Insights into the segmental identity of post-oral commissures and pharyngeal nerves in Onychophora based on retrograde fills
© Martin and Mayer. 2015
Received: 19 February 2015
Accepted: 12 August 2015
Published: 25 August 2015
While the tripartite brain of arthropods is believed to have evolved by a fusion of initially separate ganglia, the evolutionary origin of the bipartite brain of onychophorans—one of the closest arthropod relatives—remains obscure. Clarifying the segmental identity of post-oral commissures and pharyngeal nerves might provide useful insights into the evolution of the onychophoran brain. We therefore performed retrograde fills of these commissures and nerves in the onychophoran Euperipatoides rowelli.
Our fills of the anterior and posterior pharyngeal nerves revealed groups of somata that are mainly associated with the deutocerebrum. This resembles the innervation pattern of other feeding structures in Onychophora, including the jaws and several lip papillae surrounding the mouth. Our fills of post-oral commissures in E. rowelli revealed a graded arrangement of anteriorly shifted somata associated with post-oral commissures #1 to #5. The number of deutocerebral somata associated with each commissure decreases posteriorly, i.e., commissure #1 shows the highest and commissure #5 the lowest numbers of associated somata, whereas none of the subsequent median commissures, beginning with commissure #6, shows somata located in the deutocerebrum.
Based on the graded and shifted arrangement of somata associated with the anteriormost post-oral commissures, we suggest that the onychophoran brain, which is a bipartite syncerebrum, might have evolved by a successive anterior/anterodorsal migration of neurons towards the protocerebrum in the last onychophoran ancestor. This implies that the composite brain of onychophorans and the compound brain of arthropods might have independent evolutionary origins, as in contrast to arthropods the onychophoran syncerebrum is unlikely to have evolved by a fusion of initially separate ganglia.
The two widely separated ventral nerve cords of onychophorans do not show any segmental ganglia but instead are linked with each other by numerous median and ring commissures in an orthogon-like fashion [16–19]. The nerve cords are connected to the brain via a pair of medullary (i.e. non-ganglionated) structures, the so-called connecting cords  (“circumoesophageal connectives” sensu Henry ; “medullary connectives” sensu Whitington and Mayer  “connecting pieces” sensu Martin and Mayer ). The frequently used term “circumoesophageal connectives” is unsuitable for designating these cords, as connectives are somata-free structures connecting ganglia , whereas the connecting cords resemble the ventral nerve cords. Nevertheless, the connecting cords differ from the ventral nerve cords in that they are not associated with ring commissures. The connecting cords are situated on each side of the pharynx and linked with each other via a number of post-oral (=post-pharyngeal) commissures. Due to the medullary organization of the connecting cords, their segmental identity as well as that of the associated commissures is unclear.
In addition to the post-oral commissures, Henry  described a pair of “stomodaeal nerves” emanating from the connecting cords and supplying the pharynx. As with the post-oral commissures associated with the connecting cords, the segmental identity of this pair of pharyngeal nerves is unknown because the corresponding neuronal somata have not been identified. An additional prominent “loop nerve” is also associated with the onychophoran pharynx . Although this nerve shows a medullary organization, with at least some somata located in the dorsolateral pharyngeal wall, it is unclear whether or not there are additional neurons supplying this nerve located within the brain. Clarifying this issue would help to determine the innervation pattern and segmental identity of the onychophoran pharynx, which remains ambiguous.
During ontogeny, the onychophoran pharynx arises from the walls of the embryonic stomodeum [15, 21]. This would suggest that the pharynx, like the stomodeum itself, belongs to the second body segment. Accordingly, the first post-oral commissure of onychophorans, which is closely associated with the ventral pharyngeal wall , might also belong to the second body segment. Alternatively, this commissure might be composed of fibers from both the second (jaw) and the third (slime papilla) segments, thus resembling the situation in arthropods, in which the first post-oral commissure contains both deutocerebral and tritocerebral fibers [1, 22–26].
To clarify the composition of post-oral commissures and the segmental identity of pharyngeal nerves in Onychophora, we performed retrograde fills of these neural structures and localized the position of their supplying neurons in the onychophoran Euperipatoides rowelli.
