulder, Kuales, Pfister, Willems, Egger, Salvenmoser et al., 2009; Moreno, De Mulder, Salvenmoser, Ladurner, & Martinez, 2010). The I. pulchra nervous system has been characterized using a combination of histochemical (acetylcholinesterase), immunohistochemical (antibodies against serotonin, acetylated tubulin, and FMRFamide) and histological techniques (Achatz & Martinez, 2012; Smith & Bush, 1991). Isodiametra possesses an anterior brain with a prominent commissure, positioned slightly dorsally and posteriorly of the statocyst. Thickenings of the neuropil at the level of the dorso-posterior commissure and at a more anterior level were called the posterior lobe and anterior lobe, respectively (Smith & Bush, 1991). A conspicuous ring-shaped neuropil domain, the frontal ring, tips the anterior lobe. The brain neuropil emits four paired nerve cords, all of which contain serotonin-immunoreactive axons; they include a dorsal, lateral, ventral and a medio-ventral cord. The dorsal and ventral cords connect to the posterior lobe, whereas lateral and medio-ventral cords enter the anterior lobe (Achatz & Martinez, 2012). FMRFamide IR-positive neurons and axons are located in the brain and peripheral neural plexus; they do not overlap with serotonin-immunoreactive elements, as also previously reported by Raikova and collaborators (1998). None of the antibody markers detected neurons associated with the pharynx or gut, which confirms earlier reports for other acoels (Raikova et al., 2004b).
The family Mecynostomidae, represented by the genus Childia (Figure 4E), has been studied immunohistochemically by Raikova and collaborators (1998). In this group, C. cycloposthium shows what Raikova et al. (1998) call the “primitive state,” a barrel-like brain and several ring-shaped commissures. C. brachiposthium, C. macroposthium, and C. crissum display the most developed, or complex, symmetrical dorsal brain. Studies on the genus Paraphanostoma (now renamed and included within the taxon Mecynostomidae) exemplify the considerable variability that exists in details of neural architecture, such as the number and placement of nerve cords, and the position of the brain (Raikova et al., 2004b).
Within the Convolutidae, the best-studied species are Convolutriloba longifissura and Symsagittifera roscoffensis (Figure 4F). Using serotonin-immunoreactivity, Gaerber, Salvenmoser, Rieger, and Gschwentner (2007) showed that C. longifissura has a bilobed brain with a commissure-like neuropil, located beneath the body-wall musculature at a ventral position. Several, predominantly ventral, nerve cords, as well as a peripheral neural plexus were also described. A similar organization exists in C. retrogemma, which has a bilobed brain plus two lateral nerve cords and four medial nerve cords (Sikes & Bely, 2008), as well as Neochildia fusca (Ramachandra et al., 2002), which has an anterior compact brain organized as a layer of neural somata (2–3 diameters thick) surrounding a central neuropile free of cell bodies.
The Symsagittifera nervous system has been analyzed using antibodies against the neurotransmitters serotonin and FMRF-amide (Semmler, Chiodin, Bailly, Martinez, & Wanninger, 2010; anti-tubulin tyrosine, Bery, Cardona, Martinez, & Hartenstein, 2010; Semmler et al., 2010; and anti-dSap47 [generated against the Drosophila synaptic protein 47], Sprecher et al., 2015). In addition, acetylcholinesterase histochemistry (Bery & Martínez, 2011) and transmission electron microscopy (TEM; Bery et al., 2010) was employed. GABA immunoreactivity was used in S. psammophila (Bedini, Lanfranchi, & Santerini, 2001). These studies show an anterior compact brain (already suggested by Delage in 1886 (Delage, 1886), formed by a bilobed mass of neuronal cell bodies surrounding a neuropil that surrounds the statocyst (a similar arrangement is seen in a TEM analysis of S. psammophila, where a clear brain can be detected around the statocyst; Bedini & Lanfranchi, 1991). The brain neuropil can be subdivided into a posterior and anterior commissural compartment, flanking the statocyst. Thickenings at the level of these commissures form the paired dorso-anterior and dorso-posterior compartments, respectively. The neuropil on either side continues ventrally and anteriorly (ventro-anterior compartment) and terminates in a ring-shaped, commissural compartment (ventro-anterior commissure) that surrounds the thick bundle of gland necks that penetrate the brain to open in the frontal pore. Three pairs of nerve cords (dorsal, lateral, ventral) run along most of the length of the body. The dorsal cords emanate from the dorso-posterior compartment; the lateral and ventral cord from the ventro-anterior compartment (Sprecher et al., 2015). The cords are connected by numerous commissures arranged irregularly; the only prominent commissures are the ones located in the brain area. In the posterior region of the animal, commissures are barely visible.
In 2015, species-specific antibodies were raised against the proteins synaptotagmin and ELAV (Gavilán, Perea-Atienza, & Martínez, 2016; Perea-Atienza, Gavilán, Chiodin, Abril, Hoff, Poustka et al., 2015), using data obtained from genomic and transcriptomic projects, and have revealed a pattern very similar to that given by the use of other antibodies. However, some differences arise when using different antibodies, with some antibodies staining nervous system subdomains more prominently than others. For instance, anti-ELAV labeling stains mostly the anterior (brain) domain or the dorsal nerve cords. Moreover, Semmler and collaborators (2010) observed that areas of the brain (neuronal subsets) in Symsagittifera were also stained differently by antibodies against serotonin and FMRF-amide, demonstrating the functional heterogeneity of the nervous system. Interestingly S. roscoffensis is the first acoel for which we have behavioral data (Franks, Worley, Grant, Gorman, Vizard, Plackett et al., 2016; Keeble, 1910; Nissen, Shcherbakov, Heyer, Brummer, & Schill, 2015; Sprecher et al., 2015) suggesting the possibility that this species could be a good model to test the functionality of genes and neural circuits in the near future.
In conclusion, this survey of acoel neural architecture reveals an evolutionary “trend” toward greater complexity, as well as a number of common elements that unify all members of the clade. If we compare the nervous systems of acoels in the most basal family, the Diopisthoporidae, with those in the most divergent families, such as the class Crucimusculata, we see more compact brains developing in more derived acoel lineages compared to the simpler “commissural brains” at the anterior end of the older lineages. We and others (see Northcutt, 2012) have called this evolutionary change towards a more compact centralized neural structure “centralization/cephalization” (Gavilán et al., 2016). Among the “higher” acoela (Crucimusculata), numerous conserved structural elements can be observed. For example, the serotonin immunoreactivity pattern in I. pulchra resembles that in Avagina incola, another member of the Isodiametridae (Reuter et al., 2001). A dorsal posterior commissure, located in a similar position relative to the statocyst and with a similar pattern of connectivity to other parts of the brain and nerve cords, exists in A. incola, childiids (Raikova et al., 1998; Reuter et al., 2001) and convolutids (Bery et al., 2010; Semmler et al., 2010). Likewise, a frontal commissure (ring-like in I. pulchra and S. roscoffensis) is also present in all the aforementioned taxa. Aside from these common characters, variability exists in terms of the exact number and distribution of nerve cords in some clades (Kotikova & Raikova, 2008; Raikova et al., 1998). In particular, authors frequently noted the variable depth within the body at which cords (or even the brain) resided within the body, from “deeply embedded in the parenchyma” over “submuscular,” “subepidermal” to “basiepidermal.” Again, (see above the section “Clarifying the Terminology”) it is noted that in the absence of a basement membrane, and given subsequent intermingling of different cell types (epidermal cell bodies typically lying deeper than muscle fibers), the precise location of neuronal cell bodies or nerve fibers may not be of a profound discriminative significance. Moreover, the developmental stage of the animal investigated may play a role as well. In “higher” acoels (e.g., S. roscoffensis), the brain of the freshly hatched juvenile accounts for at least 25% of the body in terms of volume and cell number. This ratio declines significantly with postembryonic growth. It is also possible that the location of brain and nerve cords changes during development. Systematic and quantitative studies that follow nervous system development will be of importance in the efforts to resolve these issues and to establish a more coherent picture of acoel neural architecture. In fact, it is important to point out that the pioneering work of Bery and collaborators (2010) is allowing us to understand some basic, quantitative, aspects of the S. roscoffensis (juvenile) nervous system, providing such facts as the presence of approximately 700–1000 anterior (brain) cells, the thickness of the cords at different AP positions (~ 50–100 neurites/cord; anterior levels or approximately 15–30 neurites/cord; posterior levels) or details of the peripheral plexus, such as the realization that there are approximately 1–3 neurites/bundle. These studies provide us with a view of the acoel nervous system in exquisite detail. Needless to say, similar approaches should be implemented for the other members of Xenacoelomorpha.
