- Research article
- Open Access
A revision of brain composition in Onychophora (velvet worms) suggests that the tritocerebrum evolved in arthropods
© Mayer et al; licensee BioMed Central Ltd. 2010
- Received: 13 May 2010
- Accepted: 21 August 2010
- Published: 21 August 2010
The composition of the arthropod head is one of the most contentious issues in animal evolution. In particular, controversy surrounds the homology and innervation of segmental cephalic appendages by the brain. Onychophora (velvet worms) play a crucial role in understanding the evolution of the arthropod brain, because they are close relatives of arthropods and have apparently changed little since the Early Cambrian. However, the segmental origins of their brain neuropils and the number of cephalic appendages innervated by the brain - key issues in clarifying brain composition in the last common ancestor of Onychophora and Arthropoda - remain unclear.
Using immunolabelling and neuronal tracing techniques in the developing and adult onychophoran brain, we found that the major brain neuropils arise from only the anterior-most body segment, and that two pairs of segmental appendages are innervated by the brain. The region of the central nervous system corresponding to the arthropod tritocerebrum is not differentiated as part of the onychophoran brain but instead belongs to the ventral nerve cords.
Our results contradict the assumptions of a tripartite (three-segmented) brain in Onychophora and instead confirm the hypothesis of bipartite (two-segmented) brain composition. They suggest that the last common ancestor of Onychophora and Arthropoda possessed a brain consisting of protocerebrum and deutocerebrum whereas the tritocerebrum evolved in arthropods.
- Nerve Cord
- Ventral Nerve Cord
- Cerebral Ganglion
- Benzyl Benzoate
- Segmental Identity
The head of arthropods is a specialised anterior body region, which is distinguished by fused segments and several pairs of modified appendages [1, 2]. These appendages serve for swimming, feeding, defence, or sensory perception, and their movements are coordinated by a complex brain situated within the head. Despite over a century of intense research in this area, the ancestral composition of the arthropod head remains obscure and is one of the most controversial topics in zoology [2–8]. Fossils have contributed much to our knowledge [1, 4, 8], but their limited preservation constrains definitive conclusions about the degree of cephalisation in the last common ancestor of Panarthropoda (Onychophora + Tardigrada + Arthropoda).
Based on various studies of embryology [14–20], including the expression data of the anterior Hox genes labial, proboscipedia, Hox3 and Deformed , the onychophoran "head" appendages can therefore be aligned with the corresponding appendages of arthropods (Figure 1C). According to this alignment, the onychophoran antennae are either serial homologues of the arthropod labrum or, alternatively, the corresponding pair of appendages may have been lost in arthropods - an issue that is still controversial [5, 27–29]. (It has also been argued that the arthropod labrum is a modified appendage of the third body segment . However, the Hox gene expression data referred to above, together with the common expression of the anterior marker six3 in the insect labrum and onychophoran antenna , speak against this possibility.) Since the onychophoran antennae belong to the anterior-most body segment bearing the eyes [19, 20], they cannot be homologised with the chelicerae of chelicerates or the (first) antennae of crustaceans, insects, and myriapods, which belong to the second body segment [2, 3, 31]. The chelicerae and the (first) antennae of arthropods are instead serially homologous to the onychophoran jaws (Figure 1C). The onychophoran slime papillae are, in turn, serially homologous to the pedipalps of chelicerates and to the second antennae of crustaceans whereas the corresponding pair of appendages was lost in hexapods and myriapods [review ].
One feature that has previously been used to determine the segmental organisation of the brain in Onychophora is the position and number of transverse neuropils in the adult . Three major neuropils have been identified, leading to the conclusion that the onychophoran brain is tripartite. However, this rests on the assumption that each neuropil arises from a separate segment during development - an issue, which has not been clarified thus far. An additional feature that could be used to identify the degree of segmentation of the onychophoran brain is the position of neuronal cell bodies innervating the head appendages. If the cell bodies of neurons innervating the tritocerebrum were found to lie within the brain (Figure 2B), the hypothesis of tripartite organisation  would be supported. In contrast, a position of these neuronal cell bodies found outside the brain (Figure 2C) would speak against the existence of the tritocerebrum in Onychophora.