Arrangement and innervation pattern of the anterior post-oral commissures
Neuronal tracing of nerves supplying the pharynx
Like the anterior pair of nerves supplying the pharynx, the posterior pair also shows neuronal somata located within the deutocerebrum (Fig. 6b). However, apart from these, each posterior pharyngeal nerve has a few additional somata (~10) in each connecting cord that are grouped around the basis of the nerve (Fig. 6b). The group of somata within the deutocerebrum comprises ~30 cells that are all situated in the ipsilateral brain hemisphere. A number of fibers associated with the posterior pharyngeal nerve terminate in the connecting cord as well as in the contralateral half of the brain (arrowhead in Fig. 6b).
Deutocerebral innervation of feeding structures in Onychophora
Segmental identity of post-oral commissures in Onychophora
The median commissures that link the two widely separated nerve cords of onychophorans do not show any obvious segmental arrangement but are instead organized in a ladder-like fashion along the body (reviewed by Whitington and Mayer  and Mayer ). Using the position of leg nerves as segmental landmarks, previous studies have shown that the number of median commissures varies between 8 and 10 per segment [16–19, 27]. This variation in number and the lack of segmental ganglia in onychophorans generally render it difficult to assign each median commissure to a corresponding body segment.
Our neuroanatomical data from E. rowelli further revealed that the first post-oral commissure differs from commissures #2 to #5 in that it is thicker and longer and lies relatively further anteriorly, as it is associated with the connecting cords rather than with the anterior portions of nerve cords supplying the slime papillae (cf. Fig. 2a, b). Moreover, the first post-oral commissure is the only commissure with somata in the contralateral brain hemispheres relative to the fill site (Figs. 3b, 8a). Despite our detailed data on the position of supplying neurons, the first post-oral commissure of onychophorans is difficult to assign to a particular head segment, as numerous associated somata are located in the connecting cords, the segmental identity of which is unknown. However, since most commissural somata are situated in the anterior portions of the connecting cords as well as in the deutocerebrum and none of them is associated with the regions of nerve cords innervating the slime papillae, we suggest that the first post-oral commissure mainly, if not exclusively, receives fibers from neurons that belong to the second (jaw) segment (Fig. 9b). Clarifying the segmental identity and embryonic origin of the connecting cords will be crucial for substantiating this hypothesis.
In summary, our findings suggest that the first post-oral commissure of onychophorans is most likely associated with the second (deutocerebral) part of the brain supplying the jaws . Interestingly, Henry  reported a close association of the first post-oral commissure with the ventral pharyngeal wall, which was also evident in our preparations of the nervous system in E. rowelli. We therefore cannot exclude that at least some commissural fibers might project into the pharynx. If true, this would imply that the first post-oral commissure is involved in the control of pharyngeal function, i.e., it might play a role in controlling the ingestion of food.
Conclusion: implications for the evolution of the onychophoran brain
The overall organization of the onychophoran brain differs from that of arthropods in that it has only two segmental regions, the protocerebrum and the deutocerebrum  (Fig. 1a). In contrast, representatives of arthropods typically have at least one additional segmental brain region, the tritocerebrum (e.g. [1–3, 6, 28]; but note also that branchiopod crustaceans have a bipartite brain since the tritocerebrum is spatially separated from the proto- and deutocerebrum ). The typical arthropod brain is a compound structure, which is formed by the morphological fusion of separate embryonic ganglion anlagen . Thus, the ontogeny of the arthropod brain might recapitulate evolutionary changes that have taken place in the arthropod lineage, as the tripartite brain of arthropods might have evolved from separate ganglia that have fused to form a syncerebrum  (Additional file 1). A segmentally ganglionated nervous system is also present in tardigrades [30–34], one of the closest relatives of arthropods. However, developmental studies  argue against a multisegmented brain in tardigrades. Additionally, the position of the stomatogastric ganglion in the second trunk segment  suggests that this segment in tardigrades is homologous to the arthropod tritocerebrum. Accordingly, the ganglion of the first trunk segment corresponds to the arthropod deutocerebrum and therefore the tardigrade brain consists of only a single segmental region corresponding to the arthropod protocerebrum. Thus, Tardigrada might represent the ancestral “rope ladder-like” condition, i.e. one ganglion per segment, suggesting that ganglionization occurred before the fusion of brain neuromeres, as is found in arthropods. Such a ganglionic fusion is unlikely to have occurred in onychophorans because their last common ancestor most likely did not possess any segmental ganglia and because no segmental ganglion anlagen exist in the onychophoran embryo [17–19]. Hence, the bipartite brain of onychophorans might have evolved by an entirely different process.