The Cytology of Xenacoelomorph Neural Components
Understanding the structure and function of nervous systems relies on the detailed characterization of their constitutive cellular components. The most relevant components contributing to the sensory and processing activities of the xenacoelomorph neural systems are described in the next few paragraphs.
The body wall of Xenacoelomorpha is formed by a layer of multiciliated epidermal cells (Figure 5A, B, C). Similar to cilia in platyhelminths, the basal body underlying each cilium possesses one (Acoela and Nemertodermatida) or two (Xenoturbellida) anteriorly directed rootlets, and one posteriorly directed rootlet. Ciliary rootlets represent evolutionarily conserved cytoskeletal elements whose main constituent protein, rootletin, polymerizes to form long, striated fibrils that connect the basal body underlying the cilium to other cytoskeletal fibers within the cytoplasm (Yang, Liu, Yue, Adamian, Bulgakov, & Li, 2002). Ciliary rootlets of xenacoelomorphs are organized in a complex pattern, whereby rootlets of neighboring cilia form an interconnected network; Ax, 1996; Lundin, 1997; Lundin, 1998). This interconnected rootlet pattern sets xenacoelomorphs apart from platyhelminths and other metazoans.
Epidermal cells in Nemertodermatida and Xenoturbella form squamous or cuboideal epithelia (Lundin & Hendelberg, 1995; Pedersen & Pedersen, 1988). Subapically, a junctional complex of adherens junctions and septate junctions interconnects cells. Dense arrays of microfilaments, oriented parallel to the apical cell membrane and anchored in the belt-like adherens junctions, form the so-called terminal web. The epidermis of most acoels is formed by an insunk (or infranucleate) epithelium. Here, only thin apical processes of the epidermal cells, interconnected by a junctional complex and stabilized by a terminal web, form a continuous body wall (Rieger et al., 1991). Underneath the outer epidermal sheet, processes of muscle cells form a system of circular, longitudinal, and diagonally oriented fibers. Cell bodies of epidermal cells lie underneath the muscle net, and muscle somata are even deeper, forming a loose layer of cells surrounding the neuropil, nerve cords, and digestive system. In this deep layer of cells, somata of different cell types, including sensory and central neurons, muscle cells and gland cells are intermingled (Figure 6B). The insunk epidermal epithelium and, more generally, the absence of discrete tissue layers observed in acoels are correlated with the absence of basement membrane. This prominent sheet of extracellular matrix material demarcates a smooth boundary between tissues, including epidermis, muscle, nervous system, and gut in other animal taxa. However, the lack of basement membranes in acoels cannot be considered the only determining factor responsible for the insunk epidermal epithelium and intermingling of cell types, since nematodermatids, possessing a regular cuboidal epidermis, also lack basement membranes (Lundin & Hendelberg, 1995). Further, some platyhelminth taxa that do have basement membranes show an insunk epidermal epithelium (Tyler, 1984).
Sensory receptor cells in many invertebrate taxa, including Xenacoelomorpha, are bipolar neurons whose apical, dendritic processes are integrated in the epidermis. The dendritic process carries a sensory cilium, which is responsible for receiving the stimulus. Cell bodies of sensory receptors are typically sunk underneath the epidermis; in many cases, they are intermingled with cell bodies of central neurons and other cells in a loose cortex surrounding the neuropil (Bedini, Ferrero, & Lanfranchi, 1975; Bery et al.,2010; Todt & Tyler, 2006; Figure 5B, C; Figure 6B). Aside from the apical dendrite, sensory receptors form a central process that invades the neuropil, where it synapses with processes of central neurons and, possibly, muscle cells. In addition, figures suggestive of synaptic contacts between dendritic processes and peripheral nerve fibers have been described (Bedini, Ferrero, & Lanfranchi, 1973; Figure 6B). Overall, it has to be stated that the pathways by which sensory stimulation reaches target neurons and muscles are essentially unknown.
Acoel sensory receptors are predominantly monociliary; in a few cases, receptors with multiple (2–4) cilia have been described (Ehlers, 1992; Ehlers & Ehlers, 1977). Multiciliary receptors are much more common in platyhelminths (Bedini et al., 1975; Wright, 1992). Ciliary structure is relatively uniform, featuring an axoneme of 9 + 2 microtubules characteristic of locomotor cilia, from which sensory cilia are thought to be derived. Different classes of sensory receptors can be defined by the presence or absence of a microvillar collar surrounding the cilium, as well as the structure of the ciliary rootlet. Todt and Tyler (2006) propose a scheme that distinguishes between three types of non-collared receptors (type 1–3), and two types of collared receptors (type 4–5; Figure 6A). They suggest homology between a given type found in different acoel taxa.
Type 1 describes non-collared receptors with a single long, straight rootlet. They occur singly or in groups, and were described for several basal acoel clades (genera Hofstenia, Proporus, Paratomella; Todt & Tyler, 2006), but also in Xenoturbella (Israelsson, 1999) and several platyhelminths (Ehlers & Ehlers, 1977; Rohde & Watson, 1995).
Type 2 sensory cells are non-collared and have an enlarged main rootlet, as well as an additional horizontally oriented posterior rootlet. This type of receptor was found only in several basal acoel clades (genera Hofstenia, Solenofilomorpha; Todt & Tyler, 2006, and not outside the acoels.
Type 3 cells (“Type I” of Bedini et al., 1973) are also without a collar, but possess a more complex, hollow rootlet apparatus that contains a granulated core. The apical membrane lies above the level of the surrounding epidermal cells. The rootlet apparatus splits apart toward proximal levels where it surrounds a core of granulated material. Another distinguishing feature of the rootlet is its knee-like proximal bent that resembles the shape of the rootlet seen in locomotor cilia. Type 3 cells appear to be restricted to more derived acoel clades (genera Convoluta, Isodiametra, Mecynostomum, Symsagittifera; Bedini et al., 1973; Bery et al., 2010; Todt & Tyler, 2006). It is possible that they represent an apomorphy of these higher acoels, evolving (independently from existing type 1 receptors) directly from locomotor cilia (Todt & Tyler, 2006).
Type 4 represents a type of collared receptors, where the central cilium is surrounded by a ring (“collar”) of thickened microvilli that each contains a bundle of microfilaments protruding basally into the apical cytoplasm (Figure 6). The ciliary root is split into numerous parallel, striated fibers. In addition, oblique fibers radially connect the apical membrane adjacent to the base of the cilium with the zonula adherens (Figure 6). Type 4 receptors were described for higher acoel clades (Convoluta, Symsagittifera; Bery et al., 2010; Todt & Tyler, 2006), but also more basal clades (Paratomella, Diophistoporus; Ehlers, 1992)
Type 5 cells (“Type II” of Bedini et al., 1973) represent the main type of collared receptors which occurs at high density, singly or in small groups together with other types. The long central single cilium is surrounded by an outer ring of microvilli, numbering between 12 and 20, each one containing a bundle of microfilaments. In addition, two to three inner microvilli, longer and thicker than the outer ones, flank the central cilium. Microfilament bundles exit the microvilli and project basally into the cytoplasm, converging towards each other and merging at a level underneath the rootlet apparatus (Figure 6). The rootlet apparatus is highly modified: instead of striated fibers, it consists of a granulated, electron dense core (granular body) that has the peculiar shape of a “swallow’s nest” (and was called accordingly by numerous authors; Figure 6). Collared receptors with granular bodies are characteristic of higher acoels (Todt & Tyler, 2006); in Symsagittifera roscoffensis, they form the most prominent type of sensillum, distributed in rows along the nerve cords (Bery et al., 2010). Similar types of receptors occur also outside the acoels, in platyhelminths, annelids and cnidarians; however, based on subtle, yet significant structural differences of these receptors, Todt and Tyler (2006) argue against homologizing them with the acoel collared receptors.
Electron microscopic accounts of the central nervous system and sensory organs of the other members of the xenacoelomorph clade are rare. Monociliary receptors have been described for both nemertodermatids and Xenoturbella (Ehlers, 1992; Raikova, 2004), but it is unknown how they fit into the classification scheme developed for the Acoela.
Sagittocytes are a specialized type of gland cell that occurs in the derived clade Sagittiferidae. These cells are large and flask-shaped, with a stout apical neck integrated in the epidermal epithelium. Each cell forms in its cytoplasm a large, electron-dense granule, the sagittocyst (Gschwentner, Ladurner, Salvenmoser, Rieger, & Tyler, 1998; Yamasu, 1991). Perforating the center of the sagittocyst is a long filament. The sagittocyte is surrounded by a thick, multi-layered mantle of muscle fibers, which is thought to contract and thereby rapidly expel the sagittocyst upon stimulation. The appropriate stimulus for and function of sagittocyte “firing” is not known. In sexually reproducing species, sagittocytes are present only in mature animals, where they are clustered around the genital pores, and are thought to be involved in reproductive behavior; in the mainly asexual genus Convolutriloba, sagittocytes occur at the dorsal and antero-ventral body surface and are more likely involved in defense or prey capture or both (reviewed in Gschwentner et al., 1998).