To clarify the segmental composition of the onychophoran brain, we combined two approaches. First, we studied brain development to determine the embryonic origin of transverse neuropils. Second, we analysed the position of neuronal cell bodies innervating the cephalic appendages. Our results show that the major transverse neuropils of the onychophoran brain arise from only one (the anterior-most) body segment, and that only the antennae and jaws are innervated by the brain. These findings suggest that the onychophorans show a lower degree of cephalisation in relation to their brain organisation than the arthropods and that the tritocerebrum was not integrated into the brain in the last common ancestor of Onychophora and Arthropoda.
The formation of onychophoran brain neuropils involves only one segment
Despite two recent and extensive studies of brain development in Onychophora [19, 40], the embryonic origin and segmental identities of transverse brain neuropils, other than the first ("antennal") commissure, remain unclear. Strausfeld et al.  subdivided the adult onychophoran brain into protocerebrum, deutocerebrum and tritocerebrum by analysing series of histological and silver- and osmium-stained sections and assessing the number and spatial separation of brain neuropils. To clarify whether these brain neuropils have independent origins from different segments, we examined brain development in onychophoran embryos using an antibody raised against acetylated α-tubulin. This antibody labels mainly nerve tracts and neuropils in the developing nervous system [19, 40–42].
Retrograde axonal tracing reveals that the tritocerebrum is absent from the onychophoran brain
The position of neurons that project out the segmental nerves within the onychophoran head might be a key feature for determining the segmental identity of different brain regions. We therefore performed retrograde axonal tracing studies (backfills) of segmental cephalic nerves in adult onychophorans, using dextran coupled to different fluorochromes as a tracer .
In summary, our findings suggest an increase in the number of segmental brain regions in the (pan)arthropod lineage, from two in the last common ancestor of Onychophora and Arthropoda, to at least three in various arthropods [e.g. [2, 27, 31, 32]]. This evolutionary sequence may help clarify the phylogenetic position of Tardigrada (water bears), which is still controversial. Currently, tardigrades are regarded as either the sister group of arthropods, of onychophorans, of onychophorans plus arthropods, or of one of the cycloneuralian taxa (nematodes, kinorhynchs, and allies) [10, 11, 41, 46–54]. Our findings suggest that the number of segments in the tardigrade brain, which remains unclear [48, 55–58], will be a key feature in elucidating the position of this animal group within the Ecdysozoa.
Furthermore, our suggestion of a two-segmented brain in the last common ancestor of Onychophora and Arthropoda challenges the hypothesis that a tripartite brain existed in the last common ancestor of the bilaterally symmetrical animals, the so-called "urbilaterian" [59, 60]. Such a brain is absent in all protostomes apart from arthropods. Moreover, the closest relatives of chordates, including hemichordates and echinoderms , lack a centralised brain. We therefore suggest that similar gene expression patterns in the anterior body region of arthropods and vertebrates [59, 60] are not related to brain segmentation but rather to a general patterning of the antero-posterior body axis in these animals.
Specimens of Euperipatoides rowelli Reid, 1996 and Epiperipatus isthmicola (Bouvier, 1902) were collected and handled and the embryos staged and labelled with an antibody raised against acetylated α-tubulin as described previously [41, 42]. For neuronal tracing, adult brain nerves were dissected in physiological saline based on onychophoran blood composition . Retrograde fills of the antennal nerves (n = 3), jaw nerves (n = 9), and slime papillae nerves (n = 7) were carried out with dextran (MW 3000) coupled to either tetramethylrhodamine or fluorescein according to standard procedures used for arthropods . Scanning electron microscopy and immunohistochemistry were performed as described previously . Stained specimens were dehydrated through a methanol series and mounted between two cover slips in a 2:1 mixture of benzyl benzoate and benzyl alcohol. Confocal laser-scanning microscopy and image processing were carried out as described previously [41, 42].
This work was supported by a grant from the German Research Foundation (DFG) to GM (Ma 4147/3-1). GM is a Research Group Leader supported by the Emmy Noether Programme of the DFG.