Our retrograde fills revealed a graded arrangement of anteriorly shifted somata associated with post-oral commissures #1 to #5 in E. rowelli (cf. Fig. 8a–e). The number of deutocerebral somata associated with each commissure decreases posteriorly, i.e., commissure #1 shows the highest and commissure #5 the lowest numbers, whereas none of the subsequent commissures, beginning with commissure #6, exhibits somata located in the deutocerebrum. Based on this graded arrangement of commissural neurons, we assume that the deutocerebral somata might have migrated anteriorly along each nerve cord, i.e., towards the protocerebrum, in the onychophoran lineage (Additional file 2). The medullary organization of the ventral nervous system in onychophorans [7, 16–19, 36] might have allowed the neuronal somata to change their position along the antero-posterior body axis in their last common ancestor. This hypothesis does not require an assumption of pre-existing segmental ganglia in the onychophoran ancestor that might have fused to a compound brain, as in arthropods.
These findings imply that the bipartite syncerebrum of onychophorans and the tripartite brain of arthropods might have evolved convergently by two different processes: an anterior migration of neurons in the onychophoran lineage, and a fusion of pre-existing segmental ganglia in the arthropod lineage (Additional files 1, 2). To distinguish between the two types of brain, Richter et al.  suggested the terms “compound” for the arthropod brain and “composite” for the onychophoran brain—a suggestion that receives support from our observations.
Alternatively, assuming that Onychophora form the sister group to tardigrades and arthropods, the last common ancestor of panarthropods might have possessed a monopartite brain and medullary ventral nerve cords (Fig. 10b). Within the onychophoran lineage, neurons of the second body segment migrated towards the protocerebrum and gave rise to a bipartite, composite brain. The last common ancestor of tardigrades and arthropods, on the other hand, evolved separate ganglia. The three anteriormost ganglia fused within the arthropod lineage, thus forming the arthropod compound brain (Fig. 10b).
As neither of the two onychophoran groups (Peripatidae nor Peripatopsidae) show any evidence of a loss of ganglia [17, 18], we favor the second scenario over the first one. Furthermore, our presented findings support the hypothesis that the onychophoran brain evolved by an anterior migration of deutocerebral somata rather than the fusion of two initially separate ganglia.
Specimen collection and maintenance
Specimens of Euperipatoides rowelli Reid, 1996 (Onychophora, Peripatopsidae) were collected from rotted logs in the Tallaganda State Forest (New South Wales, Australia; 35°26′S, 149°33′E, 954 m) in January 2013. Permission for specimen collection was obtained from the Forestry Commission of New South Wales (permit no. SPPR0008). The animals were kept in plastic jars (diameter 55 mm, height 70 mm) with perforated lids at 18 °C as described previously  and fed with decapitated crickets every 4 weeks.
Retrograde fills of selected nerves
Number of fills performed for each commissure and pharyngeal nerve in specimens of Euperipatoides rowelli
Number of fills
Anterior pharyngeal nerve
Posterior pharyngeal nerve
Confocal microscopy, light microscopy and image processing
Whole-mount preparations of the central nervous system were analyzed with a fluorescence microscope (Leica Leitz DMR; Leica Microsystems, Wetzlar, Germany) and a confocal laser-scanning microscope (Leica TCS STED; Leica Microsystems). Confocal image stacks were processed with Leica ASAF v2.3.5 (Leica Microsystems), IMARIS 7.2.1 (Bitplane, Zurich, Switzerland), and ImageJ 1.48v (National Institutes of Health, Bethesda, MD, USA). Final panels and diagrams were designed using Adobe (San Jose, CA, USA) Photoshop CS5 and Illustrator CS5. Animations were designed using Adobe Flash CS5.