Frontal Organ and Frontal Glandular Complex
Gland cells, aside from muscles, constitute a major effector organ targeted by the nervous system in the Xenacoelomorpha. Gland cells fall into a large variety of different categories that will not be considered in detail (for review, see Rieger, Tyler, Smith, & Rieger, 1991). Similar to sensory receptors, gland cells have cell bodies sunk into the body cavity, and elongated necks that protrude into epidermal epithelium. Many gland cells are large, with cell bodies located posterior to the brain, and long necks projecting anteriorly on either side of the neuropil, or right through the neuropil, to terminate at front end of the head. In particular, a subset of these long-necked glands that secrete mucus (mucoid glands, recognizable by their floccular, electron lucent inclusions) terminate in a depressed region of the epidermis (“frontal pore”) at the tip of the animal. Other types of glands (“accessory frontal glands”; Klauser, Smith, & Tyler, 1986) of unknown function, and named simply after the shape or texture of their inclusions (e.g., “ellipsoid” glands, “target glands,” “alcian blue-positive rhabdoid glands”) terminate close to, but outside the frontal pore. In addition, many densely clustered sensory receptors surround the frontal pore and accessory frontal glands. This entire ensemble, called the “frontal organ,” is widely considered an apomorphic trait of the acoels; it was found in all representatives of this clade, excepting Paratomella, that were investigated electron microscopically (Ehlers, 1992; Klauser et al., 1986; Smith & Tyler, 1986). In Paratomella, as well as in nemertodermatids and many platyhelminth taxa, one finds a somewhat different arrangement of long-necked mucoid and other types of glands, termed “frontal glandular complex” (Ehlers, 1992). Unlike the frontal organ, the frontal glandular complex lacks the convergence of mucoid gland necks in a frontal pore; instead, one finds regularly spaced sets of glandular cells, interspersed with sensory receptors. Sensory neurons and frontal glands alike have central processes in the brain neuropil (Volker Hartenstein, unpublished observation), suggesting that the frontal organ and frontal glandular complex of Acoelomorpha acts as an integrated sensory-effector organ.
The acoelomorph brain, also termed “cerebral ganglion,” “cerebral mass,” “commissural brain,” or “statocyst ganglion,” is formed by an irregular layer of cell bodies (cortex) surrounding a central neuropil. Neuronal somata, processes (neurites), and synaptic contacts were analyzed at the electron microscopic level in a number of studies (Achatz & Martinez, 2012; Bedini & Lanfranchi, 1991; Bery et al., 2010; Gaerber et al., 2007). However, these studies did not use series of contiguous sections, which made it impossible to reconstruct neurons with all of their processes or contacts. Many of the cells described as neurons were likely muscle cells or gland cells which, according to ongoing serial Electron Microscopy are intermingled with neurons in the cortex. Furthermore, classifications of “neurons” as unipolar, bipolar, or multipolar have to be taken with reservations, given the limitations of analyzing representative sections (rather than complete series of sections) of neurons.
Neuronal cell bodies of the acoelomorph brain are generally described as small, scant in cytoplasm, and in possession of a heterochromatin-rich nucleus (Bedini & Lanfranchi, 1991; Figure 5A, B). Voluminous organelles, such as endoplasmic reticulum (ER) and associated Golgi apparatus, are restricted to pockets of cytoplasm located at the position where a neurite exits the cell body (termed “axon hillock” by Bedini & Lanfranchi, 1991). Frequently, multivesicular bodies, consisting of a swarm of small vesicles surrounded by a double membrane, were observed close to the Golgi apparatus. In addition, vesicles of different sizes, shapes, and electron density can be found in cell bodies, as well as neurites and synapses located in the neuropil; these vesicles were used to classify neurons. Both, Bedini and Lafranchi (1991) studying Convoluta psammophila, Otocelis rubropunctata, and Amphiscolops sp.) and Bery and collaborators (2010), who similarly studied Symsagittifera roscoffensis, distinguish in their papers four classes of vesicles and neurons, including (1) small clear vesicles (20–40 nm; Figure 5D); (2) dense vesicles (70–90 nm; Figure 5D); (3) dense-core vesicles (60–120 nm); (4) large and irregularly shaped clear vesicles (20–400 nm):
(1) Small clear vesicles across the animal kingdom are associated with neurons signaling by classical, fast-acting transmitters (acetylcholine, glutamate, GABA); in the acoelomorph nervous system, they were also taken to signify this type of neuron. Bedini and collaborators (2001) documented the presence of GABA in small clear vesicles by immune electron microscopy. Small clear vesicles are predominant at synaptic sites, either homogenously, or interspersed with dense vesicles.
(2) Large, (uniformly) electron-dense vesicles store neuropeptides, slow-acting neurotransmitters that are released at synaptic sites or extra-synaptically (volume transmission). It stands to reason that these vesicles, which, in acoelomorph neurons do occur both at synaptic sites and diffusely along the neurites and in the cell body, are also associated with neuropeptides. Light microscopic studies have documented the high prevalence of peptidergic neurons in the acoel nervous system.
(3) Dense-core vesicles, as defined for acoelomorph or platyhelminth neurons, differ from the “uniformly dense” vesicles by their more variable size and shape, and by the fact that a light halo surrounds the inner dense core (Figure 5B, inset). The core itself is of different sizes, and it can be located in the center of the vesicle, or eccentrically. It is not yet clear how to interpret this type of dense-core vesicle. Note that in the vertebrate literature, the term “dense-core vesicle” (associated with neuropeptide content) refers to a type of vesicle that is called “uniformly dense vesicle” in acoelomorphs or platyhelminths. Synaptic vesicles that resemble the “dense-core” vesicles sensu Acoela are rarely described for vertebrates. Importantly, it was shown for vertebrate chromaffin cells that the same content can be stored in two types of dense vesicles (both called dense core; Grabner, Price, Lysakowski, & Fox, 2005): small ones that resemble the “uniformly dense” vesicles described for acoelomorphs, as well as larger ones in which the dense content was surrounded by a light halo, and which thereby resemble the “dense-core” vesicles of acoelomorphs. It is therefore possible that both types of vesicles maybe associated with peptidergic or aminergic neurotransmission or both in acoelomorphs.
(4) Large, clear pleomorphic vesicles are frequent in neuronal cell bodies, and were associated with sensory neurons (Bedini & Lanfranchi, 1991). The functional significance of this type of vesicle is not apparent.
The center of the brain is filled with densely packed, branched neurites of different diameters (Bery et al., 2010). The diameters of neurites in a given section range from less than 0.1 µm to approximately 1 µm (Figure 5C, D). Small-diameter profiles (0.05–0.2 μm) outnumber thicker profiles (0.4–1 μm) by a large margin, similar to what has been described for neuropil of other invertebrate species (e.g., Cardona, Saalfeld, Preibisch, Schmid, Cheng, Pulokas et al., 2010). Profiles with large diameters (0.4–1 µm) are the ones that mostly carry presynaptic (axonal) sites (Figure 5D, E). Thin processes (0.05–0.2 μm) belong to terminal postsynaptic (dendritic) branches. Large “globular” axonal terminals (larger than 1.5 μm) that are characteristic for the brain of insects (where they represent, for example, sensory endings of olfactory or gustatory receptors) and other taxa are rare or absent in the acoel brain. Neurite profiles containing vesicles in conjunction with membrane densities have been identified as synapses (Bedini & Lanfranchi, 1991). As in vertebrates (Colonnier, 1968), symmetric and asymmetric synapses can be found in acoels. Clusters of small and/or dense-core vesicles confined to the presynaptic element characterize asymmetric synapses. The presynaptic membrane sometimes has a specialization called the synaptic ribbon (Bedini & Lanfranchi, 1991) (Figure 5D). Symmetric synapses have synaptic vesicle on either side of the cleft. Most synapses are polyadic, meaning that the (often large) presynaptic site contacts multiple (2–6) postsynaptic partners. Many synapses are of the “en passant” type, whereby neurites form multiple swellings (“boutons” or “varicosities”) along their path that are filled with mitochondria and carry the presynaptic sites (Figure 5D, E).