- Budd GE: A palaeontological solution to the arthropod head problem. Nature. 2002, 417: 271-275. 10.1038/417271a.View ArticlePubMedGoogle Scholar
- Scholtz G, Edgecombe GD: The evolution of arthropod heads: reconciling morphological, developmental and palaeontological evidence. Dev Genes Evol. 2006, 216: 395-415. 10.1007/s00427-006-0085-4.View ArticlePubMedGoogle Scholar
- Scholtz G, Edgecombe GD: Heads, Hox and the phylogenetic position of trilobites. Crustacea and Arthropod Phylogeny. Edited by: Koenemann S, Jenner R, Vonk R. 2005, Boca Raton: CRC Press, 16: 139-165. full_text.View ArticleGoogle Scholar
- Chen J, Waloszek D, Maas A: A new "great appendage" arthropod from the Lower Cambrian of China and homology of chelicerate chelicerae and raptorial antero-ventral appendages. Lethaia. 2004, 37: 3-20.Google Scholar
- Budd GE, Telford MJ: The origin and evolution of arthropods. Nature. 2009, 457: 812-817. 10.1038/nature07890.View ArticlePubMedGoogle Scholar
- Jager M, Murienne J, Clabaut C, Deutsch J, Le Guyander H, Manuel M: Homology of arthropod anterior appendages revealed by Hox gene expression in a sea spider. Nature. 2006, 441: 506-508. 10.1038/nature04591.View ArticlePubMedGoogle Scholar
- Maxmen A, Browne WE, Martindale MQ, Giribet G: Neuroanatomy of sea spiders implies an appendicular origin of the protocerebral segment. Nature. 2005, 437: 1144-1148. 10.1038/nature03984.View ArticlePubMedGoogle Scholar
- Waloszek D, Maas A, Chen J, Stein M: Evolution of cephalic feeding structures and the phylogeny of Arthropoda. Palaeogeography, Palaeoclimatology, Palaeoecology. 2007, 254: 273-287. 10.1016/j.palaeo.2007.03.027.View ArticleGoogle Scholar
- Kusche K, Ruhberg H, Burmester T: A hemocyanin from the Onychophora and the emergence of respiratory proteins. Proc Natl Acad Sci USA. 2002, 99: 10545-10548. 10.1073/pnas.152241199.PubMed CentralView ArticlePubMedGoogle Scholar
- Mallatt J, Giribet G: Further use of nearly complete 28 S and 18 S rRNA genes to classify Ecdysozoa: 37 more arthropods and a kinorhynch. Mol Biol Evol. 2006, 40: 772-794.Google Scholar
- Dunn CW, Hejnol A, Matus DQ, Pang K, Browne WE, Smith SA, Seaver E, Rouse GW, Obst M, Edgecombe GD, et al: Broad phylogenomic sampling improves resolution of the animal tree of life. Nature. 2008, 452: 745-749. 10.1038/nature06614.View ArticlePubMedGoogle Scholar
- Budd GE: Why are arthropods segmented?. Evol Dev. 2001, 3: 332-342. 10.1046/j.1525-142X.2001.01041.x.View ArticlePubMedGoogle Scholar
- Maas A, Mayer G, Kristensen RM, Waloszek D: A Cambrian micro-lobopodian and the evolution of arthropod locomotion and reproduction. Chin Sci Bull. 2007, 52: 3385-3392. 10.1007/s11434-007-0515-3.View ArticleGoogle 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
- von Kennel J: Entwicklungsgeschichte von Peripatus edwardsii Blanch. und Peripatus torquatus n.sp. II. Theil. Arb Zool -Zootom Inst Würzburg. 1888, 8: 1-93.Google Scholar
- Sedgwick A: The development of the Cape species of Peripatus. Part III. On the changes from stage A to stage F. Q J Microsc Sci. 1887, 27: 467-550.Google Scholar
- Evans R: On the Malayan species of Onychophora. Part II. - The development of Eoperipatus weldoni. Q J Microsc Sci. 1901, 45: 41-88.Google Scholar
- Walker M, Campiglia S: Some aspects of segment formation and post-placental development in Peripatus acacioi Marcus and Marcus (Onychophora). J Morphol. 1988, 195: 123-140. 10.1002/jmor.1051950202.View ArticleGoogle Scholar