CM and GM conceived and designed the experiments. CM performed the experiments. CM and GM analyzed the data and wrote the manuscript. Both authors read and approved the final manuscript.
We are thankful to Noel Tait for organizing the collecting permits, to Paul Sunnucks, David Rowell, Noel Tait, Ivo de Sena Oliveira, Franziska Anni Franke, Sandra Treffkorn, and Michael Gerth for their help with specimen collection and to Vladimir Gross for proofreading the manuscript. The staff members of the State Forests NSW (New South Wales, Australia) are gratefully acknowledged for providing the collecting permits. The Open Access Office of the University of Leipzig is thanked for financial support. This work was supported by a grant from the German Research Foundation (DFG; grant Ma 4147/3-1) to GM, who is a Research Group Leader supported by the Emmy Noether Programme of the DFG.
Compliance with ethical guidelines
Competing interests The authors declare that they have no competing interests.
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- Fanenbruck M, Harzsch S, Wägele JW. The brain of the Remipedia (Crustacea) and an alternative hypothesis on their phylogenetic relationships. Proc Natl Acad Sci USA. 2004;101:3868–73.PubMedView ArticlePubMed CentralGoogle Scholar
- Homberg U. Neuroarchitecture of the central complex in the brain of the locust Schistocerca gregaria and S. americana as revealed by serotonin immunocytochemistry. J Comp Neurol. 1991;303:245–54.PubMedView ArticleGoogle Scholar
- Loesel R, Wolf H, Kenning M, Harzsch S, Sombke A. Architectural principles and evolution of the arthropod central nervous system. In: Minelli A, Boxshall G, Fusco G, editors. Arthropod Biology and Evolution. Heidelberg: Springer; 2013. p. 299–342.View ArticleGoogle Scholar
- Mayer G, Kauschke S, Rüdiger J, Stevenson PA. Neural markers reveal a one-segmented head in tardigrades (water bears). PLoS One. 2013;8:e59090.PubMedView ArticlePubMed CentralGoogle Scholar
- Richter S, Loesel R, Purschke G, Schmidt-Rhaesa A, Scholtz G, Stach T, et al. Invertebrate neurophylogeny: suggested terms and definitions for a neuroanatomical glossary. Front Zool. 2010;7:29.PubMedView ArticlePubMed CentralGoogle Scholar
- Scholtz G, Edgecombe GD. The evolution of arthropod heads: reconciling morphological, developmental and palaeontological evidence. Dev Genes Evol. 2006;216:395–415.PubMedView ArticleGoogle Scholar
- Mayer G. Onychophora. In: Schmidt-Rhaesa A, Harzsch S, Purschke G, editors. Structure and Evolution of Invertebrate Nervous Systems. Oxford: Oxford University Press; 2015 (in press).Google Scholar
- Mayer G, Whitington PM, Sunnucks P, Pflüger H-J. A revision of brain composition in Onychophora (velvet worms) suggests that the tritocerebrum evolved in arthropods. BMC Evol Biol. 2010;10:255.PubMedView ArticlePubMed CentralGoogle Scholar
- Strausfeld NJ, Strausfeld C, Stowe S, Rowell D, Loesel R. The organization and evolutionary implications of neuropils and their neurons in the brain of the onychophoran Euperipatoides rowelli. Arthropod Struct Dev. 2006;35:169–96.PubMedView ArticleGoogle Scholar
- Cong P, Ma X, Hou X, Edgecombe GD, Strausfeld NJ. Brain structure resolves the segmental affinity of anomalocaridid appendages. Nature. 2014;513:538–42.PubMedView ArticleGoogle Scholar
- Eriksson BJ, Samadi L, Schmid A. The expression pattern of the genes engrailed, pax6, otd and six3 with special respect to head and eye development in Euperipatoides kanangrensis Reid 1996 (Onychophora: Peripatopsidae). Dev Genes Evol. 2013;223:237–46.PubMedView ArticlePubMed CentralGoogle Scholar