The presence of glial cells in basal bilaterian phyla, including Acoelomorpha, is controversial. One problem rests on the definition of this cell type, which even in the taxa where they are clearly present, is not clear. For example, glial cells are not only involved indirectly in neuronal interaction by controlling the ionic milieu and transmitter reuptake around synapses, but are direct synaptic partners of neurons in numerous cases (Carlson & Saint Marie, 1990). In general, glial cells described for invertebrates are structurally defined by producing large, flattened, lamellar processes wrapping around the surface of the brain, around individual neuronal cell bodies, around the neuropil, or around individual axon bundles. Cells with these properties are mentioned in several electron microscopic studies of the nervous system of platyhelminths (e.g., Biserova, 2000, 2008; and acoelomorphs, Bedini & Lanfranchi, 1991). However, as stated above, in the absence of serial electron microscopy, it is not possible to evaluate the size and architecture of such lamellar processes. What is clear is that continuous sheaths around neuronal cell bodies, or the brain surface/neuropil surface as a whole, are missing in acoels (Bedini & Lanfranchi, 1991; Bery et al., 2010). The advent of specific markers is needed to settle the question of glia in acoelomorphs and other basal bilaterian clades.
The ultrastructure of the central nervous system of Xenoturbellida and Nemertodermatida has so far received very little attention. In the former, Raikova, Reuter, Jondelius, and Gustafsson (2000) described two distinct types of neurons characterized as “light” neurons, with an electron-lucent cytoplasm and large nuclei, and “dark” neurons, which contain an electron-dense cytoplasm and smaller nuclei. Light neurons are more frequently observed than dark neurons. Light neurons contain predominantly light-clear vesicles and only few dense-core vesicles. Dark neurons contain neurons of variable electron-density. Synapses resemble their counterparts in the acoel neuropil (Raikova et al., 2000).
Nerve Cords and Peripheral Nerve Net
The nerve cords of the acoelomorph central nervous system are formed by axons of neurons located in the brain, as well as other neurons scattered along the entire length of the cords. The ultrastructure of the somata and neurites associated with the nerve cords is indistinguishable from that of the brain neuropil, with small diameter axons forming the majority of profiles, and polyadic synapses distributed along the length of the cord. Processes of muscle cells and gland cells frequently contact or even transect the cords and the brain neuropil, suggesting that efferent contact on these effector cells is made within the neuropil.
Light microscopic observations, using global or more specific markers for neurons, suggests the presence of a peripheral network of fibers located between the nerve cords and the epidermis, similar to that described for platyhelminths TEM reveals individual, scattered fibers or thin bundles (2–3 profiles) of fibers of 0.2–0.6 µm in diameter (Figures 5B, C; 6B). The fibers contain the same types of vesicles observed in the central neuropil (small, dense core, uniformly dense). Neuronal cell bodies giving rise to these fibers do not form part of the peripheral net, but are most likely part of the cortical cell mass surrounding the central neuropil (Bedini & Lanfranchi, 1991; Bery et al., 2010). The nerve net has both superficial (“subepidermal”) components, which are in direct contact with the membrane of the epidermis and sensory receptors, and deep (“submuscular”) components interspersed among muscle fibers (Bedini & Lanfranchi, 1991; Bery et al., 2010). Synapses between neurites or neurites and muscle processes are frequent within the peripheral plexus.
Statocysts are receptive to gravity, whereby one or more ciliated sensory neurons, integrated into the wall of a liquid-filled capsule, are stimulated mechanically by a heavy particle (statolith) that shifts in position within the capsule when the animal moves. Statocysts in xenacoelomorphs differ from this description, first and foremost because no ciliated sensory neurons are associated with the statocyst (Ehlers, 1991; Ferrero, 1973; Israelsson, 2007). In acoels, the statocyst has a capsule formed by two parietal cells including a cavity that houses a specialized statolith cell (lithocyte), which contains in its cytoplasm a crystalline inclusion (Figures 5A, 6D). The parietal cells are surrounded by a layer of extracellular matrix material, the only remnant of a basement membrane observed in acoels. A stereotyped set of muscle cell inserts at the capsule (Figure 6D). Nerve fibers are close to the statocyst, which, after all, is embedded in the brain neuropil; however, a clear connection to nerve endings reaching the lumen of the capsule, and acting as potential sensory receptors stimulated by the statolith, has not been described. Instead, it has been speculated that movement of the statolith could indirectly have an effect on the muscles from which the statocyst is suspended.
The statocyst of nemertodermatids resembles that of acoels, except for the fact that it is composed of two chambers, each harboring one lithocyte with its statolith inclusion. The number of parietal cells of the statocyst is variable. As in some acoels, the statocyst of Nemertodermatida can be located within the brain, and thus was described as intracerebral (Ehlers, 1991).
In Xenoturbella the statocyst harbors several mobile ciliary cells within the cavity. These mobile ciliary cells contain statolith-like inclusions. It was reported that in the living animal these ciliated cells are motile and freely moving within the capsule. It is unclear how, if at all, this architecture lends itself to the reception of gravity; some authors have speculated that, its name notwithstanding, the statocyst of Xenoturbella acts as an endocrine organ (Israelsson, 2007).
Similar to the statocyst, the eye (ocelli) found in several acoel species shows ultrastructural features that are not observed in any other clade. The eye is embedded in the brain, typically on either side of the statocyst. Characteristic features are a pigment cell, partially enclosing a small number of receptor cells that have axons connecting to the neuropil (Popova & Mamkaev, 1985; Yamasu, 1991; Figure 6C). The pigment cell possesses cytoplasmic pigment granules, in addition to a large membrane-bound organelle that includes numerous crystalline inclusions (“platelets”), and may act as a reflector. The receptor cells lack microvilli or cilia; in some of the species investigated, it was observed that the cell body of a large, primary sensory cell was penetrated by irregular processes formed by two or more neighboring secondary sensory cells (Yamasu, 1991). The eyespot of Otocelis rubropunctata is integrated in the epidermis, formed by a single, multiciliated cell containing pigment granules, and contacting adjacent nerve fibers of the peripheral plexus (Lanfranchi, 1990).
A (Preliminary) Genomic View of the Xenacoelomorph Nervous System
Very little is known concerning the genes controlling neurogenesis in Xenacoelomorpha, and thus efforts should be made to identity and characterize many more genes involved in this process. Such data would yield an insight into how neurogenic gene networks have evolved within this phylum, and how these changes relate to the inherent complexity of their nervous systems. Similar approaches have been taken in the context of sequencing other animal genomes (see, for instance, Burke, Angerer, Elphick, Humphrey, Yaguchi, Kiyama et al., 2006; Simionato, Kerner, Dray, Le Gouar, Ledent, Arendt et al., 2008).
Over the last few years, Pedro Martínez, working within an international consortium responsible for sequencing diverse Xenacoelomorpha genomes, has been able to annotate and characterize several families of transcription factors and other developmental regulators potentially involved in the construction of xenacoelomorph nervous systems. Supplementing genomic with transcriptomic data has enabled a thorough characterization. The genome of S. roscoffensis has a size of 1.4 Gb, much larger than that of Xenoturbella bocki, which is about 200 Mb (unpublished data). These two genomes have provided the best sequences, and our current data are mostly based on these two species
In order to collect preliminary data for this new project, a thorough search for genes of two major groups of transcription factors, the homeobox- and the bHLH-containing families (Jones, 2004) was performed. Initial analyses have shown that Xenacoelomorpha have a reduced complement of the bHLH families, all involved in bilaterian’s tissue specification, mainly of neural and mesodermal tissues. Seventeen different members in the acoel Symsagittifera and 33 in Xenoturbella were identified (see Table 1). Interestingly, orthologues have been detected in both animals of the major neurogenic-regulatory families, including Achaete-Scute and NeuroD. However, an absence has been observed in Symsagittifera of some well-known relatives, such as those belonging to the families Atonal, Hand, and Neurogenin, suggesting a less-complex regulatory apparatus responsible for patterning the acoel nervous system. This is surprising, since superficially, the nervous system of Xenoturbella is simpler than that of Symsagittifera. Whereas Xenoturbella NS is organized as a subepithelial net (without obvious cell aggregates; ganglia), the anterior domain of the nervous system of Symsagittifera is organized as a compact brain (see below for a more detailed description of those nervous systems). This preliminary observation would suggest that “simplicity” in the architectural organization of the nervous systems is not necessarily paralleled by a similar complexity of neural regulatory families (however, see Dupre & Yuste, 2017, for an insightful assessment of what a “simple nerve net” might mean).
In a study carried out by Brauchle and collaborators during 2016 (Simon G. Sprecher, unpublished data), using transcriptomes from different acoels, 11 classes of homeobox genes have been identified (including ANTP, PRD, POU, TALE, etc.). Most of those classes are known in all bilaterian phyla and carry out different regulatory functions, from axis specification to the control of growth and differentiation of different tissues. Since the Acoela belong to the earliest offshoot of the Bilateria, the main implication of this study is that Xenacoelomorpha should be the first bilaterian phylum with a full representation of the major homeobox classes. Needless to say, at this point very little is known about their roles in the acoel nervous system (besides the Hox family; see, for instance, Moreno et al., 2010; Moreno & Martínez, 2010). Testing the role of those genes should prove a fruitful avenue for future research.