- Eriksson BJ, Tait NN, Budd GE: Head development in the onychophoran Euperipatoides kanangrensis. With particular reference to the central nervous system. J Morphol. 2003, 255: 1-23. 10.1002/jmor.10034.View ArticlePubMedGoogle Scholar
- Mayer G, Koch M: Ultrastructure and fate of the nephridial anlagen in the antennal segment of Epiperipatus biolleyi (Onychophora, Peripatidae) - evidence for the onychophoran antennae being modified legs. Arthropod Struct Dev. 2005, 34: 471-480. 10.1016/j.asd.2005.03.004.View ArticleGoogle Scholar
- Henry LM: The nervous system and the segmentation of the head in the Annulata. Microentomology. 1948, 13: 27-48.PubMedGoogle Scholar
- Fedorow B: Zur Anatomie des Nervensystems von Peripatus. II. Das Nervensystem des vorderen Körperendes und seine Metamerie. Zool Jb Anat. 1929, 50: 279-332.Google Scholar
- Pflugfelder O: Entwicklung von Paraperipatus amboinensis n. sp. Zool Jb Anat. 1948, 69: 443-492.Google Scholar
- Pflugfelder O: Onychophora. Grosses Zoologisches Praktikum. Edited by: Czihak G. 1968, Stuttgart: Gustav Fischer, 1-42. 13aGoogle Scholar
- Eriksson BJ, Tait NN, Budd GE, Akam M: The involvement of engrailed and wingless during segmentation in the onychophoran Euperipatoides kanangrensis (Peripatopsidae: Onychophora) (Reid 1996). Dev Genes Evol. 2009, 219: 249-264. 10.1007/s00427-009-0287-7.View ArticlePubMedGoogle Scholar
- Eriksson BJ, Tait NN, Budd GE, Janssen R, Akam M: Head patterning and Hox gene expression in an onychophoran and its implications for the arthropod head problem. Dev Genes Evol. 2010Google Scholar
- Harzsch S: Neurophylogeny: Architecture of the nervous system and a fresh view on arthropod phyologeny. Integr Comp Biol. 2006, 46: 162-194. 10.1093/icb/icj011.View ArticlePubMedGoogle Scholar
- Liu Y, Maas A, Waloszek D: Early development of the anterior body region of the grey widow spider Latrodectus geometricus Koch, 1841 (Theridiidae, Araneae). Arthropod Struct Dev. 2009, 38: 401-416. 10.1016/j.asd.2009.04.001.View ArticlePubMedGoogle Scholar
- Posnien N, Bashasab F, Bucher G: The insect upper lip (labrum) is a nonsegmental appendage-like structure. Evol Dev. 2009, 11: 480-488. 10.1111/j.1525-142X.2009.00356.x.View ArticlePubMedGoogle Scholar
- Boyan GS, Williams JLD, Posser S, Bräunig P: Morphological and molecular data argue for the labrum being non-apical, articulated, and the appendage of the intercalary segment in the locust. Arthropod Struct Dev. 2002, 31: 65-76. 10.1016/S1467-8039(02)00016-6.View ArticlePubMedGoogle 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: 169-196. 10.1016/j.asd.2006.06.002.View ArticlePubMedGoogle Scholar
- Urbach R, Technau GM: Early steps in building the insect brain: neuroblast formation and segmental patterning in the developing brain of different insect species. Arthropod Struct Dev. 2003, 32: 103-123. 10.1016/S1467-8039(03)00042-2.View ArticlePubMedGoogle Scholar
- Harzsch S: The tritocerebrum of Euarthropoda: a "non-drosophilocentric" perspective. Evol Dev. 2004, 6: 303-309. 10.1111/j.1525-142X.2004.04038.x.View ArticlePubMedGoogle Scholar
- Boyan GS, Williams JLD, Hirth F: Commissural organization and brain segmentation in insects. Theories, Development, Invertebrates. Edited by: Striedter GF, Rubenstein JLR. 2007, Oxford: Academic Press, 1: 349-359.Google Scholar
- Urbach R, Technau GM: Segmental organization of cephalic ganglia in arthropods. Theories, Development, Invertebrates. Edited by: Striedter GF. 2007, Rubenstein JLR. Oxford: Academic Press, 1: 337-348.Google Scholar
- Holmgren NF: Zur vergleichenden Anatomie des Gehirns von Polychaeten, Onychophoren, Xiphosuren, Arachniden, Crustaceen, Myriapoden, und Insekten. Vorstudien zu einer Phylogenie der Arthropoden. K Svenska Vet Handl [Ser 2]. 1916, 56: 1-303.Google Scholar
- Hanström B: Onychophora. Vergleichende Anatomie des Nervensystems der Wirbellosen Tiere unter Berücksichtigung seiner Funktion. 1928, Berlin, Germany: J. Springer, 341-351.Google Scholar
- Schürmann FW: Histology and ultrastructure of the onychophoran brain. Arthropod Brain, its Evolution, Development, Structure, and Functions. Edited by: Gupta AP. 1987, New York: John Wiley & Sons, 159-180.Google Scholar
- Lane NJ, Campiglia SS: The lack of a structured blood-brain barrier in the onychophoran Peripatus acacioi. J Neurocytol. 1987, 16: 93-104. 10.1007/BF02456701.View ArticlePubMedGoogle Scholar
- Eriksson BJ, Budd GE: Onychophoran cephalic nerves and their bearing on our understanding of head segmentation and stem-group evolution of Arthropoda. Arthropod Struct Dev. 2000, 29: 197-209. 10.1016/S1467-8039(00)00027-X.View ArticlePubMedGoogle 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: 263-275. 10.1016/j.ydbio.2009.08.011.View ArticlePubMedGoogle Scholar
- Mayer G, Whitington PM: Velvet worm development links myriapods with chelicerates. Proc R Soc B. 2009, 276: 3571-3579. 10.1098/rspb.2009.0950.PubMed CentralView ArticlePubMedGoogle Scholar
- Pflüger H-J, Field LH: A locust chordotonal organ coding for proprioceptive and acoustic stimuli. J Comp Physiol A. 1999, 184: 169-183. 10.1007/s003590050316.View ArticleGoogle Scholar
- Strausfeld NJ, Strausfeld CM, Loesel R, Rowell D, Stowe S: Arthropod phylogeny: onychophoran brain organization suggests an archaic relationship with a chelicerate stem lineage. Proc R Soc B. 2006, 273: 1857-1866. 10.1098/rspb.2006.3536.PubMed CentralView ArticlePubMedGoogle Scholar
- Hanström B: Bemerkungen über das Gehirn und die Sinnesorgane der Onychophoren. Lunds Univ Årsskrift NF. 1935, 31: 1-37.Google Scholar
- Budd GE: Tardigrades as 'stem-group arthropods': The evidence from the Cambrian fauna. Zool Anz. 2001, 240: 265-279. 10.1078/0044-5231-00034.View ArticleGoogle Scholar
- Maas A, Waloszek D: Cambrian derivatives of the early arthropod stem lineage, Pentastomids, Tardigrades and Lobopodians - an "Orsten" perspective. Zool Anz. 2001, 240: 451-459. 10.1078/0044-5231-00053.View ArticleGoogle Scholar
- Nielsen C: Animal Evolution: Interrelationships of the Living Phyla. 2001, Oxford: Oxford University PressGoogle Scholar
- Telford MJ, Bourlat SJ, Economou A, Papillon D, Rota-Stabelli O: The evolution of the Ecdysozoa. Phil Trans R Soc B. 2008, 363: 1529-1537. 10.1098/rstb.2007.2243.PubMed CentralView ArticlePubMedGoogle Scholar
- Jenner RA, Scholtz G: Playing another round of metazoan phylogenetics: Historical epistemology, sensitivity analysis, and the position of Arthropoda within the Metazoa on the basis of morphology. Crustacea and Arthropod Relationships. Edited by: Koenemann S, Jenner RA. 2005, Boca Raton: CRC Press, 16: 355-385. full_text.View ArticleGoogle Scholar
- Park J-K, Rho HS, Kristensen RM, Kim W, Giribet G: First molecular data on the phylum Loricifera - an investigation into the phylogeny of Ecdysozoa with emphasis on the positions of Loricifera and Priapulida. Zool Sci. 2006, 23: 943-954. 10.2108/zsj.23.943.View ArticlePubMedGoogle Scholar
- Lartillot N, Philippe H: Improvement of molecular phylogenetic inference and the phylogeny of Bilateria. Phil Trans R Soc B. 2008, 363: 1463-1472. 10.1098/rstb.2007.2236.PubMed CentralView ArticlePubMedGoogle Scholar
- Hejnol A, Obst M, Stamatakis A, Ott M, Rouse GW, Edgecombe GD, Martinez P, Baguñà J, Bailly X, Jondelius U, et al: Assessing the root of bilaterian animals with scalable phylogenomic methods. Proc R Soc B. 2009, 276: 4261-4270. 10.1098/rspb.2009.0896.PubMed CentralView ArticlePubMedGoogle Scholar
- Rota-Stabelli O, Kayal E, Gleeson D, Daub J, Boore J, Telford M, Pisani D, Blaxter M, Lavrov D: Ecdysozoan mitogenomics: Evidence for a common origin of the legged invertebrates, the Panarthropoda. Genome Biology and Evolution. 2010Google Scholar
- Kristensen RM, Higgins RP: Revision of Styraconyx (Tardigrada: Halechiniscidae) with descriptions of two new species from Disko Bay, West Greenland. Smithson Contrib Zool. 1984, 391: 1-40.View ArticleGoogle Scholar
- Dewel RA, Dewel WC: The brain of Echiniscus viridissimus Peterfi, 1956 (Heterotardigrada): a key to understanding the phylogenetic position of tardigrades and the evolution of the arthropod head. Zool J Linn Soc. 1996, 116: 35-49. 10.1111/j.1096-3642.1996.tb02331.x.View ArticleGoogle Scholar
- Hejnol A, Schnabel R: What a couple of dimensions can do for you: Comparative developmental studies using 4 D microscopy - examples from tardigrade development. Integr Comp Biol. 2006, 46: 151-161. 10.1093/icb/icj012.View ArticlePubMedGoogle 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. 10.1007/s00435-007-0045-1.View ArticleGoogle Scholar
- Hirth F, Kammermeier L, Frei E, Walldorf U, Noll M, Reichert H: An urbilaterian origin of the tripartite brain: developmental genetic insights from Drosophila. Development. 2003, 130: 2365-2373. 10.1242/dev.00438.View ArticlePubMedGoogle Scholar
- Urbach R: A procephalic territory in Drosophila exhibiting similarities and dissimilarities compared to the vertebrate midbrain/hindbrain boundary region. Neural Dev. 2007, 2: 23-10.1186/1749-8104-2-23.PubMed CentralView ArticlePubMedGoogle Scholar
- Robson EA, Lockwood APM, Ralph R: Composition of the blood in Onychophora. Nature. 1966, 209: 533-10.1038/209533a0.View ArticleGoogle Scholar
- Mayer G: Structure and development of onychophoran eyes - what is the ancestral visual organ in arthropods?. Arthropod Struct Dev. 2006, 35: 231-245. 10.1016/j.asd.2006.06.003.View ArticlePubMedGoogle Scholar
- Edgecombe GD: Arthropod phylogeny: An overview from the perspectives of morphology, molecular data and the fossil record. Arthropod Struct Dev. 2010, 39: 74-87. 10.1016/j.asd.2009.10.002.View ArticlePubMedGoogle Scholar
- Regier JC, Shultz JW, Zwick A, Hussey A, Ball B, Wetzer R, Martin JW, Cunningham CW: Arthropod relationships revealed by phylogenomic analysis of nuclear protein-coding sequences. Nature. 2010, 463: 1079-1083. 10.1038/nature08742.View ArticlePubMedGoogle Scholar
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