- Franke FA, Mayer G. Controversies surrounding segments and parasegments in Onychophora: insights from the expression patterns of four “segment polarity genes” in the peripatopsid Euperipatoides rowelli. PLoS One. 2014;9:e114383.PubMedView ArticlePubMed CentralGoogle Scholar
- Mayer G, Martin C, de Sena Oliveira I, Franke FA, Gross V. Latest anomalocaridid affinities challenged. Nature. 2014;516:E1–2.PubMedView ArticleGoogle Scholar
- Martin C, Mayer G. Neuronal tracing of oral nerves in a velvet worm—implications for the evolution of the ecdysozoan brain. Front Neuroanat. 2014;8(7):1–13.Google Scholar
- Ou Q, Shu D, Mayer G. Cambrian lobopodians and extant onychophorans provide new insights into early cephalization in Panarthropoda. Nat Commun. 2012;3:1261.PubMedView ArticlePubMed CentralGoogle Scholar
- Whitington PM, Mayer G. The origins of the arthropod nervous system: insights from the Onychophora. Arthropod Struct Dev. 2011;40:193–209.PubMedView ArticleGoogle Scholar
- Mayer G, Harzsch S. Immunolocalization of serotonin in Onychophora argues against segmental ganglia being an ancestral feature of arthropods. BMC Evol Biol. 2007;7:118.PubMedView ArticlePubMed CentralGoogle Scholar
- Mayer G, Harzsch S. Distribution of serotonin in the trunk of Metaperipatus blainvillei (Onychophora, Peripatopsidae): implications for the evolution of the nervous system in Arthropoda. J Comp Neurol. 2008;507:1196–208.PubMedView ArticleGoogle Scholar
- Mayer G, Whitington PM. Neural development in Onychophora (velvet worms) suggests a step-wise evolution of segmentation in the nervous system of Panarthropoda. Dev Biol. 2009;335:263–75.PubMedView ArticleGoogle Scholar
- Henry LM. The nervous system and the segmentation of the head in the Annulata. Microentomology. 1948;13:27–48.PubMedGoogle Scholar
- von Kennel J. Entwicklungsgeschichte von Peripatus edwardsii Blanch. und Peripatus torquatus n.sp. I. Theil. Arb Zool-Zootom Inst Würzburg. 1885;7:95–229.Google Scholar
- Boyan GS, Reichert H, Hirth F. Commissure formation in the embryonic insect brain. Arthropod Struct Dev. 2003;32:61–77.PubMedView ArticleGoogle Scholar
- Fischer AHL, Scholtz G. Axogenesis in the stomatopod crustacean Gonodactylaceus falcatus (Malacostraca). Invertebr Biol. 2010;129:59–76.View ArticleGoogle Scholar
- Harzsch S. The tritocerebrum of Euarthropoda: a “non-drosophilocentric” perspective. Evol Dev. 2004;6:303–9.PubMedView ArticleGoogle Scholar
- Hirth F, Loop T, Egger B, Miller DFB, Kaufman TC, Reichert H. Functional equivalence of Hox gene products in the specification of the tritocerebrum during embryonic brain development of Drosophila. Development. 2001;128:4781–8.PubMedGoogle Scholar
- Mittmann B, Scholtz G. Development of the nervous system in the “head” of Limulus polyphemus (Chelicerata: Xiphosura): morphological evidence for a correspondence between the segments of the chelicerae and of the (first) antennae of Mandibulata. Dev Genes Evol. 2003;213:9–17.PubMedGoogle Scholar
- Fedorow B. Zur Anatomie des Nervensystems von Peripatus. I. Das Neurosomit von Peripatus tholloni. Zool Jahrb Abt Anat Ontog Tiere. 1926;48:273–310.Google Scholar
- Altman JS, Kien J. Functional organization of the subesophageal ganglion in arthropods. In: Gupta AP, editor. Arthropod brain, its evolution, development, structure, and functions. New York: Wiley; 1987. p. 265–301.Google Scholar
- Kirsch R, Richter S. The nervous system of Leptodora kindtii (Branchiopoda, Cladocera) surveyed with Confocal Scanning Microscopy (CLSM), including general remarks on the branchiopod neuromorphological ground pattern. Arthropod Struct Dev. 2007;36:143–56.PubMedView ArticleGoogle Scholar
- Marcus E. Tardigrada. Dr. H. G. Bronns Klassen und Ordnungen des Tier-Reichs wissenschaftlich dargestellt in Wort und Bild. Leipzig: Akademische Verlagsgesellschaft; 1929. p. 1–609.Google Scholar
- Mayer G, Martin C, Rüdiger J, Kauschke S, Stevenson PA, Poprawa I, et al. Selective neuronal staining in tardigrades and onychophorans provides insights into the evolution of segmental ganglia in panarthropods. BMC Evol Biol. 2013;13:230.PubMedView ArticlePubMed CentralGoogle Scholar
- Persson DK, Halberg KA, Jørgensen A, Møbjerg N, Kristensen RM. Neuroanatomy of Halobiotus crispae (Eutardigrada: Hypsibiidae): Tardigrade brain structure supports the clade Panarthropoda. J Morphol. 2012;273:1227–45.PubMedView ArticleGoogle Scholar
- Schulze C, Neves RC, Schmidt-Rhaesa A. Comparative immunohistochemical investigation on the nervous system of two species of Arthrotardigrada (Heterotardigrada, Tardigrada). Zool Anz. 2014;253:225–35.View ArticleGoogle Scholar
- Zantke J, Wolff C, Scholtz G. Three-dimensional reconstruction of the central nervous system of Macrobiotus hufelandi (Eutardigrada, Parachela): implications for the phylogenetic position of Tardigrada. Zoomorphology. 2008;127:21–36.View ArticleGoogle Scholar
- Gross V, Mayer G. Neural development in the tardigrade Hypsibius dujardini based on anti-acetylated α-tubulin immunolabeling. EvoDevo. 2015;6:12.PubMedView ArticlePubMed CentralGoogle Scholar
- Schürmann FW. Common and special features of the nervous system of Onychophora: a comparison with arthropoda, annelida and some other invertebrates. In: Breidbach O, Kutsch W, editors. The Nervous System of Invertebrates: an evolutionary and comparative approach. Basel: Birkhäuser; 1995. p. 139–58.View ArticleGoogle Scholar
- Campbell LI, Rota-Stabelli O, Edgecombe GD, Marchioro T, Longhorn SJ, Telford MJ, et al. MicroRNAs and phylogenomics resolve the relationships of Tardigrada and suggest that velvet worms are the sister group of Arthropoda. Proc Natl Acad Sci USA. 2011;108:15920–4.PubMedView ArticlePubMed CentralGoogle Scholar
- Dunn CW, Hejnol A, Matus DQ, Pang K, Browne WE, Smith SA, et al. Broad phylogenomic sampling improves resolution of the animal tree of life. Nature. 2008;452:745–9.PubMedView ArticleGoogle Scholar
- Rota-Stabelli O, Kayal E, Gleeson D, Daub J, Boore J, Telford M, et al. Ecdysozoan mitogenomics: evidence for a common origin of the legged invertebrates, the Panarthropoda. Genome Biol Evol. 2010;2:425–40.PubMedView ArticlePubMed CentralGoogle Scholar
- Telford MJ, Bourlat SJ, Economou A, Papillon D, Rota-Stabelli O. The evolution of the Ecdysozoa. Philos Trans R Soc B Biol Sci. 2008;363:1529–37.View ArticleGoogle Scholar
- Baer A, Mayer G. Comparative anatomy of slime glands in Onychophora (velvet worms). J Morphol. 2012;273:1079–88.PubMedView ArticleGoogle Scholar
- Robson EA, Lockwood APM, Ralph R. Composition of the blood in Onychophora. Nature. 1966;209:533.View ArticleGoogle Scholar
- Pflüger HJ, Field LH. A locust chordotonal organ coding for proprioceptive and acoustic stimuli. J Comp Physiol A. 1999;184:169–83.View ArticleGoogle Scholar