In order to obtain a better understanding of the downstream effectors involved in nervous system function, a thorough characterization of the different members of the GPCRs superfamily was carried out (Schiöth & Fredriksson, 2005), because this group of proteins controls the transduction of sensory information (vision, smell, taste, etc.). The complex families of GPCRs in both Xenoturbella and Symsagittifera were characterized (see Table 1). Interestingly, a complement of 258 different GPCR genes was detected in the Xenoturbella genome, while in the case of Symsagittifera, this complement is reduced to 225 different sequences (Perea-Atienza et al., 2015). Signaling molecules related to the formation of the primary axis, and thus to the position of the nervous system along the body, were also explored. In this context, Gavilán and collaborators (2016) identified the entire complement of Wnt ligands in the two xenacoelomorphs. Strikingly, the authors discovered that while xenoturbellids have Wnt genes that are clearly classifiable within bilaterian groups, the acoels seem to have several genes derived from lineage-specific duplications. A proper understanding of the roles of these and other candidate genes will require extensive use of in situ hybridization methods to enable a study of their expression patterns in space and time.
Table 1. Complement of genes of different families in X. bocki and S. roscoffensis.
Even though these are the best examples of the characterization (still partial) of xenacoelomorph genomes, other studies have been conducted using high-throughput transcriptomic approaches. These have resulted in the identification of many regulatory genes involved in different developmental processes, including patterning of the nervous system. A noteworthy study using transcriptomic data was performed on the acoel Hofstenia miamia (Srivastava et al., 2014), in the context of characterizing dorso-ventral pattern regulators. Other projects of a similar nature are underway in several laboratories.
We would like to thank Matz Berggren (Kristineberg, Sweden), Inga Martinek (České Budějovice, Czech Republic), Elena Perea-Atienza (Barcelona, Spain), Olga Raikova (Saint Petersburg, Russia) and Mansi Srivastava (Cambridge, Massachusetts) for providing us with pictures. Elena Perea-Atienza and Anna Aragay (Barcelona) are also acknowledged for their help in figure sketching.
P.M. acknowledges the support from the Spanish Ministry of Economy (MINECO), Grant Number BFU2009-07383. V.K. acknowledges the support from NIH, Grant R01 NS054814. S.G.S. acknowledges the support by the Swiss National Science foundation (Grant 31003A_169993) and the European Research Council (ERC-2012-StG 309832-PhotoNaviNet). P.M. and S.G.S. would like to thank the University of Gothenburg for the funds provided by the program to support internationalization and scientific renewal at the Sven Lovén Centre for Marine Sciences (Kristineberg).
We are also especially grateful for the constructive critique and advice from the anonymous reviewers.
Achatz, J. G., Chiodin, M., Salvenmoser, W., Tyler, S., & Martinez, P. (2013). The Acoela: On their kind and kinships, especially with nemertodermatids and xenoturbellids (Bilateria incertae sedis). Organisms, Diversity and Evolution, 13(2), 267–286.Find this resource:
Achatz, J. G., Hooge, M., Wallberg, A., Jondelius, U., & Tyler, S. (2010). Systematic revision of acoels with 9+0 sperm ultrastructure (Convolutida) and the influence of sexual conflict on morphology. Journal of Zoological Systematics and Evolutionary Research, 48(1), 9–32.Find this resource:
Achatz, J. G., & Martinez, P. (2012). The nervous system of Isodiametra pulchra (Acoela) with a discussion on the neuroanatomy of the Xenacoelomorpha and its evolutionary implications. Frontiers in Zoology, 9, 27.Find this resource:
Apelt, G. (1969). Fortpflanzungsbiologie, Entwicklungszyklen und vergleichende Frähenwicklung acoeler Turbellarien. Marine Biology, 4, 267–325.Find this resource:
Arroyo, A. S., López-Escardó, D., de Vargas, C., & Ruiz-Trillo, I. (2016). Hidden diversity of Acoelomorpha revealed through metabarcoding. Biology Letters, 12(9).Find this resource:
Ax, P. (1996). Multicellular animals: A new approach to the phylogenetic order in nature. Berlin: Springer Verlag.Find this resource:
Baguñà, J., & Riutort, M. (2004). The dawn of bilaterian animals: The case of acoelomorph flatworms. Bioessays, 26(10), 1046–1057.Find this resource:
Bedini, C., Ferrero, E., & Lanfranchi, A. (1973). The ultrastructure of ciliary sensory cells in two turbellaria acoela. Tissue and Cell, 5(3), 359–372.Find this resource:
Bedini, C., Ferrero, E., & Lanfranchi, A. (1975). Fine structural observations on the ciliary receptors in the epidermis of three otoplanid species (Turbellaria proseriata). Tissue and Cell, 7(2), 253–266.Find this resource:
Bedini, C., & Lanfranchi, A. (1991). The central and peripheral nervous system of Acoela (Plathelminthes). An electron microscopical study. Acta Zoologica, 72(2), 101–106.Find this resource:
Bedini, C., Lanfranchi, A., & Santerini, D. (2001). Is GABA present in the nervous system of acoel plathelminthes? An electron immunocytochemical study. Italian Journal of Zoology, 68(1), 23–27.Find this resource:
Beltagi, S., & Mandura, A. S. (1991). Hofstenia arabiensis nov. sp. (Hofsteniidae): A new species of acoelan turbellaria from the Red Sea North of Jeddah. Journal of King Abdulaziz University: Science, 3, 65–90.Find this resource:
Bery, A., Cardona, A., Martinez, P., & Hartenstein, V. (2010). Structure of the central nervous system of a juvenile acoel. Symsagittifera roscoffensis. Development Genes and Evolution, 220, 61–76.Find this resource:
Bery, A., & Martínez, P. (2011). Acetylcholinesterase activity in the developing and regenerating nervous system of the acoel. Symsagittifera roscoffensis. Acta Zoologica, 92(4), 383–392.Find this resource:
Biserova, N. (2000). The ultrastructure of glia-like cells in lateral nerve cords of adult Amphilina foliacea (Amphilinida). Acta Biologica Hungarica, 51(2–4), 439–442.Find this resource:
Biserova, N. (2008). Do glial cells exist in the nervous system of parasitic and free-living flatworms? An ultrastructural and immunocytochemical investigation. Acta Biologica Hungarica, 59 (Suppl. 2), 209–219.Find this resource:
Bock, S. (1923). Eine neue Turbellariengattung aus Japan. Uppsala Universitets Arsskrift, 1, 1–52.Find this resource:
Børve, A., & Hejnol, A. (2014). Development and juvenile anatomy of the nemertodermatid Meara stichopi (Bock) Westblad 1949 (Acoelomorpha). Frontiers in Zoology, 11, 50.Find this resource:
Bourlat, S. J., Juliusdottir, T., Lowe, C. J., Freeman, R., Aronowicz, J., Kirschner, M., et al. (2006). Deuterostome phylogeny reveals monophyletic chordates and the new phylum Xenoturbellida. Nature, 444, 85–88.Find this resource:
Bourlat, S. J., Nielsen, C., Lockyer, A. E., Littlewood, D. T., & Telford, M. J. (2003). Xenoturbella is a deuterostome that eats molluscs. Nature, 424, 925–928.Find this resource:
Boyer, B. C. (1971). Regulative development in a spiralian embryo as shown by cell deletion experiments on the Acoel, Childia. Journal of Experimental Zoology, 176, 97–106.Find this resource:
Bresslau, E. (1909). Die Entwicklung der Acoelen. Verhandlungen der Deutschen Zoologischen Gesellschaft, 19, 314–324.Find this resource:
Brusca, R. C., Moore, W., & Shuster, S. M. (2016). Invertebrates (3d ed.). Sunderland, MA: Sinauer Associates.Find this resource:
Burke, R. D., Angerer, L. M., Elphick, M. R., Humphrey, G. W., Yaguchi, S., Kiyama T., et al. (2006). A genomic view of the sea urchin nervous system. Developmental Biology, 300, 434–460.Find this resource:
Cannon, J. T., Vellutini, B. C., Smith, J., Ronquist, F., Jondelius, U., & Hejnol, A. (2016). Xenacoelomorpha is the sister group to Nephrozoa. Nature, 530, 89–93.Find this resource:
Cardona, A., Saalfeld, S., Preibisch, S., Schmid, B., Cheng, A., Pulokas, J., et al. (2010). An integrated micro- and macroarchitectural analysis of the Drosophila brain by computer-assisted serial section electron microscopy. PLoS Biology, 8(10).Find this resource:
Carlson, S. D., & Saint Marie, R. L. (1990). Structure and function of insect glia. Annual Review of Entomology, 35, 597–621.Find this resource:
Carranza, S., Baguna, J., & Riutort, M. (1997). Are the Platyhelminthes a monophyletic primitive group? An assessment using 18S rDNA sequences. Molecular Biology and Evolution, 14, 485–497.Find this resource:
Colonnier, M. (1968). Synaptic patterns on different cell types in the different laminae of the cat visual cortex: An electron microscope study. Brain Research, 9(2), 268–287.Find this resource:
Corrêa, D. D. (1960). Two new marine Turbellaria from Florida. Bulletin of Marine Science of the Gulf and Caribbean, 10, 208–216.Find this resource:
Crezee, M. (1978). Paratomella rubra Rieger and Ott, an amphiatlantic acoel turbellarian. Cahiers de Biologie Marine, 19, 1–9.Find this resource:
Crezée, M. (1975). Monographof the Solenofilomorphidae (Turbellaria: Acoela). Internationale Revue der Gesamten Hydrobiologie, 60, 769–845.Find this resource:
Crezée, M., & Tyler, S. (1976). Hesiolicium gen.n. (Turbellaria Acoela) and observations on its ultrastructure. Zoologica Scripta, 5, 207–216.Find this resource:
De Mulder, K., Kuales, G., Pfister, D., Willems, M., Egger, B., Salvenmoser, W., et al. (2009). Characterization of the stem cell system of the acoel Isodiametra pulchra. BMC Developmental Biology, 9, 69.Find this resource:
Delage, Y. (1886). Etudes histologiques sur les planaires rhabdocoeles acoeles (Convoluta Schultzii [O. Schm.]). Archives de zoologie expérimentale et générale (Ser 2), 4, 109–162.Find this resource:
Dörjes, J. (1968). Die Acoela (Turbellaria) der Deutschen Nordseekste und ein neues System der Ordnung. Zeitschrift für zoologische Systematik und Evolutionsforschung, 6, 56–452.Find this resource:
Dupre, C., & Yuste, R. (2017) Non-overlapping neural networks in Hydra vulgaris. Current Biology, 27(8), 1085–1097.Find this resource:
Egger, B., Steinke, D., Tarui, H., De Mulder, K., Arendt, D., Borgonie, G., et al. (2009). To be or not to be a flatworm: The acoel controversy. PloS One, 4(5), e5502.Find this resource:
Ehlers, U. (1985). Das Phylogenetische System der Plathelminthes. Stuttgart, Germany: Gustav Fischer.Find this resource:
Ehlers, U. (1991). Comparative morphology of statocysts in the Plathelminthes and the Xenoturbellida. Hydrobiologia, 227, 263–271.Find this resource:
Ehlers, U. (1992). Frontal glandular and sensory structures in Nemertoderma (Nemertodermatida) and Paratomella (Acoela): Ultrastructure and phylogenetic implications for the monophyly of the Euplathelminthes (Plathelminthes). Zoomorphology, 112, 227–236.Find this resource:
Ehlers, U., & Ehlers, B. (1977). Monociliary receptors in interstitial Proseriata and Neorhabdocoela (Turbellaria Neoophora). Zoomorphologie, 86(3), 197–222.Find this resource:
Ehlers, U., & Sopott-Ehlers, B. (1997). Ultrastructure of the subepidermal musculature of Xenoturbella bocki, the adelphotaxon of the Bilateria. Zoomorphology, 117, 71–79.Find this resource:
Ferrero, E. (1973). A fine structural analysis of the Statocyst in Turbellaria Acoela. Zoologica Scripta, 2, 5–16.Find this resource:
Franks, N. R., Worley, A., Grant, K. A. J., Gorman, A. R., Vizard, V., Plackett, H., et al. (2016). Social behaviour and collective motion in plant-animal worms. Proceedings of the Royal Society B: Biological Sciences, 283(1825).Find this resource:
Franzen, A., Afzelius, B. A., & Franzkn, A. (1987). The ciliated epidermis of Xenoturbella bocki (Platyhelminthes, Xenoturbellida) with some phylogenetic considerations. Zoologica Scripta, 16, 9–17.Find this resource:
Fritzsch, G., Böhme, M., Thorndyke, M., Nakano, H., Israelsson, O., Stach, T., et al. (2007). PCR survey of Xenoturbella bocki Hox genes. Journal of Experimental Zoology Part B: Molecular and Developmental Evolution. 310(3), 278–284.Find this resource:
Gaerber, C. W., Salvenmoser, W., Rieger, R. M., & Gschwentner, R. (2007). The nervous system of Convolutriloba (Acoela) and its patterning during regeneration after asexual reproduction. Zoomorphology, 126, 73–87.Find this resource:
Gavilán, B., Perea-Atienza, E., & Martínez, P. (2016). Xenacoelomorpha: A case of independent nervous system centralization? Philosophical Transactions of the Royal Society of London. Series B, Biological Sciences, 371(1685).Find this resource:
Gegenbaur, C. (1859). Grundzüge der Vergleichenden Anatomie. Leipzig, Germany: W. Engelmann.Find this resource:
Georgévitvch, J. (1899). Étude sur le développement de la Convoluta roscoffensis Graff. Archives de zoologie expérimentale et générale, 7, 343–361.Find this resource:
Grabner, C. P., Price, S. D., Lysakowski, A., & Fox, A. P. (2005). Mouse chromaffin cells have two populations of dense core vesicles. Journal of Neurophysiology, 94, 2093–2104.Find this resource:
Gschwentner, R., Ladurner, P., Salvenmoser, W., Rieger, R., & Tyler, S. (1998). Fine structure and evolutionary significance of sagittocysts of Convolutriloba longifissura (Acoela, Platyhelminthes). Invertebrate Biology, 118, 332.Find this resource:
Hanson, E. D. (1967). Regeneration in acoelous flatworms: The role of the peripheral parenchyma. Wilhelm Roux’ Archiv für Entwicklungsmechanik der Organismen, 159, 298–313.Find this resource:
Haszprunar, G. (2015). Review of data for a morphological look on Xenacoelomorpha (Bilateria incertae sedis). Organisms Diversity and Evolution, 16, 363–389.Find this resource:
Hejnol, A. (2016). Acoelomorpha. In A. Schmidt-Rhaesa, S. Harzsch, & G. Purschke (Eds.), Structure and evolution of the invertebrate nervous systems (pp. 56–61). Oxford: Oxford University Press.Find this resource:
Hejnol, A., Obst, M., Stamatakis, A., Ott, M., Rouse, G. W., Edgecombe, G.D., et al. (2009). Assessing the root of bilaterian animals with scalable phylogenomic methods. Proceedings of the Royal Society B: Biological Sciences, 276, 4261–4270.Find this resource:
Hejnol, A., & Pang, K. (2016). Xenacoelomorpha’s significance for understanding bilaterian evolution. Current Opinion in Genetics and Development, 39, 48–54.Find this resource:
Henry, J. Q., Martindale, M. Q., & Boyer, B.C. (2000). The unique developmental program of the acoel flatworm, Neochildia fusca. Developmental Biology, 220, 285–295.Find this resource:
Hooge, M. D. (2001). Evolution of body-wall musculature in the Platyhelminthes (Acoelomorpha, Catenulida, Rhabditophora). Journal of Morphology, 249, 171–194.Find this resource:
Hooge, M. D., Haye, P. A., Tyler, S., Litvaitis, M. K., & Kornfield, I. (2002). Molecular systematics of the Acoela (Acoelomorpha, Platyhelminthes) and its concordance with morphology. Molecular Phylogenetics and Evolution, 24(2), 333–342.Find this resource:
Hyman, L. H. (1951). The Invertebrates. Vol 2: Platyhelminthes and Rhynchocoela; the acoelomate Bilateria. New York: McGraw Hill.Find this resource:
Israelsson, O. (1999). New light on the enigmatic Xenoturbella (phylum uncertain): Ontogeny and phylogeny. Proceedings of the Royal Society B: Biological Sciences, 266, 835.Find this resource:
Israelsson, O. (2007). Ultrastructural aspects of the “statocyst” of Xenoturbella (Deuterostomia) cast doubt on its function as a georeceptor. Tissue and Cell, 39, 171–177.Find this resource:
Jagersten, G. (1959). Further remarks on the early phylogeny of Metazoa. Zoologiska Bidrag fran Uppsala, 33, 79–108.Find this resource:
Jondelius, U., Larsson, K., & Raikova, O. (2004). Cleavage in Nemertoderma westbladi (Nemertodermatida) and its phylogenetic significance. Zoomorphology, 123, 221–225.Find this resource:
Jondelius, U., Ruiz-Trillo, I., Baguñà, J., & Riutort, M. (2002). The Nemertodermatida are basal bilaterians and not members of the Platyhelminthes. Zoologica Scripta, 31, 201–215.Find this resource:
Jondelius, U., Wallberg, A., Hooge, M., & Raikova, O. I. (2011). How the worm got its pharynx: Phylogeny, classification and Bayesian assessment of character evolution in Acoela. Systematic Biology, 60, 845–871.Find this resource:
Jones, S. (2004). An overview of the basic helix-loop-helix proteins. Genome Biology, 5(6), 226.Find this resource:
Keeble, F. (1910). Plant-animals: A study in symbiosis. Cambridge, U.K.: Cambridge University Press.Find this resource:
Klauser, M. D., Smith, J. P. S., & Tyler, S. (1986). Ultrastructure of the frontal organ in Convoluta and Macrostomum spp.: Significance for models of the turbellarian archetype. Hydrobiologia, 132(1), 47–52.Find this resource:
Kotikova, E. A., & Raikova, O. I. (2008). Architectonics of the central nervous system of Acoela, Platyhelminthes, and Rotifera. Journal of Evolutionary Biochemistry and Physiology, 44(1), 95–108.Find this resource:
Ladurner, P., & Rieger, R. (2000). Embryonic muscle development of Convoluta pulchra (Turbellaria-acoelomorpha, platyhelminthes). Developmental Biology, 222, 359–375.Find this resource:
Lanfranchi, A. (1990). Ultrastructure of the epidermal eyespots of an acoel platyhelminth. Tissue and Cell, 22, 541–546.Find this resource:
Lundin, K. (1997). Comparative ultrastructure of the epidermal ciliary rootlets and associated structures in species of the Nemertodermatida and Acoela (Plathelminthes). Zoomorphology, 117, 81–92.Find this resource:
Lundin, K. (1998). The epidermal ciliary rootlets of Xenoturbella bocki (Xenoturbellida) revisited: New support for a possible kinship with the Acoelomorpha (Platyhelminthes). Zoologica Scripta, 27, 263–270.Find this resource:
Lundin, K. (2000). Phylogeny of the Nemertodermatida (Acoelomorpha, Platyhelminthes). A cladistic analysis. Zoologica Scripta, 29, 65–74.Find this resource:
Lundin, K., & Hendelberg, J. (1995). Ultrastructure of the epidermis of Meara stichopi (Platyhelminthes, Nemertodermatida) and associated extra-epidermal bacteria. In L.R.G. Cannon (Ed.), Biology of turbellaria and some related flatworms (pp. 161–165). Dordrecht, The Netherlands: Springer.Find this resource:
Meyer-Wachsmuth, I., Curini-Galletti, M., & Jondelius, U. (2014). Hyper-cryptic marine meiofauna: Species complexes in Nemertodermatida. PloS One, 9(9), e107688.Find this resource:
Meyer-Wachsmuth, I., Raikova, O. I., & Jondelius, U. (2013). The muscular system of Nemertoderma westbladi and Meara stichopi (Nemertodermatida, Acoelomorpha). Zoomorphology, 132, 239–252.Find this resource:
Meyer, N. P., & Seaver, E. C. (2009). Neurogenesis in an annelid: Characterization of brain neural precursors in the polychaete Capitella sp. I. Developmental Biology, 335, 237–252.Find this resource:
Moreno, E., De Mulder, K., Salvenmoser, W., Ladurner, P., & Martinez, P. (2010). Inferring the ancestral function of the posterior Hox gene within the Bilateria: Controlling the maintenance of reproductive structures, the musculature and the nervous system in the acoel flatworm Isodiametra pulchra. Evolution and Development, 12(3), 258–266.Find this resource:
Moreno, E., & Martínez, P. (2010, December 15). Origin of Bilaterian Hox patterning system. Encyclopedia of Life Sciences. Online publication.Find this resource:
Mwinyi, A., Bailly, X., Bourlat, S. J., Jondelius, U., Littlewood, D. T. J., & Podsiadlowski, L. (2010). The phylogenetic position of Acoela as revealed by the complete mitochondrial genome of Symsagittifera roscoffensis. BMC Evolutionary Biology, 10, 309.Find this resource:
Nakano, H. (2015). What is Xenoturbella? Zoological Letters, 1, 22.Find this resource:
Nakano, H., Lundin, K., Bourlat, S. J., Telford, M. J., Funch, P., Nyengaard J. R., et al. (2013). Xenoturbella bocki exhibits direct development with similarities to Acoelomorpha. Nature Communications, 4, 1537.Find this resource:
Nielsen, C. (2010). After all: Xenoturbella is an acoelomorph! Evolution and Development, 12, 241–243.Find this resource:
Nielsen, C. (2012) Animal evolution: Interrelationships of the living phyla (3d ed.). Oxford: Oxford University Press.Find this resource:
Nissen, M., Shcherbakov, D., Heyer, A., Brummer, F., & Schill, R. O. (2015). Behaviour of the plathelminth Symsagittifera roscoffensis under different light conditions and the consequences for the symbiotic algae Tetraselmis convolutae. Journal of Experimental Biology, 218, 1693–1698.Find this resource:
Noren, M., & Jondelius, U. (1997). Xenoturbella’s molluscan relatives. Nature, 390, 31–32.Find this resource:
Northcutt, R. G. (2012). Evolution of centralized nervous systems: Two schools of evolutionary thought. Proceedings of the National Academy of Sciences, 109(Suppl. 1), 10626–10633.Find this resource:
Obst, M., Nakano, H., Bourlat, S. J., Thorndyke, M. C., Telford, M. J., Nyengaard, J. R., et al. (2011). Spermatozoon ultrastructure of Xenoturbella bocki (Westblad 1949). Acta Zoologica, 92(2), 109–115.Find this resource:
Paps, J., Baguña, J., & Riutort, M. (2009). Bilaterian phylogeny: A broad sampling of 13 nuclear genes provides a new Lophotrochozoa phylogeny and supports a paraphyletic basal Acoelomorpha. Molecular Biology and Evolution, 26(10), 2397–2406.Find this resource:
Pedersen, K. J. (1964). The cellular organization of Convoluta convoluta, an acoel turbellarian: A cytological, histochemical and fine structural study. Zeitschrift Für Zellforschung Und Mikroskopische Anatomie (Vienna, Austria: 1948), 64, 655–687.Find this resource:
Pedersen, K. J., & Pedersen, L. R. (1988). Ultrastructural observations on the epidermis of Xenoturbella bocki Westblad, 1949; with a discussion of epidermal cytoplasmic filament systems of invertebrates. Acta Zoologica, 69(4), 231–246.Find this resource:
Perea-Atienza, E., Botta, M., Salvenmoser, W., Gschwentner, R., Egger, B., Kristof, A., et al. (2013). Posterior regeneration in Isodiametra pulchra (Acoela, Acoelomorpha). Frontiers in Zoology, 10(1), 64.Find this resource:
Perea-Atienza, E., Gavilán, B., Chiodin, M., Abril, J. F., Hoff, K. J., Poustka, A. J., et al. (2015). The nervous system of Xenacoelomorpha: A genomic perspective. Journal of Experimental Biology, 218, 618–628.Find this resource:
Philippe, H., Brinkmann, H., Copley, R. R., Moroz, L. L., Nakano, H., Poustka A.J., et al. (2011). Acoelomorph flatworms are deuterostomes related to Xenoturbella. Nature, 470, 255–258.Find this resource:
Popova, N.V., & Mamkaev, Y. (1985). Ultrastructure and primitive features of the eyes of Convoluta convoluta (Turbellaria Acoela). Doklady Akademii Nauk SSSR, 283, 756–759.Find this resource:
Raikova, O. I. (2004). Neuroanatomy of basal bilaterians (Xenoturbellida, Nemertodermatida, Acoela) and its phylogenetic implications (PhD thesis). Åbo Akademi University, Åbo, Finland.Find this resource:
Raikova, O. I., Meyer-Wachsmuth, I., & Jondelius, U. (2016). The plastic nervous system of Nemertodermatida. Organisms Diversity and Evolution, 16, 85–104.Find this resource:
Raikova, O. I., Reuter, M., Gustafsson, M. K. S., Maule, A. G., Halton, D. W., & Jondelius, U. (2004a). Basiepidermal nervous system in Nemertoderma westbladi (Nemertodermatida): GYIRFamide immunoreactivity. Zoology, 107(1), 75–86.Find this resource:
Raikova, O. I., Reuter, M., Gustafsson, M. K. S., Maule, A. G., Halton, D. W., & Jondelius, U. (2004b). Evolution of the nervous system in Paraphanostoma (Acoela). Zoologica Scripta, 33, 71–88.Find this resource:
Raikova, O. I., Reuter, M., Jondelius, U., & Gustafsson, M. K. S. (2000). An immunocytochemical and ultrastructural study of the nervous and muscular systems of Xenoturbella westbladi (Bilateria inc. sed.). Zoomorphology, 120, 107–118.Find this resource:
Raikova, O. I., Reuter, M., Kotikova, E. A., & Gustafsson, M. K. S. (1998). A commissural brain! The pattern of 5-HT immunoreactivity in Acoela (Plathelminthes). Zoomorphology, 118, 69–77.Find this resource:
Ramachandra, N. B., Gates, R. D., Ladurner, P., Jacobs, D. K., & Hartenstein, V. (2002). Embryonic development in the primitive bilaterian Neochildia fusca: Normal morphogenesis and isolation of POU genes Brn-1 and Brn-3. Development Genes and Evolution, 212(2), 55–69.Find this resource:
Reisinger, E. (1960). Was ist Xenoturbella? Zeitschrift für wissenschaftliche Zoologie, 164, 188–198.Find this resource:
Reuter, M., Raikova, O. I., & Gustafsson, M. K. S. (1998). An endocrine brain? The pattern of FMRF-amide immunoreactivity in Acoela (Plathelminthes). Tissue and Cell, 30, 57–63.Find this resource:
Reuter, M., Raikova, O. I., Jondelius, U., Gustafsson M. K. S., Maule, A. G., & Halton, D. W. (2001). Organisation of the nervous system in the Acoela: An immunocytochemical study. Tissue and Cell, 33, 119–128.Find this resource:
Richter, S., Loesel, R., Purschke, G., Schmidt-Rhaesa, A., Scholtz, G., Stach T., et al. (2010). Invertebrate neurophylogeny: Suggested terms and definitions for a neuroanatomical glossary. Frontiers in Zoology, 7(1), 29.Find this resource:
Rieger, R.M., Tyler, S., Smith J. P. S., III, & Rieger G. (1991). Platyhelminthes: Turbellaria. In B. B. Harrison (Ed.), Microscopic anatomy of invertebrates. New York: Wileys-Liss.Find this resource:
Rohde, K., & Watson, N. A. (1995). Ultrastructure of the buccal complex of Polylabroides australis (Monogenea, Polyopisthocotylea, Microcotylidae). International Journal for Parasitology, 25(3), 307–318.Find this resource:
Rouse, G. W., Wilson, N. G., Carvajal, J. I., & Vrijenhoek, R. C. (2016). New deep-sea species of Xenoturbella and the position of Xenacoelomorpha. Nature, 530, 94–97.Find this resource:
Ruiz-Trillo, I., & Paps, J. (2015). Acoelomorpha: Earliest branching bilaterians or deuterostomes? Organisms Diversity and Evolution, 16, 391–399.Find this resource:
Ruiz-Trillo, I., Riutort, M., Littlewood, D. T., Herniou, E. A., & Baguña, J. (1999). Acoel flatworms: Earliest extant bilaterian Metazoans, not members of Platyhelminthes. Science, 283, 1919–1923.Find this resource:
Schiöth, H. B., & Fredriksson, R. (2005). The GRAFS classification system of G-protein coupled receptors in comparative perspective. General and Comparative Endocrinology, 142(1–2), 94–101.Find this resource:
Semmler, H., Chiodin, M., Bailly, X., Martinez, P., & Wanninger, A. (2010). Steps towards a centralized nervous system in basal bilaterians: Insights from neurogenesis of the acoel Symsagittifera roscoffensis. Development, Growth and Differentiation, 52, 701–713.Find this resource:
Sikes, J. M., & Bely, A. E. (2008). Radical modification of the A-P axis and the evolution of asexual reproduction in Convolutriloba acoels. Evolution and Development, 10, 619–631.Find this resource:
Simionato, E., Kerner, P., Dray, N., Le Gouar, M., Ledent, V., Arendt, D., et al. (2008). Atonal- and achaete-scute-related genes in the annelid Platynereis dumerilii: Insights into the evolution of neural basic-Helix-Loop-Helix genes. BMC Evolutionary Biology, 8(1), 170.Find this resource:
Smith, J. P. S., & Tyler, S. (1986). Frontal organs in the Acoelomorpha (Turbellaria): ultrastructure and phylogenetic significance. Hydrobiologia, 132, 71–78.Find this resource:
Smith, J. P. S., & Bush, L. (1991). Convoluta pulchra n. sp. (Turbellaria: Acoela) from the East Coast of North America. Transactions of the American Microscopical Society, 110(1), 12.Find this resource:
Smith, J. P. S., & Tyler, S. (1985). Fine-structure and evolutionary implications of the frontal organ in Turbellaria Acoela. 1 Diopisthoporus gymnopharyngeus sp.n. Zoologica Scripta, 14, 91–102.Find this resource:
Smith, J. P. S., & Tyler, S. (1986). Frontal organs in the Acoelomorpha (Turbellaria): Ultrastructure and phylogenetic significance. Hydrobiologia, 132(1), 71–78.Find this resource:
Sopott-Ehlers, B., & Ehlers, U. (1997). Ultrastructure of the subepidermal musculature of Xenoturbella bocki, the adelphotaxon of the Bilateria. Zoomorphology, 117, 71–79.Find this resource:
Sprecher, S. G., Bernardo-Garcia, F. J., van Giesen, L., Hartenstein, V., Reichert, H., Neves R., et al. (2015). Functional brain regeneration in the acoel worm Symsagittifera roscoffensis. Biology Open, 4, 1688–1695.Find this resource:
Srivastava, M., Mazza-Curll, K. L., van Wolfswinkel, J. C., & Reddien, P. W. (2014). Whole-body acoel regeneration is controlled by Wnt and Bmp-Admp signaling. Current Biology, 24, 1107–1113.Find this resource:
Stach, T., Dupont, S., Israelson, O., Fauville, G., Nakano, H., Kånneby, T., et al. (2005): Nerve cells of Xenoturbella bocki (phylum uncertain) and Harrimania kupfferi (Enteropneusta) are positively immunoreactive to antibodies raised against echinoderm neuropeptides. Journal of the Marine Biological Association of the United Kingdom, 85, 1519–1524.Find this resource:
Stach, T. (2016). Xenoturbella. In A. Schmidt-Rhaesa, S. Harzsch, & G. Purschke (Eds.), Structure and evolution of invertebrate nervous systems (pp. 62–66). Oxford University Press.Find this resource:
Steinböck, Os. (1966). Die Hofsteniiden (Turbellaria Acoela). Grundsäzliches zur Evolution der Turbellarien. Zeitschrift für Zoologische Systematik und Evolutionsforschung, 4, 58–195.Find this resource:
Sterrer, W. (1998). New and known Nemertodermatida (Platyhelminthes-Acoelomorpha): A revision. Belgian Journal of Zoology, 128(1), 55–92.Find this resource:
Telford, M. J., Budd, G. E., & Philippe, H. (2015). Phylogenomic insights into animal evolution. Current Biology, 25, R876–887.Find this resource:
Telford, M. J., Lockyer, A. E., Cartwright-Finch, C., & Littlewood, D. T. J. (2003). Combined large and small subunit ribosomal RNA phylogenies support a basal position of the acoelomorph flatworms. Proceedings. Biological Sciences/The Royal Society, 270, 1077–1083.Find this resource:
Todt, C. (2009). Structure and evolution of the pharynx simplex in acoel flatworms (Acoela). Journal of Morphology, 270, 271–290. http://dx.doi.org/10.1002/jmor.10682Find this resource:
Todt, C., & Tyler, S. (2006). Ciliary receptors associated with the mouth and pharynx of Acoela (Acoelomorpha): A comparative ultrastructural study. Acta Zoologica, 88(1), 41–58.Find this resource:
Tyler, S. (1984). Turbellarian Platyhelminths. In J. Bereiter-Hahn, A.G. Matoltsky, & K.S. Richards (Eds.), Biology of the Integument. Vol 1: Invertebrates (pp. 112–131). Berlin: Springer Verlag.Find this resource:
Wallberg, A., Curini-Galletti, M., Ahmadzadeh, A., & Jondelius, U. (2007). Dismissal of Acoelomorpha: Acoela and Nemertodermatida are separate early bilaterian clades. Zoologica Scripta, 36, 509–523.Find this resource:
Westblad, E. (1940). Studien über skandinavische Turbellaria Acoela. I. Arkiv För Zoologi, 32A(20), 1–28.Find this resource:
Westblad, E. (1949). Xenoturbella bocki n.g., n. sp., a peculiar, primitive turbellarian type. Arkiv För Zoologi, 1, 3–29.Find this resource:
Wright, K. A. (1992). Peripheral sensilla of some lower invertebrates: The platyhelminthes and nematoda. Microscopy Research and Technique, 22(3), 285–297.Find this resource:
Yamasu, T. (1991). Fine structure and function of ocelli and sagittocysts of acoel flatworms. Hydrobiologia, 227(1), 273–282.Find this resource:
Yang, J., Liu, X., Yue, G., Adamian, M., Bulgakov, O., & Li, T. (2002). Rootletin, a novel coiled-coil protein, is a structural component of the ciliary rootlet. Journal of Cell Biology, 159, 431–440.Find this resource: